U.S. patent application number 10/634165 was filed with the patent office on 2005-05-05 for detection of hiv-1 dna.
This patent application is currently assigned to Government of the USA, represented by the Secretary, Dept. of Healthe & Human Services. Invention is credited to Chang, Nancy T., Gallo, Robert C., Wong-Staal, Flossie.
Application Number | 20050095581 10/634165 |
Document ID | / |
Family ID | 27417706 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050095581 |
Kind Code |
A1 |
Chang, Nancy T. ; et
al. |
May 5, 2005 |
Detection of HIV-1 DNA
Abstract
The determination of the nucleotide sequence of HTLV-III DNA;
identification, isolation and expression of HTLV-III sequences
which encode immunoreactive polypeptides by recombinant,DNA methods
and production of viral RNA are disclosed. Such polypeptides can be
employed in immunoassays to detect HTLV-III.
Inventors: |
Chang, Nancy T.; (Houston,
TX) ; Gallo, Robert C.; (Bethesda, MD) ;
Wong-Staal, Flossie; (San Diego, CA) |
Correspondence
Address: |
LEYDIG, VOIT & MAYER, LTD.
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
Government of the USA, represented
by the Secretary, Dept. of Healthe & Human Services
Office of Technology Tranfer 6011 Executive Boulevard, Suite
325
Rockville
MD
20852
|
Family ID: |
27417706 |
Appl. No.: |
10/634165 |
Filed: |
August 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10634165 |
Aug 5, 2003 |
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08463028 |
Jun 5, 1995 |
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6610476 |
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08463028 |
Jun 5, 1995 |
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06693866 |
Jan 23, 1985 |
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06693866 |
Jan 23, 1985 |
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06659339 |
Oct 10, 1984 |
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06659339 |
Oct 10, 1984 |
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06643306 |
Aug 22, 1984 |
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Current U.S.
Class: |
435/5 ; 435/325;
435/456; 435/69.3; 530/350; 536/23.72 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 2740/14022 20130101; C07K 16/1054 20130101; C12Q 1/702
20130101; C07K 14/005 20130101; C12Q 1/703 20130101; A61K 38/00
20130101; C12N 2740/16122 20130101; C07K 16/1063 20130101 |
Class at
Publication: |
435/005 ;
435/069.3; 435/456; 435/325; 530/350; 536/023.72 |
International
Class: |
C12Q 001/70; C07H
021/02; C07K 014/16; C12N 015/86 |
Claims
1. Immunoreactive HTLV-III polypeptide expressed by cells
transformed with a recombinant vector containing HTLV-III cDNA.
2. A polypeptide of claim 1 wherein said HTLV-III cDNA encodes an
env gene sequence.
3. A polypeptide of claim 2 which is immunoreactive with sera of
patients with acquired immunodeficiency syndrome or sera containing
antibodies to HTLV-III.
4. A polypeptide of claim 1 wherein said HTLV-III cDNA encodes an
env-lor gene sequence.
5. A polypeptide of claim 4 which is immunoreactive with sera of
patients with acquired immunodeficiency syndrome or sera containing
antibodies to HTLV-III.
6. A polypeptide of claim 1 wherein said HTLV-III cDNA is an EcoRI
restriction fragment.
7. A polypeptide of claim 6 which is immunoreactive with sera of
patients with acquired immunodeficiency syndrome or sera containing
antibodies to HTLV-III.
8. Isolated HTLV-III envelope polypeptide.
9. Isolated HTLV-III polypeptide encoded by an env-lor gene
sequence.
10. Isolated cDNA encoding an HTLV-III gene.
11. cDNA of claim 10 encoding the HTLV-III env gene.
12. cDNA of claim 10 encoding the HTLV-III env-lor gene
sequence.
13. cDNA of claim 10 encoding an EcoRI restriction fragment of
HITLV-III cDNA which encodes a polypeptide immunoreactive with sera
of patients with acquired immunodeficiency syndrome or sera
containing antibodies to HTLV-III.
14. Isolated cDNA encoding for an HTLV-III polypeptide which is
immunoreactive.
15. Isolated cDNA of claim 14 encoding for an envelope polypeptide
which is immunoreactive with sera of patients with acquired
immunodeficiency syndrome or sera containing antibodies to
HTLV-III.
16. Isolated cDNA of claim 14 which is an EcoRI restriction
fragment.
17. A DNA probe comprising a DNA sequence which is essentially
homologous to a portion of the HTLV-III genome unique to the
virus.
18. A DNA probe of claim 17 wherein the DNA sequence is essentially
homologous to a portion of the HTLV III genome which encodes a
polypeptide immunoreactive with sera of AIDS patents.
19. A hybrid protein comprising an HTLV-III polypeptide linked to
at least one other polypeptide.
20. A hybrid protein of claim 19 comprising an HTLV-III polypeptide
linked to an indicator polypeptide.
21. A hybrid protein of claim 20 wherein said indicator polypeptide
comprises beta-galactosidase.
22. An isolated RNA transcript of the env gene of HTLV-III.
23. An isolated RNA transcript of claim 22 having a label which
emits a detectable signal.
24. An isolated RNA transcript of claim 23 wherein said label
comprises a radioisotope.
25. A recombinant vector containing HTLV-III DNA capable of
expression upon insertion into host cells.
26. OmpA vector containing HTLV-III cDNA.
27. pMR 100 vector containing HTLV-III cDNA.
28. A method of producing HTLV-III polypeptide, comprising the
steps of: a. cleaving HTLV-III cDNA to produce cDNA fragments; b.
inserting the cDNA fragments into an expression vector to form a
recombinant vector; c. transforming an appropriate host cell with
the recombinant vector; and d. culturing the transformed host cell
under conditions sufficient for expression of the polypeptide
encoded for by the inserted HTLV-III cDNA.
29. A method of claim 28 wherein the cleaving step comprises
digesting the HTLV-III cDNA with restriction endonucleases to
produce restriction fragments of cDNA.
30. A method of claim 28 wherein the cleaving step comprises
shearing the HTLV-III cDNA to produce cDNA fragments.
31. A method of producing HTLV-III envelope polypeptide, comprising
the steps of: a. cleaving HTLV-III genomic cDNA with the
restriction endonuclease Sstl; b. digesting the cleaved cDNA with
restriction endonucleases sufficient to generate restriction
fragments which encompass at least a portion of the env gene; c.
isolating the restriction fragments; d. producing DNA fragments of
about 200-500 base pairs in length from the restriction fragments;
e. isolating the DNA fragments of about 200-500 base pairs; f.
inserting the isolated fragments into the open reading frame
expression vector pMR100 for production of hybrid proteins
comprising an env gene product and beta-galactosidase; g.
transforming lac z E. coli cells with the vector; h. plating the
transformed cells on MacConkey agar plates, maintaining the plates
under conditions sufficient for the formation of colonies and
selecting cell colonies exhibiting a red color; i. culturing
transformed cells from the selected colonies under conditions which
allow expression of the hybrid protein; j. obtaining cellular
protein from the cultured transformed cells; k. separating the
cellular protein obtained; l. contacting the separated protein with
sera from AIDS patients to identify protein which is immunoreactive
with the sera; and m. isolating the immunoreactive protein.
32. A fusion protein produced by the method of claim 31.
33. A method of claim 31, further comprising the step of separating
the env gene expression product from the remainder of the hybrid
protein.
34. A HTLV-III envelope polypeptide produced by the method of claim
33.
35. Antibody specifically reactive with HTLV-III envelope
polypeptide.
36. An antibody of claim 35 which is monoclonal.
37. Anibody specifically reactive with HTLV-III polypeptide
produced by recombinant DNA techniques.
38. An antibody of claim 37 which is monoclonal.
39. An immunoassay for the detection of HTLV-III employing antibody
which reacts specifically with HTLV-III polypeptide produced by
recombinant DNA techniques.
40. An immunoassay for the detection of HTLV-III employing antibody
which reacts specifically with HTLV-III envelope polypeptide.
41. An immunoassay of claim 40 wherein said antibody is
monoclonal.
42. A sandwich type immunoradiometric assay for the detection of
HTLC III employing an immobilized antibody which reacts with
HTLV-III polypeptide and a soluble antibody which reacts with
HTLV-III polypeptide.
43. An assay kit comprising an antibody which reacts specifically
with HTLV-III polypeptide bound to a solid phase and a labeled
soluble antibody which reacts specifically with HTLV-III
polypeptide.
44. A method of detecting antibodies against HTLV-III in a bodily
fluid comprising the steps of: a. contacting an immunoadsorbent
comprising an HTLV-III polypeptide bound to a solid phase with a
bodily fluid until antibodies against HTLV-III polypeptide in the
bodily fluid bind the solid phase polypeptide; b. separating the
immunoadsorbent from the bodily fluid; c. contacting the
immunoadsorbent with a labeled HTLV-III polypeptide or labeled
antibody against human immunoglobulin; and d. determining the
amount of labeled polypeptide bound to immunoadsorbent as an
indication of antibody to HTLV-III.
45. A kit for determining the presence of antibody against HTLV-III
in a bodily fluid comprising: a. an immunoadsorbent comprising a
HTLV-III polypeptide bound to a solid phase; and b. labeled
HLTV-III polypeptide or a labeled antibody against human
immunoglobulin.
46. A method of detecting HTLV-III nucleic acid in a bodily fluid
comprising the steps of: a. adsorbing the nucleic acid in a bodily
fluid onto an adsorbent; b. denaturing the adsorbed nucleic acid;
c. contacting the adsorbed nucleic acid with a HTLV-III DNA or RNA
probe; and d. determining if the probe hybridizes with the adsorbed
nucleic acid.
47. A method of claim 46 wherein the bodily fluid is a cell
lysate.
48. A hybridoma cell line which produces antibody specifically
reactive with HTLV-III envelope polypeptide.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 659,339, filed Oct. 10, 1984.
TECHNICAL FIELDS
[0002] This invention is in the fields of molecular biology and
virology and in particular relates to human T cell leukemia
virus--type III (HTLV-III).
BACKGROUND
[0003] The term human T cell leukemia-lymphoma virus (HTLV) refers
to a unique family of T cell tropic retroviruses. These viruses
play an important role in the pathogenesis of certain T cell
neoplasms. There are presently three known types of HTLVs. One
subgroup of the family, HTLV-type I (HTLV-I), is linked to the
cause of adult T-cell leukemia-lymphoma (ATLL) that occurs in
certain regions of Japan, the Caribbean and Africa. HTLV-type II
(HTLV-II) has been isolated from a patient with a T-cell variant of
hairy cell leukemia. M. Popovic et al., Detection, Isolation, and
Continuous Production of Cytopathic Retroviruses (HTLV-III) from
Patients with AIDS and Pre-AIDS. Science, 224:497-500 (1984).
[0004] HTLV-type III (HTLV-III) has been isolated from many
patients with acquired immunodeficiency syndrome (AIDS). HTLV-III
refers to prototype virus isolated from AIDS patients. Groups
reported to be at greatest risk for AIDS include homosexual or
bisexual males; intravenous drug users and Haitian immigrants to
the United States. Hemophiliacs who receive blood products pooled
from donors and recipients of multiple blood transfusions are also
at risk. Clinical manifestations of AIDS include severe,
unexplained immune deficiency which generally involves a depletion
of helper T lymphocytes. These may be accompanied by malignancies
and infections. The mortality rate for patients with AIDS is high.
A less severe form of AIDS also exists, in which there may be
lymphadenopathy and depressed helper T cell counts; there is not,
however, the devastating illness characteristic of full-blown AIDS.
There are many individuals, who are classified as having early AIDS
(pre-AIDS), who exhibit these signs. It is not now possible to
predict who among them will develop the more serious symptoms.
[0005] Much of the evidence implicates HTLV-III as the etiological
agent of the infectious AIDS. First, there is consistent
epidemiology; greater than 95% of the patients with AIDS have
antibodies specific for HTLV-III. Second, there has been
reproducible identification and isolation of virus in this disease;
more than 100 variants of HTLV-III have been isolated from AIDS
patients. Third, there has been transmission of the disease to
normal healthy individuals who received blood transfusions from
infected blood donors.
[0006] HTLV-III has been shown to share several properties with
HTLV-I and HTLV-II but also to be morphologically, biologically and
antigenically distinguishable. R. C. Gallo et al., Frequent
Detection and Isolation of Cytopathic Retroviruses (HTLV-III) from
Patients with AIDS and At Risk for AIDS. Science, 224:500-503.
(1984). For example, HTLV-III has been shown to be antigenically
related to HTLV-I and HTLV-II by demonstrating cross-reactivity
with antibodies to HTLV-I and HTLV-II core proteins, p24 and p19,
and envelope antigens and by nucleic acid cross-hybridization
studies with cloned HTLV-I and HTLV-II DNAs. However, unlike HTLV-I
and HTLV-II, it lacked the ability to infect and transform T cells
from normal umbilical cord blood and bone marrow in vitro, and has
the cytopathic effect on infected cells only.
[0007] Like the RNA genome of other retroviruses, the RNA genome of
HTLV-III contains three genes which encode viral proteins: 1) the
gag gene, which encodes the internal structural (nucleocapsid or
core) proteins; 2) the pol gene, which encodes the RNA-directed DNA
polymerase (reverse transcriptase); and 3) the env gene, which
encodes the envelope glycoproteins of the virion. In addition, the
HTLV-III genome contains a region designated Px, located between
the env gene and the 3' LTR, which appears to be involved in
functional killing of the virus.
[0008] At this time, AIDS is still difficult to diagnose before the
onset of clinical manifestations. There is no method presently
available for the prevention of the disease. Treatment of those
with AIDS is generally not successful and victims succumb to the
devastating effects HTLV-III has on the body.
