Chiron Corporation, Emeryville, California 94608,1 and Cancer Research Institute and Department ofMedicine,. University of California, School ofMedicine, San ...
Vol. 64, No. 8
JOURNAL OF VIROLOGY, Aug. 1990, p. 40164020 0022-538X/90/084016-05$02.00/0 Copyright X) 1990, American Society for Microbiology
Human Immunodeficiency Virus Type 1 Cellular Host Range, Replication, and Cytopathicity Are Linked to the Envelope Region of the Viral Genome DEBORAH YORK-HIGGINS,1 CECILIA CHENG-MAYER,2 DIANE BAUER,1 JAY A. LEVY,2* AND DINO DINA Chiron Corporation, Emeryville, California 94608,1 and Cancer Research Institute and Department of Medicine, University of California, School of Medicine, San Francisco, California 9414301282 Received 19 January 1990/Accepted 15 May 1990
Human immunodeficiency virus type 1 (HIV-1) isolates vary in their in vitro biologic characteristics such as cellular host range, replication kinetics, and cytopathicity. In this study, we molecularly exchanged equivalent regions between two cloned HIV-1 isolates with differing replicative and cytopathic properties. To facilitate generation of recombinant viruses, we used a method involving cotransfection of human monolayer cells with plasmid constructs containing half of the biologically active viral genome. The two halves of the genome were subsequently ligated by intracellular processes to form the complete proviral genome. This method simplifies plasmid construction, since new infectious virus particles can be produced easily from the individual constructs that are correctly ligated in vivo. Results obtained by using recombinant viruses generated in this manner indicate that the ability of HIV to replicate in specific cell types and cytopathicity segregate with the env region of the viral genome. in the MT-4 cell line (33). Both isolates have been molecularly cloned. The molecular cloning of HIV-lsF2 has been previously reported (20). HIV-1sF33 was molecularly cloned by the same procedure (20) from a library of recombinant phages constructed from the EcoRI partially digested genomic DNA of HIV-lSF33-infected HUT 78 cells. The molecular clones of HIV-lsF2 and HIV-lsF33 were found to be biologically active upon transfection into human rhabdomyosarcoma (RD-4) and HUT 78 cells. The biologic properties of the molecular clones showed characteristics identical to those of the parental viruses (Table 1). The genomic sequences of genes in the 3' region of the HIV-lSF33 genome and their predicted amino acid sequences were determined and compared to those of HIV-1sF2 (28) (Table 2). For all genes compared, the two isolates showed 90.2 to 96.4% homology of the nucleotide sequence and 82.5 to 88.1% homology of the predicted amino acid sequence. The amino acid sequence homology in the gpl20 region of env is 82.5%, while the gp4l region shows 86.7% homology
The genomes of the human immunodeficiency virus type 1 (HIV-1) viral isolates are highly heterogeneous (2, 4). This well-recognized genomic diversity most likely reflects some of the biologic differences observed among HIV-1 isolates (5, 9, 17, 27). These include the ability of an HIV-1 isolate to replicate in different cell types and to cause cytopathic changes in infected host cells. Previous studies on a panel of transectional and sequential HIV-1 isolates have provided evidence for a role of fast-replicating and highly cytopathic variants in the progression of acquired immune deficiency syndrome (5, 9, 34). Defining the functional gene(s) of HIV-1 that controls these biologic properties could therefore help in the understanding of HIV pathogenesis and in designing strategies for antiviral approaches. In this study, recombinant viruses generated between molecular clones of two HIV-1 isolates (HIV-1sF2 and HIV-lsF33) showing differences in biologic properties were used to map the region of the HIV-1 genome that controls host range, viral replication, and cytopathicity. The HIV-lsF2 strain (formerly called acquired immune deficiency syndrome-associated retrovirus, ARV-2) was initially isolated from the peripheral mononuclear cells (PMC) of a patient with oral candidiasis (15). HIV-lSF33 was obtained from the PMC of a patient with thrombocytopenia (16). HIV-1sF2 replicates efficiently in PMC from seronegative donors and the established T-cell line HUT 78, reaching peak virus production at 12 to 14 days postinfection. This isolate does not productively infect the CD4-negative human osteosarcoma (HOS) cells (32) and replicates inefficiently in the CEM cell line. Reverse transcriptase (RT) activity is not detected (13) in infected CEM cultures until -30 days postinfection; however, cocultivation at 10 to 14 days postinoculation with PMC, a sensitive target cell for HIV-1, demonstrates a low level of virus replication. In contrast, the HIV-1sF33 strain replicates rapidly and to high titers in the HUT 78 and CEM cell lines and productively infects HOS cells. This isolate is also very cytopathic and forms plaques *
TABLE 1. Biologic properties of HIV-1sF2 and HIV-lSF33 molecular clones clone
cytopathol°gYa Cytopathologya
Plaque b formation
SF2 SF33
+ ++
+
HIV-1
clone
-
~~PMC
Replication HUT
in':
CEM
HOS
2.7 1.0 873.3 749.0 1267.7 1034.0 246.6 626.9
a Cytopathology was assessed by the presence of balloon cell degeneration and syncytial formation in HUT 78 and MT-4 cells at peak RT activity: day 12 to 14 postinfection for HIV-1sF2, day 10 for HIV-1S33. +, -50% infected cells showing cytopathology; + +, >90% infected cells showing cytopathology. b Plaque formation was assayed in MT-4 cells as described previously (33). +, Plaque-forming ability at 5 days postinfection; -, no plaque-forming ability exhibited. PMC, Peripheral blood mononuclear cells; HUT, HUT 78 cells; HOS, human osteosarcoma cells. For HIV-1 replication in PMC, HUT 78, and CEM cells, RT activity (103 cpm/ml) of culture supernatant at 10 days postinfection is given (13). Infection of HOS cells with HIV-1 was performed as described previously (32). RT activity in the supernatant of PMC cultures at 10 days after cocultivation with infected HOS cells is presented.
Corresponding author. 4016
VOL. 64, 1990
NOTES
TABLE 2. Homology between HIV-1sF2 and HIV-lSF33a Region rev tat
vpu
% Nucleic acid homology
% Amino acid homology
90.2 90.4 90.9
86.2 85.1
(Fig. 1). In both HIV-lsF2 and HIV-lsF33, the vpu protein is truncated. Termination occurs in the HIV-lsF2 isolate after the 38th amino acid, whereas in the HIV-lsF33 isolate, termination immediately follows the initiating methionine. To generate recombinant viruses, a methodology was devised using two plasmid constructs which contain approximately the 5' and 3' halves of the HIV-1 genome separately. Restriction mapping identified a common internal EcoRI site at the midpoint of both viral genomes. The EcoRI fragments generated after digestion were subcloned into pUC19 plasmid vectors (Fig. 2A). The plasmids containing the 5' long terminal repeat (