SUMMARY OF THE INVENTION
[0009] This invention is based upon applicant's cloning of HTLV-III
DNA in recombinant/vector host systems capable of expressing
immunoreactive HTLV-III polypeptides. Based on the cloning of
HTLV-III DNA in systems which express immunoreactive-polypeptides,
applicant has developed methods useful in the diagnosis, treatment
and prevention of AIDS. Applicant has developed methods of
detecting HTLV-III and antibodies against HTLV-III in body fluids
(e.g., blood, saliva, semen), and methods useful in immunotherapy
(e.g., vaccination and passive immunization against AIDS). In
addition, applicant has developed methods of making HTLV-III DNA
probes and RNA probes useful in detecting HTLV-III in body
fluids.
[0010] Polypeptides encoded by segments of the HTLV-III genome have
been produced by these recombinant DNA methods. For example,
polypeptides encoded by three regions of the HTLV-III genome (an
env gene sequence, an env-lor gene sequence and a 1.1Kb EcoRI
restriction fragment from HTLV-III cDNA) have been produced. The
polypeptides expressed have been isolated. These polypeptides are
immunoreactive with sera of patients having AIDS and with
antibodies to HTLV-III and thus are useful in screening blood and
other body fluids for the presence of antibodies against HTLV-III.
Applicant's invention threfore provides a method not only for
diagnosing AIDS, but also for preventing the transmission of the
disease to others through blood or blood components harboring
HTLV-III. The latter is particularly valuable in screening donated
blood before it is transfused or used to obtain blood components
(e.g., Factor VIII for the treatment of hemophilia; Factor IX)
[0011] Polypeptides produced by the recombinant DNA methods are
employed in the production of antibodies, including monoclonal
antibodies, against the virus. Such antibodies form the basis for
immunoassay and diagnostic techniques for directly detecting
HTLV-III in body fluids such as blood, saliva, semen, etc.
Neutralizing antibodies against the virus may be used to passively
immunize against the disease.
[0012] Applicant's cloning of HTLV-III DNA in such recombinant
vector host systems also provides the basis for determination of
the nucleotide sequence of HTLV-III DNA. The DNA probes are
homologous to DNA regions which are unique to the HTLV-III genome.
DNA probes provide another method of detecting HTLV-III in blood,
saliva or other body fluids. RNA probes which contain regions
unique to the HTLV-III genome can also be formed and used for the
detection of HTLV-III in body fluids.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a representation of HTLV-III DNA. FIG. 1a shows
sites at which the genome is cut by the restriction enzyme SstI and
FIG. 1b shows the fragments of HTLV-III genome produced through the
action of restriction enzymes Kpn, EcoRI and Hind III.
[0014] FIG. 2 is a representation of HTLV-III DNA. FIG. 2a shows
the location of restriction enzyme sites in the genome and FIG. 2b
shows the location in the HTLV-III genome of DNA inserts in open
reading frame clones. The (+) and (-) indicate reactivity and lack
of reactivity, respectively, of the fusion protein expressed by
cells transformed by the ORF vectors with sera of AIDS
patients.
[0015] FIG. 3 shows the nucleotide sequence for HTLV-III DNA and
the predicted amino acid sequence of the four longest open reading
frames. Restriction enzyme sites are indicated above the nucleotide
sequence.
[0016] FIG. 4 is an immunoblot showing the position on an SDS
polyacrylamide gel of HTLV-III env-Beta-galactosidase fusion
proteins.
[0017] FIG. 5 shows sites at which the genome is cut by the
restriction enzyme EcoRI and construction of recombinant plasmids
carrying HTLV-III DNA.
[0018] FIG. 6 is an immunoblot showing the positions on
nitrocellulose blots of peptides produced by bacterial cells
transformed by recombinant constructs ompA1-R-6; ompA2-R-7 and
ompA3-R-3, into which a 1.1Kb EcoRI HTLV-III cDNA restriction
fragment had been inserted. FIG. 6a shows the nucleotide sequence
of the ompA signal peptide and the pertinent region of recombinant
plasmids ompA1-R-6; ompA2-R-7 and ompA3-R-3.
[0019] FIG. 7 is an immunoblot showing blocking of reaction between
HTLV-III antigens and an AIDS serum by lysates of E.coli containing
HTLV-III DNA recombinant plasmid ompA1-R-6 (lanes 1-5) and no
blocking of the reaction by lysates of E.coli control cells (lanes
6-10).
[0020] FIG. 8 is an immunoblot showing the presence or absence of
antibodies against the peptide encoded by the 1.1Kb EcoRI HTLV-III
restriction fragment of HTLV-III CDNA in sera from healthy
individuals (lanes 1-3) and from AIDS patients (lanes 4-11).
Purified HTLV-III virus (panel A) or total cell lysate of bacterial
clone ompA1-R-6(OlR6) were reacted with sera samples.
[0021] FIG. 9 represents the open reading frame expression vector
pMRIOO having HTLV-III DNA.
[0022] FIG. 10 represents lambdaCI-HTLV-III beta-galactosidase
fusion proteins. FIG. 10a is an immunoblot showing the position on
SDS polyacrylamide gel of lambdaCI-HTLV-III beta-galactosidase
fusion proteins, and FIG. 10b shows the immunoreactivity of such
proteins with sera from AIDS patients.
BEST MODE FOR CARRYING OUT THE INVENTION
[0023] Despite the similarity between HTLV-III and the other
members of the HTLV-bovine leukemia virus (BLV) family of viruses,
the biology and pathology of HTLV-III differs substantially. For
example, relatively little homology has been found in the HTLV-III
genome when compared with that of the HTLV-I or -II genome.
Infection with HTLV-III often results in profound immunosuppression
(AIDS), consequent to the depletion of the OKT4(+) cell population.
This effect is mirrored by a pronounced cytopathic, rather than
transforming, effect of HTLV-III infection upon the OKT4(+) cells
in lymphocyte cultures in vitro. In contrast, infection with HTLV-I
results in a low incidence of T-cell leukemia lymphoma (an OKT4(+)
cell malignancy). There is evidence for some degree of
immunodeficiency in HTLV-I patients as well. Infection of primary
lymphocytes in culture by HTLV-I and -II results in vitro
transformation of predominantly OKT4(+) cells. A cytopathic effect
of HTLV-I infection upon lymphocytes is apparent, but the effect is
not as pronounced as that observed for HTLV-III.
[0024] HTLV-III also differs from HTLV-I and -II in the extent of
infectious virion production in vivo and in vitro. High titers of
cell free, infectious virions can be obtained from AIDS patient
semen and saliva and from the supernatant of cultures infected with
HTLV-III. Very few, if any, cell free infectious virions can be
recovered from adult T-cell leukemia lymphoma (ATLL) patients or
from cultures infected with HTLV-I or -II.
[0025] Envelope glycoprotein is the major antigen recognized by the
antiserum of AIDS patients. In this respect, HTLV resembles other
retroviruses, for which the envelope glycoprotein is typically the
most antigenic viral polypeptide. In addition, the neutralizing
antibodies are generally directed toward the envelope glycoprotein
of the retrovirus. Serum samples from 88 percent to 100 percent of
those with AIDS have been shown to have antibodies reactive with
antigens of HTLV-III; the major immune reactivity was directed
against p41, the presumed envelope antigen of HTLV-III. Antibodies
to core proteins have also been demonstrated in serum of AIDS
patients, but do not appear to be as effective an indicator of
infection as is the presence of antibodies to envelope antigen.
[0026] The p41 antigen of HTLV-III has been difficult to
characterize because the viral envelope is partially destroyed
during the process of virus inactivation and purification. This
invention responds to the great need to characterize this antigenic
component of the HTLV-III virus and to determine the existence and
identity of other viral antigenic components in several ways. It
provides products, such as HTLV-III polypeptides, antibodies to the
polypeptides and RNA and DNA probes, as well as methods for their
production. These serve as the basis for screening, diagnostic and
therapeutic products and methods.
[0027] This invention relates to HTLV-III polypeptides which are
produced by translation of recombinant DNA sequences encoding
HTLV-III proteins. Polypeptides which are produced in this way and
which are immunoreactive with serum from AIDS patients or
antibodies to HTLV-III are referred to as recombinant DNA-produced
immunoreactive HTLV-III polypeptides. They include, but are not
limited to, antigenic HTLV-III core and envelope polypeptides which
are produced by translation of the recombinant DNA sequences
specific to the gag and the env DNA sequences encoding HTLV-III
core proteins and envelope glycoproteins, respectively. They also
include the polypeptides which are produced by translation of the
recombinant DNA sequences included in a 1.1Kb EcoRI restriction
fragment of HTLV-III cDNA and recombinant DNA sequences specific to
the sor gene and the Px genes of HTLV-III. The sor DNA sequence is
common to replication competent HTLV-III viruses. The Px genes
contain a coding sequence with one large open reading frame (lor),
located between the env gene and the 3' end of the HTLV-III genome.
Both the env DNA sequences and the lor DNA sequences are located
within the same open reading frame of the HTLV-III genome and this
gene region is accordingly designated env-lor.
[0028] The polypeptides encoded by these regions of the HTLV III
can be used in immunochemical assays for detecting antibodies
against HTLV-III and HTLV-VIII infection. These methods can assist
in diagnosing AIDS. In addition, they can also be employed to
screen blood before it is used for transfusions or for the
production of blood components (e.g., Factor VIII for the treatment
of hemophilia). Availability of screening technics will reduce the
risk of AIDS transmission.
[0029] Detection of antibodies reactive with the polypeptides can
be carried out by a number of established methods. For example, an
immunoreactive HTLV III polypeptide can be affixed to a solid phase
(such as polystyrene bead or other solid support). The sold phase
is then incubated with blood sample to be tested for antibody
against HTLV-III. After an appropriate incubation period the solid
phase and blood sample are separated. Antibody bound to the solid
phase can be detected with labeled polypeptide or with a labeled
antibody against human immunoglobulin.
[0030] HTLV-III polypeptides can be used in a vaccine prevention of
AIDS. For vaccination against the virus, immunogenic polypeptides
which elicit neutralizing antibody would be employed. The leading
candidates for use in vaccines are the viral envelop
polypeptides.
[0031] The polypeptides can also be used to produce antibodies,
including monoclonal antibodies, against the HTLV-III polypeptides.
These antibodies can be used in immunochemical assays for direct
detection of the virus in body fluids (such as blood, saliva
semen). Assays employing monoclonal antibody against specific HTLV
III antigenic determinants will reduce false-positive results
thereby improving accuracy of assays for the virus. Antibodies
against the virus may also be useful in immunotherapy. For example,
antibodies may be used to passively immunize against the virus.
[0032] The methods of producing the polypeptides are also a subject
of this invention, as are diagnostic methods based on these
polypeptides.
[0033] This invention also provides methods for the isolation of
genes of HTLV-III which encode immunoreactive polypeptides;
identification of the nucleotide sequence of these genes;
introduction of DNA sequences specific to these viral DNA sequences
into appropriate vectors to produce viral RNA and the formation of
DNA probes. These probes are comprised of sequences specific to
HTLV-III DNA and are useful, for example, for detecting
complementary HTLV-III DNA sequences in body fluids (e.g.,
blood).
[0034] HTLV-III Polypeptides
[0035] Genetic engineering methods are used to isolate segments of
HTLV-III DNA which encode immunoreactive HTLV-III polypeptides.
Among these are polypeptides which are immunoreactive with serum
from AIDS patients or antibodies to HTLV-III. These polypeptides
include the core protein, a 15Kd peptide encoded by a 1.1Kb EcoRI
HTLV-III restriction fragment of HTLV-III DNA and the envelope
glycoprotein. These methods are also used to sequence the fragments
which encode the polypeptides. The proviral genes integrated into
host cell DNA are molecularly cloned and the nucleotide sequences
of the cloned provirus is determined.
[0036] An E. coli expression library of HTLV-III DNA is
constructed. The HTLV-III genome is cloned and cuts are then made
in the cloned HTLV-III genome with restriction enzymes to produce
DNA fragments. (FIGS. 1 and 2) HTLV-III DNA fragments of
approximately 200-500bp are isolated from agarose gel, end repaired
with T.sub.4 polymerase and ligated to linker DNA. The linker
ligated DNA is then treated with a restriction enzyme, purified
from agarose gel and cloned in an expression vector. Examples of
the expression vectors used are: OmpA, pIN (A,B and C), lambda pL,
T7, lac, Trp, ORF and lambda gt11. In addition, mammalian cell
vectors such as pSV28pt, pSV2neo, pSVdhfr and VPV vectors, and
yeast vectors, such as GALI and GAL10, may be used.
[0037] The bacterial vectors contain the lac coding sequences, into
which HTLV-III DNA can be inserted for the generation of
B-galactosidase fusion protein. The recombinant vectors are then
introduced into bacteria (e.g., E.coli); those cells which take up
a vector containing HTLV-III DNA are said to be transformed. The
cells are then screened to identify cells which have been
transformed and are expressing the fusion protein. For example, the
bacteria are plated on MacConkey agar plates in order to verify the
phenotype of clone. If functional B-galactosidase is being
produced, the colony will appear red.
[0038] Bacterial colonies are also screened with HTLV-III DNA
probes to identify clones containing the DNA regions of interest
(e.g., HTLV-III a, pol and env DNA sequences). Clones which are
positive when screened with the DNA probe and positive on the
MacConkey agar plates are isolated.
[0039] This identification of cells harboring the HTLV-III DNA
sequences makes it possible to produce HTLV-III polypeptides which
are immunoreactive with HTLV-III specific antibody. The cells from
the selected colonies are grown in culture under conditions
allowing the expression of the hybrid protein. Cell protein is then
obtained by means known in the art. For example, the culture can be
centrifuged and the resulting cell pellet broken. Polypeptides
secreted by the host cell can be obtained (without disruption of
the cells) from the cell culture supernatant.
[0040] The total cellular protein is analysed by being run on an
SDS polyacrylamide gel electrophoresis. The fusion proteins are
identified at a position on the gel which contains no other
protein. Western blot analyses are also carried out on the clones
which screened positive. Such analyses are performed with serum
from AIDS patients, with the result that it is possible to identify
those clones expressing HTLV-III B-galactosidase fusion proteins
(antigens) that cross-react with the HTLV-III specific
antibody.
[0041] Lambda .sub.10 clones harboring HTLV-III DNA are cloned from
the replicated form of the virus. As the retrovirus is replicating,
double stranded DNA is being produced. The cloned HTLV-III DNA is
digested with the restriction enzyme SstI. (FIG. 1a) Because there
are two SstI recognition sites within the LTR of HTLV-III DNA, one
LTR region is not present in the cloned DNA sequence removed from
the lambda.sub.10 vector. As a result, a small (approximately 200
bp) fragment of the HTLV-III DNA is missing.
[0042] The resulting DNA is linearized and fragments are produced
by digesting the linearized genomic DNA spanning the env gene
region with restriction enzymes. For example, fragments are
produced using Kpn or EcoRI plus HindIII, as shown in FIG. 1b. The
resulting 2.3kb KpnI-KpnI fragments; 1.0kbEcoRI-EcoRI fragments and
2.4Kb EcoRI-HindIII fragments are isolated by gel electrophoresis
and electro-elution. These fragments are randomly sheared to
produce smaller fragments. The fragments thus produced are
separatedfrom agarose gel and DNA fragments between about 200-500
bp are eluted.
[0043] The eluted 200-500bp DNA fragments are end filled through
the use of E. coli T.sub.4 polymerase and blunt end ligated into an
open reading frame expression (ORF) vector, such as pMR100. This
ligation may occur at the SmaI site of the pMR100 vector, which
contains two promoter regions, hybrid coding sequences of lambdaCI
gene and lacI-LacZ gene fusion sequence. In the vector, these are
out of frame sequences; as a result, the vector is nonproductive.
The HTLV-III DNA is inserted into the vector; the correct DNA
fragments will correct the reading frame, with the result that
CI-HTLV-III-B-galactosidase fusion proteins are produced. The
expression of the hybrid is under the control of the lac promoter.
Based on the sequence of pMR100, it appears that if a DNA fragment
insert cloned into the SmaI site is to generate a proper open
reading frame between the lambdaCI gene fragment and the lac-Z
fragment, the inserted DNAtmust not contain any stop codons in the
reading frame set by the frame of the lambdaCI gene.
[0044] The recombinant pMIR100 vectors are then introduced into E.
coli. The bacteria are plated on MacConkey agar plates to verify
the phenotype of the clone. If functional B-galactosidase is being
produced, the colony will appear red. The colonies are also
screened with HTLV-III DNA probes, for the purpose of identifying
those clones containing the insert. Clones which are positive when
screened with the DNA probe and positive on the MacCohkey agar
plates are isolated.
[0045] The cells from the selected colonies are grown in culture.
The culture is spun down and the cell pellet broken. Total cellular
protein is analysed by being run on an SDS polyacrylamide gel. The
fusion proteins are identified at a position on the gel which
contains no other protein. (FIG. 4) Western blot analyses are also
carried out on the clones which screened positive. Sera from AIDS
patients are used, thus making it possible to identify those clones
which express the HTLV-III-B-galactosidase fusion proteins that
cross-react with the HTLV-III specific antibody. 1000 clones were
screened by this method; 6 were positive.
[0046] Because of the nature of the pMR100 cloning vehicle, a
productive DNA insert should also be expressed as a part of a
larger fusion polypeptide. HTLV-III env gene containing recombinant
clones was identified by colony hybridization. The production of
larger fusion polypeptides bearing functional B-galactosidase
activity was verified by phenotype identification on MacConkey agar
plates; by B-galactosidase enzymatic assays and by analysis on 75%
SDS-polyacrylamide gels. Immunoreactivity of the larger protein
with antibody to HTLV-III was assessed by western blot analysis
using serum from AIDS patients. These large fusion proteins also
reacted with anti-B-galactosidase and anti-CI antiserum. This
finding is consistent with the hypothesis that they are proteins of
CI-HTLV-III-lacIZ.
[0047] The open reading frame insert fragment of HTLV-III is
further analyzed by DNA sequencing analysis. Because one of the two
BamHI sites flanking the SmaI cloning site in pMR100 is destroyed
in the cloning step, positive clones are digested with restriction
enzymes HindIII and claI to liberate the inserted HTLV-III DNA
fragment. The HTLV-III ORF inserts are isolated from the fusion
recombinant and cloned into M13 sequencing cloning vector mp18 and
mp19 digested with HindIII and AccI. DNA sequences of the positive
ORF clones are then determined.
[0048] Fragments of HTLV-III DNA of approximately 200-500 bps are
isolated from agarose gel, end repaired with T.sub.4 polymerase and
ligated to EcoRI linker. The EcoRI linker ligated DNA is then
treated with EcoRI purified from 1% agarose gel and cloned in an
expression vector, lambda gt11. This vector contains lac Z gene
coding sequences into which the foreign DNA can be inserted for the
generation of B-galactosidase fusion protein. The expression of the
hybrid gene is under the control of lac repressor. The lac
repressor gene, lac I, is carried on a separate plasmid pMC9 in the
host cell, E. coli Y1090. AIDS patient serum was used to probe the
lambdagt11 library of HTLV-III genome DNA containing
1.5.times.10.sup.4 recombinant phage. In a screen of 5000
recombinants, 100 independent clones that produced strong signals
were isolated. The positive recombinant DNA clones were further
characterized for their specific gene expression. Rabbit
hyperimmune serum against P24 was also used to identify the gag
gene specific clones. Nick-translated DNA probes of specific
HTLV-III gene, specifically the gag gene, env gene and Px gene were
used to group the positive immunoreactive clones into specific gene
region.
[0049] Recombinant clones that produced strong signals with AIDS
serum and contain insert DNA spanning the HTLV-III ga, pol, sor and
env-lor gene regions were examined in detail by mapping their
insert with restriction enzymes and DNA sequencing analysis.
Determination of the Nucleotide Sequence of HTLV-III DNA
[0050] Genetic engineering methods are used to determine the
nucleotide sequence of HTLV-III DNA. One technique that can be used
to determine the sequence is a shotgun/random sequencing methods.
HTLV-III DNA is sheared randomly into fragments of about 300-500 bp
in size. The fragments are cloned, for example, using m13, and the
colonies screened to identify those having an HTLV-III DNA fragment
insert. The nucleotide sequence is then generated, with multiple
analysis producing overlaps in the sequence. Both strands of the
HTLV-III DNA are sequenced to determine orientation. Restriction
mapping is used to check the sequencing data generated.
[0051] The nucleotide sequence of one cloned HTLV-III genome (BH10)
is shown in FIG. 3, in which the position of sequences encoding gag
protein p17 and the N-terminus of gag p24 and the C-terminus of gag
p15 (which overlaps with the N-terminus of the pol protein) are
indicated. The open reading frames (ORF) for pol, sor and env-lor
are also indicated. The sequence of the remaining 182 base pairs of
the HTLV-III DNA not present in clone BH10 (including a portion of
R, U5, the tRNA primer binding site and a portion of the leader
sequence) was derived from clone HXB2. The sequences of two
additional clones (BH8 and BH5) are also shown. Restriction enzyme
sites are listed above the nucleotide sequence; sites present in
clone BH8 but not in clone BH10 are in parentheses. Deletions are
noted ([]) at nucleotides 251, 254, 5671 and 6987-7001. The
nucleotide positions (to the right of each line) start with the
transcriptional initiation site. The amino acid residues are
numbered (to the right of each line) for the four largest open
reading frames starting after the preceding termination codon in
each case except gag which is enumerated from the first methionine
codon. A proposed peptide cleavage site (V) and possible
asparagine-linked glycosylation sites are shown (*) for the env-lor
open reading frame. The sequences in the LTR derived from clones
BH8 and BH10 listed in the beginning of the figure are derived from
the 3'-portion of each clone and are assumed to be identical to
those present in the 5'-LTR of the integrated copies of these viral
genomes.
[0052] Clone HXB2 was derived from a recombinant phage library of
XbaI digested DNA from HTLV-III infected H9 cells cloned in
lambdaJ1. H9 cells are human leukemic cells infected by a pool of
HTLV-III from blood of AIDS patients, F. Wong-Staal, Nature, 312,
November, 1984. Cloning vector clones BH10, BH8, and BH5 were
derived from a library of SstI digested DNA from the Hirt
supernatant fraction of HTLV-III infected H9 cells cloned in
lambdagtWes.lambdaB. Both libraries were screened with cDNA probe
synthesized from virion RNA using oligo.dT as a primer. Clones BH8,
BH5, and a portion of HXB2 were sequenced as described by Maxam and
Gilbert. (1980) Maxam, A. M. and Gilbert, Co. Methods in
Enzymology. 65: 499-560. Clone BH10 was sequenced by the method of
Sanger modified by the use of oligonucleotides complementary to the
M13 insert sequence as primers and using Klenow fragment of DNA
polymerase I or reverse transcriptase as the polymerase.
[0053] Formation of RNA, RNA Probes and DNA Probes Specific to
HTLV-III
[0054] DNA sequences which are an entire gene or segment of a gene
from HTLV-III are inserted into a vector, such as a T7 vector. In
this embodiment, the vector has the Tceu promoter from the T cell
gene 10 promoter and DNA sequences encoding eleven amino acids from
the T cell gene 10 protein.
[0055] The vectors are then used to-transform cells, such as E.
coli. The T7 vector makes use of the T7 polymerase, which catalyzes
RNA formation and recognizes only T7 promoter, which is the site
where RNA polymerase binds for the initiation of transcription. The
T7 polymerase does not recognize E. coli promoter. As a result, if
HTLV-III DNA sequences are inserted after the promoter and
polymerase genes of the T7 vector, which recognizes them to the
exclusion of other signals, and a terminator is placed immediately
after the HTLV-III DNA sequences, the T7 vector will direct
manufacture RNA complementary to the HTLV-III DNA insert.
[0056] Determination of the nucleotide sequence of HTLV-III DNA
also provides the basis for the formation of DNA probes. Both RNA
proves and DNA HTLV-III probes must have a distinctive region of
the HTLV-III genome in order to be useful in detecting HTLV-III in
body fluids. There is relatively little homology between the
HTLV-III genome and the HTLV-I and -II genomes and probes contain
regions which are unique to HTLV-III (i.e., not shared with HTLV-I
or -II). For example, nucleotide sequences in the env gene region
of HTLV-III can be used.
[0057] Either viral RNA or DNA can be used for detecting HTLV-III
in, for example, saliva, which is known to have a very high
concentration of the virus. This can be done, for example, by means
of a dot blot, in which the saliva sample is denatured, blotted
onto paper and then screened using either type of probe. If saliva
is used as the test fluid, detection of HTLV-III is considerable
faster and easier than is the case if blood is tested.
[0058] Production of Monoclonal Antibodies Reactive with HTLV-III
Polypeptides
[0059] Monoclonal antibodies reactive with HTLV-III polypeptides
are produced by antibody-producing cell lines. The
antibody-producing cell lines may be hybridoma cell lines commonly
known as hybridomas. The hybrid cells are formed by fusion of cells
which produce antibodv to HTLV-III polypeptide and an immortalizing
cell, that is, a cell which imparts long term tissue culture
stability on the hybrid cell. In the formation of the hybrid cell
lines, the first fusion partner--the antibody-producing cell--can
be a spleen cell of an animal immunized against HTLV-III
polypeptide. Alternatively, the antibody-producing cell can be
isolated B lymphocyte which produces antibody against an HTLV-III
antigen. The lymphocyte can be obtained from the spleen, peripheral
blood, lymph nodes or other tissue. The second fusion partner--the
immortal cell--can be a lymphoblastoid cell or a plasmacytoma cell
such as a myeloma cell, itself an antibody-producing cell but also
malignant.
[0060] Murine hybridomas which produce monoclonal antibodies
against HTLV-III polypeptide are formed by the fusion of mouse
myeloma cells and spleen cells from mice immunized against the
polypeptide. To immunize the mice, a variety of different
immunization protocols may be followed. For instance mice may
receive primary and boosting immunizations of the purified
polypeptide. The fusions are accomplished by standard procedures.
Kohler and Milstein, (1975) Nature (London) 256, 495-497; Kennet,
R., (1980) in Monoclonal Antibodies (Kennet et al., Eds. pp.
365-367, Plenum Press, NY).
[0061] The hybridomas are then screened for production of antibody
reactive with the polypeptide. This can be performed by screening
procedures known in the art.
[0062] Another way of forming the antibody-producing cell line is
by transformation of antibody-producing cells. For example, a B
lymphocyte obtained from an animal immunized against HTLV-III
polypeptide may be infected and transformeed with a virus such as
the Epstein-Barr virus in the case of human B lymphocytes to give
an immortal antibody-producing cell. See, e.g., Kozbor and Rodor
(1983) Immunology Today 4(3), 72-79. Alternatively, the B
lymphocyte may be transformed by a transforming gene or
transforming gene product.
[0063] The monoclonal antibodies against HTLV-III polypeptide can
be produced in large quantities by injecting antibody-producing
hybridomas into the peritoneal cavity of mice and, after an
appropriate time, harvesting the ascites fluid which contains very
high titer of homogenous antibody and isolating the monoclonal
antibodies therefrom. Xenogeneic hybridomas should be injected into
irradiated or athymic nude mice. Alternatively, the antibodies may
be produced by culturing cells which produce HTLV-III polypeptide
in vitro and isolating secreted monoclonal antibodies from the cell
culture medium. The antibodies produced according to these methods
can be used in diagnostic assays (e.g., detecting HTLV-III in body
fluids) and in passive immunotherapy. The antibodies reactive with
HTLV-III polypeptides provide the basis for diagnostic tests for
the detection of AIDS or the presence of HTLV-III in biological
fluids (e.g., blood, semen, saliva) and for passive immunotherapy.
For example, it is possible to produce anti p 41, to attach it to a
solid phase using conventional techniques and to contact the body
fluid to be tested with the immobilized antibody. In this way,
HTLV-III (antigen) can be detected in the body fluid; this method
results in far fewer false positive test results than do tests, in
which antibody against HTLV-VIII is detected.
[0064] This invention will now be further illustrated by the
following examples.
EXAMPLE 1
[0065] Preparation of Sonicated DNA Fragments
[0066] 10 ug of gel purified HTLV-III restriction fragments were
sonicated to fragment size on average of 500 bps. After sonication,
the DNA was passed through a DEAE-cellulose column in 0.1.times.TBE
in order to reduce the volume. The DEAE-bound DNA was washed with 5
ml of 0.2 M NaCl-TE (2 M NaCl, 10 mm Tris HCl pH 7.5, 1 mM EDTA)
and then eluted with 1 M NaCl-TE, and ethanol precipitated. The
size range of the sonicated DNA was then determined on 1.2% agarose
gel. DNA fragments of desired length (200-500 bps) was eluted from
the gel. T4 DNA polymerase was used to fill in and/or trim the
single strand DNA termini generated by the sonication procedure.
DNA fragments were incubated with T4 polymerase in the absence of
added nucleotides for five minutes at 37.degree. C. to remove
nucleotides from 3' end and then all 4 nucleotide precursors were
added to a final concentration of 100 uM and the reaction mixture
was incubated another 30 minutes to repair the 5'-end single
stranded overhang. The reaction was stopped by heat inactivation of
the enzyme at 68.degree. C. for 10 minutes. DNA was phenol
extracted once, ethanol precipitated and resuspended in TE.
EXAMPLE 2
[0067] Cloning of Random Sheared DNA Fragments
[0068] The sonicated blunt end repaired HTLV-III DNA fragments were
ligated into the SmaI site of the ORF expression vector pMR100 and
transformed into host cell LG90 using standard transformation
procedures. B-galactosidase positive phenotype of the transformant
were identified by plating the transformed cell on ampicillin (25
ug/ml) containing McConkey agar plates and scoring the phenotype
after 20 hours at 37.degree. C.
EXAMPLE 3
[0069] Hybrid Protein Analysis
[0070] Ten milliliter samples of cells from an overnight saturated
culture grown in L broth containing ampicillin (25 ug/ml) were
centrifuged, the cell pellet was resuspended in 500 ul of 1.2 fold
concentrated Laemmli sample buffer. The cells were resuspended by
vortexing and boiling for 3 minutes at 100.degree. C. The lysate
was then repeated by being forced through a 22 guage needle to
reduce the lysate viscosity. Approximately 10 ul of the protein
samples were electrophoresed in 7.5% SDS-PAGE (SDS-polyacrylamide)
gels.
[0071] Electrophoretic transfer of proteins from SDS-PAGE gels to
nitrocellulose paper was carried out according to Towbin et. al..
After the transfer, the filter was incubated at 37.degree. C. for
two hours in a solution of 5% (w/v) nonfat milk in PBS containing
0.1% antifoam A and 0.0001% merthiolate to saturate all available
protein binding sites. Reactions with AIDS antisera were carried
out in the same milk buffer containing 1% AIDS patient antisera
that had been preabsorbed with E. coli lysate. Reactions were
performed in a sealed plastic bag at 4.degree. C. for 18-24 hours
on a rotatory shaker. Following this incubation, the filter was
washed three times for 20 minutes each at room temperature in a
solution containing 0.5% deoxycholic, 0.1 M NaCl, 0.5% triton
X-100, 10 mm phosphate buffer pH 7.5 and 0.1 mM PMSF.
[0072] To visualize antigen-antibody interactions, the
nitrocellulose was then incubated with the second goat antihuman
antibody that had been iodinated with .sup.125I. The reaction with
the iodinated antibody was carried out at room temperature for 30
minutes in the same milk buffer as was used for the first antibody.
The nitrocellulose was then washed as previously described and
exposed at -70.degree. C. using Kodak XAR5 film with an
intensifying screen.
EXAMPLE 4
[0073] Screening of the HTLV-III ORF Library by Colony
Hybridization
[0074] E. coli LG90 transformants were screened with HTLV-III DNA
probes containing the DNA regions of interest (e.g. HTLV-III gag,
env or Px gene specific sequences). Colonies were grown on
nitrocellulose filter and screened according to the procedure of
Grunstein and Hogness by using a nick-translated HTLV-III DNA as
hybridization probe.
[0075] The DNA fragment was in general excised by restriction
endonuclease digestion, gel purified, and .sup.32P-labeled to a
specific activity of 0.5.times.10.sup.8 cpm/ug by nick-translation
(Rigby, P. W. J. et al., J. Mol. Biol. 113, 237 (1977). Duplicate
nitrocellulose filters with DNA fixed to them were prehybridized
with 6.times.SSC (0.9 Ml NaCl/0.09 M sodium citrate, pH 7.0),
5.times. Denhardt's solution (Denhardt's solution: 0.02% each of
polyvinylpyrrolidone, Ficoll and bovine serum albumin) 10 ug of
denatured sonicated E. coli DNA per ml at 55.degree. C. for 3-5
hours. The filters were then placed in a fresh sample of the same
solution to which the denatured hybridization probe had been added.
Hybridization was permitted to take place at 68.degree. C. for 16
hours. The filters were washed repeatedly in 0.3.times.SSC at
55.degree. C., and then exposed to x-ray film.
EXAMPLE 5
[0076] Recombinant DNA Produced Peptide of HTLV-III which is
Immunoreactive with Sera From Patients with AIDS
[0077] An expression vector, pIN-III-ompA (ompA) was used. ompA has
the lipoprotein (the most abundant protein in E.coli) gene promoter
(lpp) and the lacUV5 promoter-operator (FIG. 1). ompA vectors also
contain the DNA segment encoding the lac repressor, which allows
the expression of the inserted DNA to be regulated by lac operon
inducers such as IPTG. The ompA cloning vehicles contain three
unique restriction enzyme sites EcoRI, HindIII, Bam HI in all three
reading frames and permit the insertion of DNA into any of these
restriction sites.
[0078] Various restriction fragments were excised from the
recombinant clone, lambdaBH10, which contains a 9 Kb long HTLV-III
DNA insert in the SstI site of the vector lambdagtWES lambdaB.
These restriction fragments were them inserted into the ompA
vectors at all three reading frames and used to transform E.coli
JA221 cells. Transformants were first screened for HTLV-III DNA by
in situ colony hybridization using nick-translated HTLV-III DNA
probes. The positive clones were then screened for expression of
HTLV-III antigenic peptides using HTLV-III specific antibodies. For
this, lysates of E.coli cell containing HTLV-III DNA recombinant
plasmids were electrophoresed on 12.5% SDS-polyacrylamide gel and
electroblotted onto nitrocellulose filters. The filters were then
incubated first with well-characterized sera from AIDS patients and
next with .sup.125I-labelled goat anti-human IgG antibodies. The
washed filters were autoradiographed to identify peptides reactive
with anti-HTLV-III antibodies.
[0079] Several gene segments that encode peptides showing
immunoreactivity with anti-HTLV-III antibodies were demonstrated.
Among these is a 1.1 Kb EcoRI restriction fragment. This fragment
was inserted into ompA vectors in all three reading frames (FIG.
5). Cells were grown at 37.degree. C. in L broth containing 100
mg/ml. ampicillin to an OD.sub.600 of 0.2. At this time, the cell
cultures were divided into two aliquots. IPTG was added to one
aliquot to a final concentration of 2 mM (induced). IPTG was not
added to the other aliquot (uninduced). Upon IPTG induction,
transformants of all three plasmid constructs (designated
OmpA.sub.1-R-6 (O1R6), OmpA 2-R-7 (O2R7), and OmpA.sub.3-R-3
(O3R3)) produced a 15 Kd peptide that is strongly reactive with
anti-HTLV-III antibodies in sera from AIDS patients (FIG. 6 lane 1,
purified HTLV-III virions; lanes 2 and 3, O1R6 uninduced and
induced; lanes 4 and 5, O2R7 uninduced and induced; lanes 6 and 7
O3R3 uninduced and induced). This reactivity is not detected when
sera from normal individuals is used.
[0080] DNA sequence data of the HTLV-III genome indicates that
there is an open reading frame inside the pol gene located at the
5'-end of the EcoRI fragment. DNA sequence analysis of the three
recombinant constructs, O1R6, O2R7 and P3R3, confirmed that each of
these recombinants has a different reading frame of the HTLV-III
plus strand coupled to the coding sequence of each vector. Only in
O3R3 is the reading frame of the inserted DNA in phase with that
set by the signal peptide in the ompA vector; in O1R6 and O2R7 the
pol gene segment DNA is out of phase (FIG. 6a).
[0081] There is a 6 bp ribosome binding site, AAGGAG
(Shine-Dalgarno sequence), located at nucleotide position 24-29 and
an initiation codon, ATG, located 11 bp downstream (position
41-43). The 15 Kd peptide synthesized by all three recombinants
appears to be translated from the transcripts using this internal
initiation codon. If this is true, the peptide starts from the ATG
located at position 41-43 and ends at the stop codon at position
446-448, producing a peptide of 135 amino acid residues encoded by
the 3'-end segment of the pol gene of HTLV-III.
[0082] In addition to the 15 Kd peptide, the O3R3 construct, in
which the reading frame of the HTLV-III DNA pol gene is in phase
with that set by the vector, produced two additional peptides about
19 Kd and 16.5 Kd in size (FIG. 6). It is possible that the 19 Kd
peptide contains an additional 35 amino acid residues, 21 of which
are from the signal peptide encoded by the ompA.sub.3 vector and 14
encoded by the inserted HTLV-III DNA itself. The 16.5 Kd peptide
may be the processed 19 Kd peptide in which the signal peptide is
cleaved.
[0083] The O1R6 and O2R7 constructs also produces another peptide
of about 17.5 Kd (FIG. 6) and weakly reactive with sera of AIDS
patients. The origin of this peptide is not clear. The 1.1 Kb EcoRI
fragment contains a second potential coding region designated as
the short open reading frame (SOR) extending from nucleotide
position 360 to 965 (FIG. 5). Four of the five AUG methionine
codons in this region are near the 5'-end of this open reading
frame. This DNA segment could encode peptides of 192, 185, 177 or
164 amino acid residues. However, there is no clearly recognizable
ribosome binding site at the 5'-end of this open reading frame.
[0084] Further evidence also supports the conclusion that the 15 Kd
peptide is indeed derived from the pol gene. First, deletion of the
3'-end StuI to EcoRI fragment from the 1.1 Kb EcoRI insert from
O1R6, O2R7 and O3R8 (FIG. 5) does not affect the synthesis of the
15 Kd peptide. Second, clones containing only the 5'-end EcoRI to
NdeI fragment still produce the same 15 Kd peptide. Finally,
several recombinant clones containing various DNA fragments having
the SOR coding sequence properly inserted into the open reading
frame cloning vector, pMR100, produced lambdaCI-HTLV-III
B-galactosidase tripartite fusion proteins which have very little
immunoreactivity with anti-HTLV-III antibodies present in sera from
AIDS patients.
[0085] Significant immunoreactivity against the 15 Kd peptide
derived from the viral pol gene in sera from AIDS patients was
detected. The identity of this immunoreactive peptide, with respect
to the banding pattern of HTLV-III virion antigen in
SDS-polyacrylamide gel electrophoresis, was determined by means of
a competition inhibition inmunoassay. Purified HTLV-III virions
were treated with SDS, electrophoresed, and electroblotted onto a
nitrocellulose filter. Identical filter strips containing disrupted
HTLV-III virions were incubated with well characterized serum from
an AIDS patient in the presence or absence of lysates of O1R6,
02R7, or control bacterial clones. The specific immunoreaction
between anti-HTLV-III antibodies present in sera of the AIDS
patients and the blotted virion proteins were then revealed by
.sup.125I-labeled goat anti-human antibody. As shown in FIG. 7,
lysates of O1R6 block the immunoreactivity of the viral p31 protein
with the AIDS serum, while lysates of control cells do not. This
result suggests that the recombinant 15 Kd peptide encoded by
3'-end of the-viral pol gene is also a part of another virion
protein, p31, in contrast to the view shared by some that p31 is a
cellular protein which co-purifies with HTLV-III virions.
[0086] The prevalence in the sera of AIDS patients of antibodies
against the 15 Kd peptide was also evaluated. In Western blot
analysis employing the lysate of O1R6 as the source of antigen, a
panel of coded sera from AIDS patients and normal healthy
individuals was tested. All of the 20 AIDS sera and none of the 8
normal controls reacted with the 15 Kd peptide. Representative
results are shown in (FIG. 8). These data indicate that most, if
not all, AIDS patients produce antibodies against the viral p31
protein.
EXAMPLE 6
[0087] Expression in E. coli of Open Reading Frame Gene Segments of
HTLV-III
[0088] HTLV-III DNA-was excised from lambda BH-10, which is a
previously constructed recombinant lambda phage containing a 9 Kb
segment of HTLV-III DNA inserted into the vector lambdagtwes lambda
B (FIG. 2a). This HTLV-III DNA was sonicated and DNA fragments of
about 0.5 Kb purified by gel electrophoresis, end repaired,- and
inserted into the SmaI site of the open reading frame (ORF) vector,
pMR100 (FIG. 9). This vector contains a bacterial lac promotor DNA
segment linked to a second DNA fragment containing a hybrid coding
sequence in which the N-terminus (5' segment) of the lambda CI gene
of bacteriophage lambda is fused to an N-terminal-deleted lacIZ
gene (3' segment). A short linker DNA fragment, containing a SmaI
cloning site, has been inserted between these two fragments in such
a manner that a frame shift mutation has been introduced upstream
of the lacIZ-coding DNA. As a result, pMR100 does not produce any
detectable B-galactosidase activity when introduced into cells of
the Lac host E. coli LG90. The insertion of foreign DNA containing
an open reading frame, in this case the HTLV-III DNA, at the SmaI
cloning site can reverse the frame shift mutation if the inserted
coding sequence is in the correct reading frame with respect to
both the lambdaCI leader and the lacIZ gene. Transformants were
screened on MacConkey plates to detect individual clones that
expressed B-galactosidase enzymatic activity in situ.
[0089] Among the 6000 ampicillin resistant transformants screened,
about 300 were found to express B-galactosidase activity. Colony
hybridization using .sup.32p-labelled nick-translated HTLV-III DNA
as a probe revealed that all these Lac.sup.+ clones contained
HTLV-III DNA. In the Lac.sup.+ clones the HTLV-III fragment
inserted into the Sma I site of pMR100 must contain no stop codons
in the reading frame set by the lambdaCI leader segment and the
lacIZ gene must also be in the correct translational reading frame.
The three-element-fused genes were expressed as tripartite fusion
proteins, having a portion of the lambdaCI protein at the
N-terminus, the HTLV-III segment in the middle, and the lacIZ
polypeptide at the C-terminus.
[0090] The proteins produced by the Lac clones were analyzed by
resolving cell lysates on 7.5% SDS-polyacrylamide gels along with
those of the control Lac.sup.+ clone pMR200, which produced a
lambdaCI-B-galactosidase fusion protein. The lacIZ gene in pkIR200
is identical to that in pMR100 except that it has a single base
pair deletion which brings it in phase with the lambdaCI gene to
produce an active B-galactosidase. By virtue of the very large size
of the B-galactosidase and its fusion protecins, they are separated
from the bulk of proteins in the cell lysates on the
SDS-polyacrylamide gels and can be easily identified by Coomassie
brilliant blue staining as shown in FIG. 10a. Some of the Lac.sup.+
clones containing HTLV-III DNA produce polypeptides that are larger
(15,000 to 27,000 daltons) than the lambdaCI-lacIZ fusion protein.
These findings are consistent with data that the DNA inserts are up
to 700 bp long. The B-galactosidase fusion proteins accounted for
about 1-2% of total cellular protein.
[0091] The peptides produced by the Lac.sup.+ clones were examined
by Western blot analysis for immunoreactivity with sera from AIDS
patients. After the lysates of Lac clones were electrophoresed in
SDS-polyacrylamide gels, they were electro-transferred to
nitrocellulose filters. These protein blots were first reacted with
AIDS patient sera and then with .sup.125I-labeled goat anti-human
IgG. The autoradiograph in FIG. 10b shows the immunoreactivity of a
representative fused protein with the serum from an AIDS patient.
The recombinant peptides also reacted with anti-B-galactosidase
antiserum, consistent with the proposition that they had the
general structure lambdaCI-HTLV-III peptide-LacIZ. Prom the
immunoreactivity pattern of the negative controls, pMR100 and
pMR200, which do not contain an HTLV-III DNA insert, it is evident
that this particular AIDS serum contains antibodies reactive with
several bacterial proteins of the host E. coli. This is not
surprising, since AIDS patients are usually infected with a number
of bacteria. Absorbing AIDS patient sera with Sepharose 4B
conjugated with E. coli extract reduced the background
immunoreactivity to some extent but did not completely eliminate
it.
[0092] About 300 independent HTLV-III DNA-containing Lac colonies
were analyzed in SDS polyacrylamide gels using Coomassie brilliant
blue staining and Western blotting. About half of them were found
to express fusion proteins containing extra peptides of about
100-200 amino acids, corresponding to DNA inserts of 300-600 bp
long. Of these fusion proteins, 20 were found to react specifically
with sera from AIDS patients. The unreactive clones probably
contain peptides that fold in such a way that they are not reactive
with antibodies or correspond to regions of HTLV-III protein
molecules which are not immunogenic in AIDS patients. The other
half of the Lac clones expressed fusion proteins whose sizes were
not obviously different from that of the lambdaCI B-galactosidase
protein. None from this group of fusion proteins was found to react
with sera from AIDS patients.
[0093] The HTLV-III DNA inserts from Lac ORF clones were mapped to
specific segments in the HTLV-III genome using Southern blotting
procedures. In these studies, each plasmid clone was labelled with
.sup.32P by nick-translation and hybridized to a battery of
HTLV-III DNA restriction fragments. This hybridization analysis
mapped all of the Lac ORF clones into four open reading frame
segments designated ORF-A, ORF-B, ORF-C, and ORF-D (FIG. 2a)
consistent with the DNA sequencing data. The open reading frames
ORF-A and -B, corresponding to the coding regions of the gag and
pol genes, are 1.5 Kb and 3.0 Kb long, respectively. ORF-C is about
0.6 Kb long, slightly overlaps with the ORF-B region, and is
capable of encoding a polypeptide of 21 overlaps with the ORF-B
region, and is capable of encoding a polypeptide of 21 Kd. The
location of ORF-C and its overlap with the pol gene are reminiscent
of the structure of the env genes in HTLV-I and -II. However,
ORF-C, designated as the short open reading frame (sor), is too
short to code for the entire envelope protein. The fourth open
reading frame, ORF-D, is 2.5 Kb long and could encode both a large
precursor of the major envelope glycoprotein and another protein
derived from the 39 terminus, which may be analogous to the lor
products of HTLV-I and -II. This gene region of HTLV-III,
designated env-lor, is at least twice as long as the lor of HTLV-I
and HTLV-II and it is presently unclear whether single or multiple
proteins are encoded herein.
[0094] Both Southern blotting and DNA sequencing studies were
employed to analyze a number of clones. As shown in FIG. 2b, the
Lac.sup.+ ORF clones expressing fusion proteins immunoreactive with
sera from AIDS patients were located in ORF-A (e.g. #175 and 4191),
ORF-B (e.g. #13, 31, and 162), or ORF-D (e.g. #113, 121, and 127)
and not in the sor region. Not all peptides in these regions were
immunoreactive, e.g. ORF clone #76 located in ORF-D.
[0095] Analysis of the open reading frame structures in HTLV-III
posed questions as to which open reading frame(s) corresponds to
the env gene. It is possible that the env-lor region in HTLV-III
contains all or a part of the env gene in addition to the presumed
lor gene. Recent evidence suggests that the lor in HTLV-I encodes a
42 Kd protein involved in the process of viral activation and
transformation. When the lysate of one of the ORF clones (#127 in
FIG. 2b) was tested against sera from 20 AIDS patients and 12
healthy normals in a strip radioimmunoassay based on the Western
blot technique, immunoreactivity against the
lambdaCI-HTLV-III-B-galactasidase fusion polypeptide was detected
in the sera from 19 of the AIDS patients and none from normal
controls. This result indicates that the protein encoded by the
portion of the env-lor region contained in ORF clone #127 is
produced in HTLV-III infected cells and induces antibody production
in most if not all AIDS patients.
[0096] Industrial Applicability
[0097] This invention has industrial applicability in screening for
the presence of HTLV-III DNA in body fluids and the diagnosis of
AIDS.
[0098] Equivalents
[0099] Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific substantces and procedures described
herein. Such equivalents are considered to be within the scope of
this invention and are covered by the following claims.
Sequence CWU 1
1
11 1 492 DNA T cell leukemia-lymphoma virus (HTLV) misc_feature
(1)..(492) standard_name= "Clone BH10" / note = "Corresponds to
nucleotide positions -453 to 39 in figure 3 of US 06/693,866" 1
tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
60 cacaaggcta cttccctgat tagcagaact acacaccagg gccagggatc
agatatccac 120 tgacctttgg atggtgctac aagctagtac cagttgagcc
agagaagtta gaagaagcca 180 acaaaggaga gaacaccagc ttgttacacc
ctgtgagcct gcatggaatg gatgacccgg 240 agagagaagt gttagagtgg
aggtttgaca gccgcctagc atttcatcac atggcccgag 300 agctgcatcc
ggagtacttc aagaactgct gacatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
420 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga
ccagatctga 480 gcctgggagc tc 492 2 492 DNA T cell leukemia-lymphoma
virus (HTLV) misc_feature (1)..(492) /standard_name= "Clone BH8"
/note= "Corresponds to nucleotide positions -453 to 39 in figure 3
of US 06/693,866" 2 tggaagggct aattcactcc caacgaagac aagatatcct
tgatctgtgg atccaccaca 60 cacaaggcta cttccctgat tggcagaact
acacaccagg gccaggagtc agatatccac 120 tgacctttgg atggtgctac
aagctagtac cagttgagcc agagaagtaa gaagaagcca 180 ataaaggaga
gaacaccagc ttgttacacc ctgtgagcct gcatggaatg gatgaccctg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
300 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag
ggactttccg 360 ctggggactt tccagggagg cgtggcctgg gcgggactgg
ggagtggcga gccctcagat 420 cctgcatata agcagctgct ttttgcctgt
actgggtctc tctggttaga ccagatctga 480 gcctgggagc tc 492 3 182 DNA T
cell leukemia-lymphoma virus (HTLV) misc_feature (1)..(182)
/standard_name= "Clone HXB2" /note= "Corresponds to nucleotide
positions 40 to 221 in figure 3 of US 06/693,866" 3 tctggctaac
tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa 60
gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
120 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cctgaaagcg
aaagggaaac 180 ca 182 4 8933 DNA T cell leukemia-lymphoma virus
(HTLV) misc_feature (1)..(8933) /standard_name= "Clone BH10" /note=
"Corresponds to nucleotide positions 222 to 9154 in figure 3 of EP
85307260" 4 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
cgaggggcgg 60 cgactggtga gtacgccaaa aattttgact agcggaggct
agaaggagag agatgggtgc 120 gagagcgtca gtattaagcg ggggagaatt
agatcgatgg gaaaaaattc ggttaaggcc 180 agggggaaag aaaaaatata
aattaaaaca tatagtatgg gcaagcaggg agctagaacg 240 attcgcagtt
aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca 300
gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
360 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
ctttagacaa 420 gatagaggaa gagcaaaaca aaagtaagaa aaaagcacag
caagcagcag ctgacacagg 480 acacagcagt caggtcagcc aaaattaccc
tatagtgcag aacatccagg ggcaaatggt 540 acatcaggcc atatcaccta
gaactttaaa tgcatgggta aaagtagtag aagagaaggc 600 tttcagccca
gaagtaatac ccatgttttc agcattatca gaaggagcca ccccacaaga 660
tttaaacacc atgctaaaca cagtgggggg acatcaagca gccatgcaaa tgttaaaaga
720 gaccatcaat gaggaagctg cagaatggga tagagtacat ccagtgcatg
cagggcctat 780 tgcaccaggc cagatgagag aaccaagggg aagtgacata
gcaggaacta ctagtaccct 840 tcaggaacaa ataggatgga tgacaaataa
tccacctatc ccagtaggag aaatttataa 900 aagatggata atcctgggat
taaataaaat agtaagaatg tatagcccta ccagcattct 960 ggacataaga
caaggaccaa aagaaccttt tagagactat gtagaccggt tctataaaac 1020
tctaagagcc gagcaagctt cacaggaggt aaaaaattgg atgacagaaa ccttgttggt
1080 ccaaaatgcg aacccagatt gtaagactat tttaaaagca ttgggaccag
cggctacact 1140 agaagaaatg atgacagcat gtcagggagt aggaggaccc
ggccataagg caagagtttt 1200 ggctgaagca atgagccaag taacaaatac
agctaccata atgatgcaga gaggcaattt 1260 taggaaccaa agaaagatgg
ttaagtgttt caattgtggc aaagaagggc acacagccag 1320 aaattgcagg
gcccctagga aaaagggctg ttggaaatgt ggaaaggaag gacaccaaat 1380
gaaagattgt actgagagac aggctaattt tttagggaag atctggcctt cctacaaggg
1440 aaggccaggg aattttcttc agagcagacc agagccaaca gccccaccat
ttcttcagag 1500 cagaccagag ccaacagccc caccagaaga gagcttcagg
tctggggtag agacaacaac 1560 tccccctcag aagcaggagc cgatagacaa
ggaactgtat cctttaactt ccctcagatc 1620 actctttggc aacgacccct
cgtcacaata aagatagggg ggcaactaaa ggaagctcta 1680 ttagatacag
gagcagatga tacagtatta gaagaaatga gtttgccagg aagatggaaa 1740
ccaaaaatga tagggggaat tggaggtttt atcaaagtaa gacagtatga tcagatactc
1800 atagaaatct gtggacataa agctataggt acagtattag taggacctac
acctgtcaac 1860 ataattggaa gaaatctgtt gactcagatt ggttgcactt
taaattttcc cattagccct 1920 attgagactg taccagtaaa attaaagcca
ggaatggatg gcccaaaagt taaacaatgg 1980 ccattgacag aagaaaaaat
aaaagcatta gtagaaattt gtacagaaat ggaaaaggaa 2040 gggaaaattt
caaaaattgg gcctgagaat ccatacaata ctccagtatt tgccataaag 2100
aaaaaagaca gtactaaatg gagaaaatta gtagatttca gagaacttaa taagagaact
2160 caagacttct gggaagttca attaggaata ccacatcccg cagggttaaa
aaagaaaaaa 2220 tcagtaacag tactggatgt gggtgatgca tatttttcag
ttcccttaga tgaagacttc 2280 aggaagtata ctgcatttac catacctagt
ataaacaatg agacaccagg gattagatat 2340 cagtacaatg tgcttccaca
gggatggaaa ggatcaccag caatattcca aagtagcatg 2400 acaaaaatct
tagagccttt taaaaaacaa aatccagaca tagttatcta tcaatacatg 2460
gatgatttgt atgtaggatc tgacttagaa atagggcagc atagaacaaa aatagaggag
2520 ctgagacaac atctgttgag gtggggactt accacaccag acaaaaaaca
tcagaaagaa 2580 cctccattcc tttggatggg ttatgaactc catcctgata
aatggacagt acagcctata 2640 gtgctgccag aaaaagacag ctggactgtc
aatgacatac agaagttagt ggggaaattg 2700 aattgggcaa gtcagattta
cccagggatt aaagtaaggc aattatgtaa actccttaga 2760 ggaaccaaag
cactaacaga agtaatacca ctaacagaag aagcagagct agaactggca 2820
gaaaacagag agattctaaa agaaccagta catggagtgt attatgaccc atcaaaagac
2880 ttaatagcag aaatacagaa gcaggggcaa ggccaatgga catatcaaat
ttatcaagag 2940 ccatttaaaa atctgaaaac aggaaaatat gcaagaatga
ggggtgccca cactaatgat 3000 gtaaaacaat taacagaggc agtgcaaaaa
ataaccacag aaagcatagt aatatgggga 3060 aagactccta aatttaaact
acccatacaa aaggaaacat gggaaacatg gtggacagag 3120 tattggcaag
ccacctggat tcctgagtgg gagtttgtta atacccctcc tttagtgaaa 3180
ttatggtacc agttagagaa agaacccata gtaggagcag aaaccttcta tgtagatggg
3240 gcagctaaca gggagactaa attaggaaaa gcaggatatg ttactaacaa
aggaagacaa 3300 aaggttgtcc ccctaactaa cacaacaaat cagaaaactg
agttacaagc aatttatcta 3360 gctttgcagg attcaggatt agaagtaaac
atagtaacag actcacaata tgcattagga 3420 atcattcaag cacaaccaga
taaaagtgaa tcagagttag tcaatcaaat aatagagcag 3480 ttaataaaaa
aggaaaaggt ctatctggca tgggtaccag cacacaaagg aattggagga 3540
aatgaacaag tagataaatt agtcagtgct ggaatcagga aaatactatt tttagatgga
3600 atagataagg cccaagatga acatgagaaa tatcacagta attggagagc
aatggctagt 3660 gattttaacc tgccacctgt agtagcaaaa gaaatagtag
ccagctgtga taaatgtcag 3720 ctaaaaggag aagccatgca tggacaagta
gactgtagtc caggaatatg gcaactagat 3780 tgtacacatt tagaaggaaa
agttatcctg gtagcagttc atgtagccag tggatatata 3840 gaagcagaag
ttattccagc agaaacaggg caggaaacag catattttct tttaaaatta 3900
gcaggaagat ggccagtaaa aacaatacat acagacaatg gcagcaattt caccagtgct
3960 acggttaagg ccgcctgttg gtgggcggga atcaagcagg aatttggaat
tccctacaat 4020 ccccaaagtc aaggagtagt agaatctatg aataaagaat
taaagaaaat tataggacag 4080 gtaagagatc aggctgaaca tcttaagaca
gcagtacaaa tggcagtatt catccacaat 4140 tttaaaagaa aaggggggat
tggggggtac agtgcagggg aaagaatagt agacataata 4200 gcaacagaca
tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg 4260
gtttattaca gggacagcag aaatccactt tggaaaggac cagcaaagct cctctggaaa
4320 ggtgaagggg cagtagtaat acaagataat agtgacataa aagtagtgcc
aagaagaaaa 4380 gcaaagatca ttagggatta tggaaaacag atggcaggtg
atgattgtgt ggcaagtaga 4440 caggatgagg attagaacat ggaaaagttt
agtaaaacac catatgtatg tttcagggaa 4500 agctagggga tggttttata
gacatcacta tgaaagccct catccaagaa taagttcaga 4560 agtacacatc
ccactagggg atgctagatt ggtaataaca acatattggg gtctgcatac 4620
aggagaaaga gactggcatt tgggtcaggg agtctccata gaatggagga aaaagagata
4680 tagcacacaa gtagaccctg aactagcaga ccaactaatt catctgtatt
actttgactg 4740 tttttcagac tctgctataa gaaaggcctt attaggacac
atagttagcc ctaggtgtga 4800 atatcaagca ggacataaca aggtaggatc
tctacaatac ttggcactag cagcattaat 4860 aacaccaaaa aagataaagc
cacctttgcc tagtgttacg aaactgacag aggatagatg 4920 gaacaagccc
cagaagacca agggccacag agggagccac acaatgaatg gacactagag 4980
cttttagagg agcttaagaa tgaagctgtt agacattttc ctaggatttg gctccatggc
5040 ttagggcaac atatctatga aacttatggg gatacttggg caggagtgga
agccataata 5100 agaattctgc aacaactgct gtttatccat tttcagaatt
gggtgtcgac atagcagaat 5160 aggcgttact cgacagagga gagcaagaaa
tggagccagt agatcctaga ctagagccct 5220 ggaagcatcc aggaagtcag
cctaaaactg cttgtaccaa ttgctattgt aaaaagtgtt 5280 gctttcattg
ccaagtttgt ttcataacaa aagccttagg catctcctat ggcaggaaga 5340
agcggagaca gcgacgaaga cctcctcaag gcagtcagac tcatcaagtt tctctatcaa
5400 agcagtaagt agtacatgta atgcaaccta tacaaatagc aatagtagca
ttagtagtag 5460 caataataat agcaatagtt gtgtggtcca tagtaatcat
agaatatagg aaaatattaa 5520 gacaaagaaa aatagacagg ttaattgata
gactaataga aagagcagaa gacagtggca 5580 atgagagtga aggagaaata
tcagcacttg tggagatggg ggtggagatg gggcaccatg 5640 ctccttggga
tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac agtctattat 5700
ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga tgctaaagca
5760 tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac
agaccccaac 5820 ccacaagaag tagtattggt aaatgtgaca gaaaatttta
acatgtggaa aaatgacatg 5880 gtagaacaga tgcatgagga tataatcagt
ttatgggatc aaagcctaaa gccatgtgta 5940 aaattaaccc cactctgtgt
tagtttaaag tgcactgatt tgaagaatga tactaatacc 6000 aatagtagta
gcgggagaat gataatggag aaaggagaga taaaaaactg ctctttcaat 6060
atcagcacaa gcataagagg taaggtgcag aaagaatatg cattttttta taaacttgat
6120 ataataccaa tagataatga tactaccagc tatacgttga caagttgtaa
cacctcagtc 6180 attacacagg cctgtccaaa ggtatccttt gagccaattc
ccatacatta ttgtgccccg 6240 gctggttttg cgattctaaa atgtaataat
aagacgttca atggaacagg accatgtaca 6300 aatgtcagca cagtacaatg
tacacatgga attaggccag tagtatcaac tcaactgctg 6360 ttaaatggca
gtctggcaga agaagaggta gtaattagat ctgccaattt cacagacaat 6420
gctaaaacca taatagtaca gctgaaccaa tctgtagaaa ttaattgtac aagacccaac
6480 aacaatacaa gaaaaagtat ccgtatccag agaggaccag ggagagcatt
tgttacaata 6540 ggaaaaatag gaaatatgag acaagcacat tgtaacatta
gtagagcaaa atggaataac 6600 actttaaaac agatagatag caaattaaga
gaacaatttg gaaataataa aacaataatc 6660 tttaagcagt cctcaggagg
ggacccagaa attgtaacgc acagttttaa ttgtggaggg 6720 gaatttttct
actgtaattc aacacaactg tttaatagta cttggtttaa tagtacttgg 6780
agtactaaag ggtcaaataa cactgaagga agtgacacaa tcaccctccc atgcagaata
6840 aaacaaatta taaacatgtg gcaggaagta ggaaaagcaa tgtatgcccc
tcccatcagt 6900 ggacaaatta gatgttcatc aaatattaca gggctgctat
taacaagaga tggtggtaat 6960 agcaacaatg agtccgagat cttcagacct
ggaggaggag atatgaggga caattggaga 7020 agtgaattat ataaatataa
agtagtaaaa attgaaccat taggagtagc acccaccaag 7080 gcaaagagaa
gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt 7140
gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
7200 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
ggctattgag 7260 gcgcaacagc atctgttgca actcacagtc tggggcatca
agcagctcca ggcaagaatc 7320 ctggctgtgg aaagatacct aaaggatcaa
cagctcctgg ggatttgggg ttgctctgga 7380 aaactcattt gcaccactgc
tgtgccttgg aatgctagtt ggagtaataa atctctggaa 7440 cagatttgga
ataacatgac ctggatggag tgggacagag aaattaacaa ttacacaagc 7500
ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
7560 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
ttggctgtgg 7620 tatataaaat tattcataat gatagtagga ggcttggtag
gtttaagaat agtttttgct 7680 gtactttctg tagtgaatag agttaggcag
ggatattcac cattatcgtt tcagacccac 7740 ctcccaatcc cgaggggacc
cgacaggccc gaaggaatag aagaagaagg tggagagaga 7800 gacagagaca
gatccattcg attagtgaac ggatccttag cacttatctg ggacgatctg 7860
cggagcctgt gcctcttcag ctaccaccgc ttgagagact tactcttgat tgtaacgagg
7920 attgtggaac ttctgggacg cagggggtgg gaagccctca aatattggtg
gaatctccta 7980 cagtattgga gtcaggagct aaagaatagt gctgttagct
tgctcaatgc cacagctata 8040 gcagtagctg aggggacaga tagggttata
gaagtagtac aaggagctta tagagctatt 8100 cgccacatac ctagaagaat
aagacagggc ttggaaagga ttttgctata agatgggtgg 8160 caagtggtca
aaaagtagtg tggttggatg gcctgctgta agggaaagaa tgagacgagc 8220
tgagccagca gcagatgggg tgggagcagc atctcgagac ctagaaaaac atggagcaat
8280 cacaagtagc aacacagcag ctaacaatgc tgattgtgcc tggctagaag
cacaagagga 8340 ggaggaggtg ggttttccag tcacacctca ggtaccttta
agaccaatga cttacaaggc 8400 agctgtagat cttagccact ttttaaaaga
aaagggggga ctggaagggc taattcactc 8460 ccaacgaaga caagatatcc
ttgatctgtg gatctaccac acacaaggct acttccctga 8520 ttagcagaac
tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta 8580
caagctagta ccagttgagc cagagaagtt agaagaagcc aacaaaggag agaacaccag
8640 cttgttacac cctgtgagcc tgcatggaat ggatgacccg gagagagaag
tgttagagtg 8700 gaggtttgac agccgcctag catttcatca catggcccga
gagctgcatc cggagtactt 8760 caagaactgc tgacatcgag cttgctacaa
gggactttcc gctggggact ttccagggag 8820 gcgtggcctg ggcgggactg
gggagtggcg agccctcaga tcctgcatat aagcagctgc 8880 tttttgcctg
tactgggtct ctctggttag accagatctg agcctgggag ctc 8933 5 5362 DNA T
cell leukemia-lymphoma virus (HTLV) misc_feature (1)..(5362)
/standard_name= "Clone BH5" /note= "Corresponds to nucleotide
positions 222 to 5585 in figure 3 of US 06/693,866" 5 gagctctctc
gacgcaggac tcggcttgcg agcgcgcacg gcaagaggcg aggggcggcg 60
actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
120 gagcgtcagt attaagcggg ggagaattag atcgatggga aaaaattcgg
ttaaggccag 180 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
aagcagggag ctagaacgat 240 tcgcagttaa tcctggcctg ttagaaacat
cagaaggctg tagacaaata ctgggacagc 300 tacaaccatc ccttcagaca
ggatcagaag aacttagatc attatataat acagtagcaa 360 ccctctattg
tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga 420
tagaggaaga gcaaaacaaa agtaagaaaa aagcacagca agcagcagct gacacaggac
480 acagcagtca ggtcagccaa aattacccta tagtgcagaa catccagggg
caaatggtac 540 atcaggccat atcacctaga actttaaatg catgggtaaa
agtagtagaa gagaaggctt 600 tcagcccaga agtgataccc atgttttcag
cattatcaga aggagccacc ccacaagatt 660 taaacaccat gctaaacaca
gtggggggac atcaagcagc catgcaaatg ttaaaagaga 720 ccatcaatga
ggaagctgca gaatgggata gagtgcatcc agtgcatgca gggcctatcg 780
caccaggcca gatgagagaa ccaaggggaa gtgacatagc aggaactact agtacccttc
840 aggaacaaat aggatggatg acaaataatc cacctatccc agtaggagaa
atttataaaa 900 gatggataat cctgggatta aataaaatag taaggatgta
tagtcctacc agcattctgg 960 acataagaca aggaccaaag gaacccttta
gagactatgt agaccggttc tataaaactc 1020 taagagccga gcaagcttca
caggaagtaa aaaattggat gacagaaacc ttgttggtcc 1080 aaaatgcgaa
cccagattgt aagactattt taaaagcatt gggaccagcg gctacactag 1140
aagaaatgat gacagcatgt cagggagtag gaggacccgg ccataaggca agagttttgg
1200 ctgaagcaat gagccaagta acaaattcaa ctaccataat gatgcaaaga
ggcaatttta 1260 ggaaccaaag aaaaattgtt aagtgtttca attgtggcaa
agaagggcac atagcaagaa 1320 attgcaaggc ccctagaaaa aagggctgtt
ggaaatgtgg aaaggaagga caccaaatga 1380 aagattgtac tgagagacag
gctaattttt tagggaagat ctggccttcc tacaagggaa 1440 ggccagggaa
ttttcttcag agcagaccag agccaacagc cccaccattt cttcagagca 1500
gaccagagcc aacagcccca ccagaagaga gcttcaggtc tggggtagag acaacaactc
1560 cccctcagaa gcaggagccg atagacaagg aactgtatcc tttaacttcc
ctcagatcac 1620 tctttggcaa cgacccctcg tcacaataaa gatagggggg
caactaaagg aagctctatt 1680 agatacagga gcagatgata cagtattaga
agaaatgagt ttgccaggaa gatggaaacc 1740 aaaaatgata gggggaattg
gaggttttat caaagtaaga cagtatgatc agatactcat 1800 agaaatctgt
ggacataaag ctataggtac agtattagta ggacctacac ctgtcaacat 1860
aattggaaga aatctgttga ctcagattgg ttgcacttta aattttccca ttagtcctat
1920 tgaaactgta ccagtaaaat taaagccagg aatggatggc ccaaaagtta
aacaatggcc 1980 attgacagaa gaaaaaataa aagcattagt agaaatttgt
acagaaatgg aaaaggaagg 2040 gaaaatttca aaaattgggc ctgaaaatcc
atacaatact ccagtatttg ccataaagaa 2100 aaaagacagt actaaatgga
gaaaattagt agatttcaga gaacttaata ggagaactca 2160 agacttctgg
gaagttcaat tgggaatacc acatcccgca gggttaaaaa agaaaaaatc 2220
agtaacagta ctggatgtgg gtgatgcata tttttcagtt cccttagatg aagacttcag
2280 gaagtatact gcatttacca tacctagtat aaataatgag acaccaggga
gtggatatca 2340 gtacaatgtg cttccacagg gatggaaagg atcaccagca
atattccaaa gtagcatgac 2400 aaaaatctta gagcctttta gaaaacaaaa
tccagacata gttatttatc aatacatgga 2460 tgatttgtat gtaggatctg
acttagaaat agggcagcat agaacaaaaa tagaggagct 2520 gagacaacat
ctgttgaggt ggggatttac cacaccagac aaaaaacatc agaaagaacc 2580
tccattcctt tggatgggtt atgaactcca tcctgataaa tggacgatac agcctatagt
2640 gctgccagaa aaagacagct ggactgtcaa tgacatacag aagttagtgg
gaaaattgaa 2700 ttgggcaagt cagatttatc cagggattaa agtaaggcaa
ttatgtaaac tccttagagg 2760 aaccaaagca ctaacagaag taataccact
aacagaagaa gcagagctag aactggcaga 2820 aaacagagag attctaaaag
aaccagtaca tggagtgtat tatgacccat caaaagactt 2880 aatagcagaa
atacagaagc aggggcaagg ccaatggaca tatcaaattt atcaagagcc 2940
atttaaaaat ctgaaaacag gaaaatatgc aagaatgagg ggtgcccaca ctaatgatgt
3000 aaaacaatta acagaggcag tgcaaaaaat aaccacagaa agcatagtaa
tatggggaaa 3060 gactcctaaa tttaaactac ccatacaaaa agaaacatgg
gaaacatggt ggacagagta 3120 ttggcaagcc acctggattc ctgagtggga
gtttgttaat acccctcctt tagtgaaatt 3180 atggtaccag ttagagaaag
aacccatagt aggagcagaa accttctatg tagatggggc 3240 agctagcagg
gagactaaat taggaaaagc aggatatgtt actaatagag gaagacaaaa 3300
agttgtcacc ctaactcaca caacaaatca gaagactgaa ttacaagcaa ttcatctagc
3360 tttgcaggat tcgggattag aagtaaatat agtaacagac tcacaatatg
cattaggaat 3420 cattcaagca caaccagata aaagtgaatc agagttagtc
aatcaaataa tagagcagtt 3480 aataaaaaag gaaaaggtct atctggcatg
ggtaccagca cacaaaggaa ttggaggaaa 3540 tgaacaagta gataaattag
tcagtgctgg aatcaggaaa atactatttt tagatggaat 3600 agataaggcc
caagaagaac atgagaaata tcacagtaat tggagagcaa tggctagtga 3660
ttttaacctg ccacctgtag tagcaaaaga aatagtagcc agctgtgata aatgtcagct
3720 aaaaggagaa gccatgcatg gacaagtaga ctgtagtcca ggaatatggc
aactagattg 3780 tacacattta gaaggaaaag ttatcctggt agcagttcat
gtagccagtg gatatataga 3840 agcagaagtt attccagcag aaacagggca
ggaaacagca tattttcttt taaaattagc 3900 aggaagatgg ccagtaaaaa
caatacatac agacaatggc agcaatttca ccagtgctac 3960 ggttaaggcc
gcctgttggt gggcgggaat caagcaggaa tttggaattc cctacaatcc 4020
ccaaagtcaa ggagtagtag aatctatgaa taaagaatta aagaaaatta taggacaggt
4080 aagagatcag gctgaacatc ttaagacagc agtacaaatg gcagtattca
tccacaattt 4140 taaaagaaaa ggggggattg gggggtacag tgcaggggaa
agaatagtag acataatagc 4200 aacagacata caaactaaag aattacaaaa
acaaattaca aaaattcaaa attttcgggt 4260 ttattacagg gacagcagaa
atccactttg gaaaggacca gcaaagctcc tctggaaagg 4320 tgaaggggca
gtagtaatac aagataatag tgacataaaa gtagtgccaa gaagaaaagc 4380
aaagatcatt agggattatg gaaaacagat ggcaggtgat gattgtgtgg caagtagaca
4440 ggatgaggat tagaacatgg aaaagtttag taaaacaccg tatgtatgtt
tcagggaaag 4500 ctaggggatg gttttataga catcactatg aaagccctca
tccaagaata agttcagaag 4560 tacacatccc actaggggat gctagattgg
taataacaac atattggggt ctgcatacag 4620 gagaaagaga ctggcatttg
ggtcagggag tctccataga atggaggaaa aggagatata 4680 gcacacaagt
agaccctgaa ctagcagacc aactaattca tctgcattac tttgattgtt 4740
tttcagactc tgctataaga aaggccttat taggacacat agttagccct aggtgtgaat
4800 atcaagcagg acataacaag gtaggatctc tacaatactt ggcactagca
gcattaataa 4860 caccaaaaaa ggtaaagcca cctttgccta gtgttacgaa
actgacagag gatagatgga 4920 acaagcccca gaagaccaag ggccacagag
gaagccacac aatgaatgga cactagagct 4980 tttagaggag cttaagaatg
aagctgttag acattttcct aggatttggc tccatggctt 5040 agggcaacat
atctatgaaa cttatgggga tacttgggca ggagtggaag ccataataag 5100
aattctgcaa caactgctgt ttatccattt tcagaattgg gtgtcgacat agcagaatag
5160 gcgttactca acagaggaga gcaagaaatg gagccagtag atcctagact
agagccctgg 5220 aagcatccag gaagtcagcc taaaactgct tgtaccactt
gctattgtaa aaagtgttgc 5280 tttcattgcc aagtttgttt cataacaaaa
gccttaggca tctcctatgg caggaagaag 5340 cggagacagc gacgaagagc tc 5362
6 3563 DNA T cell leukemia-lymphoma virus (HTLV) misc_feature
(1)..(3563) /standard_name= "Clone BH8" /note= "Corresponds to
nucleotide positions 5580 to 9154 in figure 3 of US 06/693,866" 6
gagctcatcg aagcagtcag actcatcaag tttctctatc aaagcagtaa gtagtacatg
60 taacgcaacc tataccaata gtaacaatag tagccttagc agtagcaata
ataatagcaa 120 tagttgtgtg gtccatagta atcatagaat ataggaaaat
attaagacaa agaaaaatag 180 acaggttaat tgatagacta atagaaagag
cagaagacag tggcaatgag agtgaaggag 240 aaatatcagc acttgtggag
atgggggtgg agatggggca ccatgctcct tgggatgttg 300 atgatctgta
gtgctacaga aaaattgtgg gtcacagtct attttggggt acctgtgtgg 360
aaggaagcaa ccaccactct attttgtgca tcagatgcta aagcatatga tacagaggta
420 cataatgttt gggccacaca tgcctgtgta cccacagacc ccaacccaca
agaagtagta 480 ttggtaaatg tgacagaaaa ttttaacatg tggaaaaatg
acatggtaga acagatgcat 540 gaggatataa tcagtttatg ggatcaaagc
ctaaagccat gtgtaaaatt aaccccactc 600 tgtgttagtt taaagtgcac
tgatttgaag aatgatacta ataccaatag tagtagcggg 660 agaatgataa
tggagaaagg agagataaaa aactgctctt tcaatatcag cacaagcaaa 720
agaggtaagg tgcagaaaga atatgcattt ttttataaac ttgatataat accaatagat
780 aatgatacta ccagctatac gttgacaagt tgtaacacct cagtcattac
acaggcctgt 840 ccaaaggtat cctttgagcc aattcccata cattattgtg
ccccggctgg ttttgcgatt 900 ctaaaatgta ataataagac gttcaatgga
acaggaccat gtacaaatgt cagcacagta 960 caatgtacac atggaattag
gccagtagta tcaactcaac tgctgttaaa tggcagtctg 1020 gcagaagaag
aggtagtaat tagatctgtc aatttcacgg acaatgctaa aaccataata 1080
gtacagctgg acacatctgt agaaattaat tgtacaagac ccaacaacaa tacaagaaaa
1140 aaaatccgta tccagagggg accagggaga gcatttgtta caataggaaa
aataggaaat 1200 atgagacaag cacattgtaa cattagtaga gcaaaatgga
atgccacttt aaaacagata 1260 gatagcaaat taagagaaca atttggaaat
aataaaacaa taatctttaa gcagtcctca 1320 ggaggggacc cagaaattgt
aacgcacagt tttaattgtg gaggggaatt tttctactgt 1380 aattcaacac
aactgtttaa tagtacttgg agtactaaag ggtcaaataa cactgaagga 1440
agtgacacaa tcaccctccc atgcagaata aaacaaatta taaacatgtg gcaggaagta
1500 ggaaaagcaa tgtatgcccc tcccatcagt ggacaaatta gatgttcatc
aaatattaca 1560 gggctgctat taacaagaga tggtggtaat agcaacaatg
agtccgagat cttcagacct 1620 ggaggaggag atatgaggga caattggaga
agtgaattat ataaatataa agtagtaaaa 1680 attgaaccat taggagtagc
acccaccaag gcaaagagaa gagtggtgca gagagaaaaa 1740 agagcagtgg
gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg 1800
ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
1860 cagcagaaca atttgctgag ggctattgag ggccaacagc atctgttgca
actcacagtc 1920 tggggcatca agcagctcca ggcaagaatc ctggctgtgg
aaagatacct aaaggatcaa 1980 cagctcctgg ggatttgggg ttgctctgga
aaactcattt gcaccactgc tgtgccttgg 2040 aatgctagtt ggagtaataa
atctctggaa cagatttgga ataacatgac ctggatggag 2100 tgggacagag
aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa 2160
aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
2220 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
gatagtagga 2280 ggcttggtag gtttaagaat agtttttgct gtactttcta
tagtgaatag agttaggcag 2340 ggatattcac cattatcgtt tcagacccac
ctcccaaacc cgaggggacc cgacaggccc 2400 gaaggaatag aagaagaagg
tggagagaga gacagagaca gatccattcg attagtgaac 2460 ggatccttag
cacttatctg ggacgatctg cggagcctgt gcctcttcag ctaccaccgc 2520
ttgagagact tactcttgat tgtaacgagg attgtggaac ttctgggacg cagggggtgg
2580 gaagccctca aatattggtg gaatctccta cagtattgga gtcaggaact
aaagaatagt 2640 gctgttaact tgctcaatgc cacagctata gcagtagctg
aggggacaga tagggttata 2700 gaattagtac aagcagctta tagagccatt
cgccacatac ctagaagaat aagacagggc 2760 ttggaaagga ttttgctata
agatgggtgg caagtggtca aaaagtagtg tggttggatg 2820 gcctgctgta
agggaaagaa tgagacgagc tgagccagca gcagatgggg tgggagcagt 2880
atctcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
2940 cgattgtgct tggctagaag cacaagagga ggaggaggtg ggttttccag
tcacacctca 3000 ggtaccttta agaccaatga cttacaaggc agctgtagat
cttagccact ttttaaaaga 3060 aaagggggga ctggaagggc taattcactc
ccaacgaaga caagatatcc ttgatctgtg 3120 gatccaccac acacaaggct
acttccctga ttggcagaac tacacaccag ggccaggagt 3180 cagatatcca
ctgacctttg gatggtgcta caagctagta ccagttgagc cagagaagta 3240
agaagaagcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatggaat
3300 ggatgaccct gagagagaag tgttagagtg gaggtttgac agccgcctag
catttcatca 3360 catggcccga gagctgcatc cggagtactt caagaactgc
tgatatcgag cttgctacaa 3420 gggactttcc gctggggact ttccagggag
gcgtggcctg ggcgggactg gggagtggcg 3480 agccctcaga tcctgcatat
aagcagctgc tttttgcctg tactgggtct ctctggttag 3540 accagatctg
agcctgggag ctc 3563 7 142 DNA T cell leukemia-lymphoma virus (HTLV)
misc_feature (1)..(142) /standard_name= "Clone HXB2" /note=
"Corresponds to nucleotide positions 9155 to 9296 in figure 3 of US
06/693,866" 7 tctggctagc tagggaaccc actgcttaag cctcaataaa
gcttgccttg agtgcttcaa 60 gtagtgtgtg cccgtctgtt gtgtgactct
ggtaactaga gatccctcag acccttttag 120 tcagtgtgga aaatctctag ca 142 8
512 PRT T cell leukemia-lymphoma virus (HTLV) MISC_FEATURE
(1)..(512) /note= "gag protein of HTLV-III" 8 Met Gly Ala Arg Ala
Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp 1 5 10 15 Glu Lys Ile
Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30 His
Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40
45 Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu
50 55 60 Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu
Tyr Asn 65 70 75 80 Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile
Glu Ile Lys Asp 85 90 95 Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu
Glu Gln Asn Lys Ser Lys 100 105 110 Lys Lys Ala Gln Gln Ala Ala Ala
Asp Thr Gly His Ser Ser Gln Val 115 120 125 Ser Gln Asn Tyr Pro Ile
Val Gln Asn Ile Gln Gly Gln Met Val His 130 135 140 Gln Ala Ile Ser
Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu 145 150 155 160 Glu
Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170
175 Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly
180 185 190 Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn
Glu Glu 195 200 205 Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala
Gly Pro Ile Ala 210 215 220 Pro Gly Gln Met Arg Glu Pro Arg Gly Ser
Asp Ile Ala Gly Thr Thr 225 230 235 240 Ser Thr Leu Gln Glu Gln Ile
Gly Trp Met Thr Asn Asn Pro Pro Ile 245 250 255 Pro Val Gly Glu Ile
Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 260 265 270 Ile Val Arg
Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly 275 280 285 Pro
Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 290 295
300 Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr
305 310 315 320 Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile
Leu Lys Ala 325 330 335 Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met
Thr Ala Cys Gln Gly 340 345 350 Val Gly Gly Pro Gly His Lys Ala Arg
Val Leu Ala Glu Ala Met Ser 355 360 365 Gln Val Thr Asn Thr Ala Thr
Ile Met Met Gln Arg Gly Asn Phe Arg 370 375 380 Asn Gln Arg Lys Met
Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His 385 390 395 400 Thr Ala
Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 405 410 415
Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 420
425 430 Phe Leu Gly Lys Ile Trp Pro Ser Tyr Lys Gly Arg Pro Gly Asn
Phe 435 440 445 Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Phe Leu
Gln Ser Arg 450 455 460 Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe
Arg Ser Gly Val Glu 465 470 475 480 Thr Thr Thr Pro Pro Gln Lys Gln
Glu Pro Ile Asp Lys Glu Leu Tyr 485 490 495 Pro Leu Thr Ser Leu Arg
Ser Leu Phe Gly Asn Asp Pro Ser Ser Gln 500 505 510 9 1015 PRT T
cell leukemia-lymphoma virus (HTLV) MISC_FEATURE (1)..(1015) /note=
"pol protein of HTLV-III" 9 Phe Phe Arg Glu Asp Leu Ala Phe Leu Gln
Gly Lys Ala Arg Glu Phe 1 5 10 15 Ser Ser Glu Gln Thr Arg Ala Asn
Ser Pro Thr Ile Ser Ser Glu Gln 20 25 30 Thr Arg Ala Asn Ser Pro
Thr Arg Arg Glu Leu Gln Val Trp Gly Arg 35 40 45 Asp Asn Asn Ser
Pro Ser Glu Ala Gly Ala Asp Arg Gln Gly Thr Val 50 55 60 Ser Phe
Asn Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Thr 65 70 75 80
Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala 85
90 95 Asp Asp Thr Val Leu Glu Glu Met Ser Leu Pro Gly Arg Trp Lys
Pro 100 105 110 Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg
Gln Tyr Asp 115 120 125 Gln Ile Leu Ile Glu Ile Cys Gly His Lys Ala
Ile Gly Thr Val Leu 130 135 140 Val Gly Pro Thr Pro Val Asn Ile Ile
Gly Arg Asn Leu Leu Thr Gln 145 150 155 160 Ile Gly Cys Thr Leu Asn
Phe Pro Ile Ser Pro Ile Glu Thr Val Pro 165 170 175 Val Lys Leu Lys
Pro Gly Met Asp Gly Pro Lys Val Lys Gln Trp Pro 180 185 190 Leu Thr
Glu Glu Lys Ile Lys Ala Leu Val Glu Ile Cys Thr Glu Met 195 200 205
Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu Asn Pro Tyr Asn 210
215 220 Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg
Lys 225 230 235 240 Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln
Asp Phe Trp Glu 245 250 255 Val Gln Leu Gly Ile Pro His Pro Ala Gly
Leu Lys Lys Lys Lys Ser 260 265 270 Val Thr Val Leu Asp Val Gly Asp
Ala Tyr Phe Ser Val Pro Leu Asp 275 280 285 Glu Asp Phe Arg Lys Tyr
Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn 290 295 300 Glu Thr Pro Gly
Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp 305 310 315 320 Lys
Gly Ser Pro Ala Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu 325 330
335 Pro Phe Lys Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met Asp
340 345 350 Asp Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg
Thr Lys 355 360 365 Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly
Leu Thr Thr Pro 370 375 380 Asp Lys Lys His Gln Lys Glu Pro Pro Phe
Leu Trp Met Gly Tyr Glu 385 390 395 400 Leu His Pro Asp Lys Trp Thr
Val Gln Pro Ile Val Leu Pro Glu Lys 405 410 415 Asp Ser Trp Thr Val
Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn 420 425 430 Trp Ala Ser
Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys 435 440 445 Leu
Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Ile Pro Leu Thr Glu 450 455
460 Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro
465 470 475 480 Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile
Ala Glu Ile 485 490 495 Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln
Ile Tyr Gln Glu Pro 500 505 510 Phe Lys Asn Leu Lys Thr Gly Lys Tyr
Ala Arg Met Arg Gly Ala His 515 520 525 Thr Asn Asp Val Lys Gln Leu
Thr Glu Ala Val Gln Lys Ile Thr Thr 530 535 540 Glu Ser Ile Val Ile
Trp Gly Lys Thr Pro Lys Phe Lys Leu Pro Ile 545 550 555 560 Gln Lys
Glu Thr Trp Glu Thr Trp Trp Thr Glu Tyr Trp Gln Ala Thr 565 570 575
Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu 580
585 590 Trp Tyr Gln Leu Glu Lys Glu Pro Ile Val Gly Ala Glu Thr Phe
Tyr 595 600 605 Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly Lys
Ala Gly Tyr 610 615 620 Val Thr Asn Lys Gly Arg Gln Lys Val Val Pro
Leu Thr Asn Thr Thr 625 630 635 640 Asn Gln Lys Thr Glu Leu Gln Ala
Ile Tyr Leu Ala Leu Gln Asp Ser 645 650 655 Gly Leu Glu Val Asn Ile
Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile 660 665 670 Ile Gln Ala Gln
Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile 675 680 685 Ile Glu
Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu Ala Trp Val Pro 690 695 700
Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser 705
710 715 720 Ala Gly Ile Arg Lys Ile Leu Phe Leu Asp Gly Ile Asp Lys
Ala Gln 725 730 735 Asp Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala
Met Ala Ser Asp 740 745 750 Phe Asn Leu Pro Pro Val Val Ala Lys Glu
Ile Val Ala Ser Cys Asp 755 760 765 Lys Cys Gln Leu Lys Gly Glu Ala
Met His Gly Gln Val Asp Cys Ser 770 775 780 Pro Gly Ile Trp Gln Leu
Asp Cys Thr His Leu Glu Gly Lys Val Ile 785 790 795 800 Leu Val Ala
Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile 805 810 815 Pro
Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu Leu Lys Leu Ala 820 825
830 Gly Arg Trp Pro Val Lys Thr Ile His Thr Asp Asn Gly Ser Asn Phe
835 840 845 Thr Ser Ala Thr Val Lys Ala Ala Cys Trp Trp Ala Gly Ile
Lys Gln 850 855 860 Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly
Val Val Glu Ser 865 870 875 880 Met Asn Lys Glu Leu Lys Lys Ile Ile
Gly Gln Val Arg Asp Gln Ala 885 890 895 Glu His Leu Lys Thr Ala Val
Gln Met Ala Val Phe Ile His Asn Phe 900 905 910 Lys Arg Lys Gly Gly
Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val 915 920 925 Asp Ile Ile
Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile 930 935 940 Thr
Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asn Pro 945 950
955 960 Leu Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala
Val 965 970 975 Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg
Arg Lys Ala 980 985 990 Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala
Gly Asp Asp Cys Val
995 1000 1005 Ala Ser Arg Gln Asp Glu Asp 1010 1015 10 203 PRT T
cell leukemia-lymphoma virus (HTLV) MISC_FEATURE (1)..(203) /note=
"sor protein of HTLV-III" 10 Cys Gln Glu Glu Lys Gln Arg Ser Leu
Gly Ile Met Glu Asn Arg Trp 1 5 10 15 Gln Val Met Ile Val Trp Gln
Val Asp Arg Met Arg Ile Arg Thr Trp 20 25 30 Lys Ser Leu Val Lys
His His Met Tyr Val Ser Gly Lys Ala Arg Gly 35 40 45 Trp Phe Tyr
Arg His His Tyr Glu Ser Pro His Pro Arg Ile Ser Ser 50 55 60 Glu
Val His Ile Pro Leu Gly Asp Ala Arg Leu Val Ile Thr Thr Tyr 65 70
75 80 Trp Gly Leu His Thr Gly Glu Arg Asp Trp His Leu Gly Gln Gly
Val 85 90 95 Ser Ile Glu Trp Arg Lys Lys Arg Tyr Ser Thr Gln Val
Asp Pro Glu 100 105 110 Leu Ala Asp Gln Leu Ile His Leu Tyr Tyr Phe
Asp Cys Phe Ser Asp 115 120 125 Ser Ala Ile Arg Lys Ala Leu Leu Gly
His Ile Val Ser Pro Arg Cys 130 135 140 Glu Tyr Gln Ala Gly His Asn
Lys Val Gly Ser Leu Gln Tyr Leu Ala 145 150 155 160 Leu Ala Ala Leu
Ile Thr Pro Lys Lys Ile Lys Pro Pro Leu Pro Ser 165 170 175 Val Thr
Lys Leu Thr Glu Asp Arg Trp Asn Lys Pro Gln Lys Thr Lys 180 185 190
Gly His Arg Gly Ser His Thr Met Asn Gly His 195 200 11 863 PRT T
cell leukemia-lymphoma virus (HTLV) MISC_FEATURE (1)..(863) /note=
"env protein of HTLV-III" 11 Lys Glu Gln Lys Thr Val Ala Met Arg
Val Lys Glu Lys Tyr Gln His 1 5 10 15 Leu Trp Arg Trp Gly Trp Arg
Trp Gly Thr Met Leu Leu Gly Met Leu 20 25 30 Met Ile Cys Ser Ala
Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly 35 40 45 Val Pro Val
Trp Lys Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp 50 55 60 Ala
Lys Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala Thr His Ala 65 70
75 80 Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Val Val Leu Val Asn
Val 85 90 95 Thr Glu Asn Phe Asn Met Trp Lys Asn Asp Met Val Glu
Gln Met His 100 105 110 Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu
Lys Pro Cys Val Lys 115 120 125 Leu Thr Pro Leu Cys Val Ser Leu Lys
Cys Thr Asp Leu Lys Asn Asp 130 135 140 Thr Asn Thr Asn Ser Ser Ser
Gly Arg Met Ile Met Glu Lys Gly Glu 145 150 155 160 Ile Lys Asn Cys
Ser Phe Asn Ile Ser Thr Ser Ile Arg Gly Lys Val 165 170 175 Gln Lys
Glu Tyr Ala Phe Phe Tyr Lys Leu Asp Ile Ile Pro Ile Asp 180 185 190
Asn Asp Thr Thr Ser Tyr Thr Leu Thr Ser Cys Asn Thr Ser Val Ile 195
200 205 Thr Gln Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His
Tyr 210 215 220 Cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys Asn Asn
Lys Thr Phe 225 230 235 240 Asn Gly Thr Gly Pro Cys Thr Asn Val Ser
Thr Val Gln Cys Thr His 245 250 255 Gly Ile Arg Pro Val Val Ser Thr
Gln Leu Leu Leu Asn Gly Ser Leu 260 265 270 Ala Glu Glu Glu Val Val
Ile Arg Ser Ala Asn Phe Thr Asp Asn Ala 275 280 285 Lys Thr Ile Ile
Val Gln Leu Asn Gln Ser Val Glu Ile Asn Cys Thr 290 295 300 Arg Pro
Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gln Arg Gly Pro 305 310 315
320 Gly Arg Ala Phe Val Thr Ile Gly Lys Ile Gly Asn Met Arg Gln Ala
325 330 335 His Cys Asn Ile Ser Arg Ala Lys Trp Asn Asn Thr Leu Lys
Gln Ile 340 345 350 Asp Ser Lys Leu Arg Glu Gln Phe Gly Asn Asn Lys
Thr Ile Ile Phe 355 360 365 Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile
Val Thr His Ser Phe Asn 370 375 380 Cys Gly Gly Glu Phe Phe Tyr Cys
Asn Ser Thr Gln Leu Phe Asn Ser 385 390 395 400 Thr Trp Phe Asn Ser
Thr Trp Ser Thr Lys Gly Ser Asn Asn Thr Glu 405 410 415 Gly Ser Asp
Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn 420 425 430 Met
Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Ser Gly 435 440
445 Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp
450 455 460 Gly Gly Asn Ser Asn Asn Glu Ser Glu Ile Phe Arg Pro Gly
Gly Gly 465 470 475 480 Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr
Lys Tyr Lys Val Val 485 490 495 Lys Ile Glu Pro Leu Gly Val Ala Pro
Thr Lys Ala Lys Arg Arg Val 500 505 510 Val Gln Arg Glu Lys Arg Ala
Val Gly Ile Gly Ala Leu Phe Leu Gly 515 520 525 Phe Leu Gly Ala Ala
Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu 530 535 540 Thr Val Gln
Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn 545 550 555 560
Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr 565
570 575 Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Val Glu
Arg 580 585 590 Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys
Ser Gly Lys 595 600 605 Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala
Ser Trp Ser Asn Lys 610 615 620 Ser Leu Glu Gln Ile Trp Asn Asn Met
Thr Trp Met Glu Trp Asp Arg 625 630 635 640 Glu Ile Asn Asn Tyr Thr
Ser Leu Ile His Ser Leu Ile Glu Glu Ser 645 650 655 Gln Asn Gln Gln
Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys 660 665 670 Trp Ala
Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr 675 680 685
Ile Lys Leu Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile 690
695 700 Val Phe Ala Val Leu Ser Val Val Asn Arg Val Arg Gln Gly Tyr
Ser 705 710 715 720 Pro Leu Ser Phe Gln Thr His Leu Pro Ile Pro Arg
Gly Pro Asp Arg 725 730 735 Pro Glu Gly Ile Glu Glu Glu Gly Gly Glu
Arg Asp Arg Asp Arg Ser 740 745 750 Ile Arg Leu Val Asn Gly Ser Leu
Ala Leu Ile Trp Asp Asp Leu Arg 755 760 765 Ser Leu Cys Leu Phe Ser
Tyr His Arg Leu Arg Asp Leu Leu Leu Ile 770 775 780 Val Thr Arg Ile
Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu 785 790 795 800 Lys
Tyr Trp Trp Asn Leu Leu Gln Tyr Trp Ser Gln Glu Leu Lys Asn 805 810
815 Ser Ala Val Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly
820 825 830 Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala
Ile Arg 835 840 845 His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg
Ile Leu Leu 850 855 860
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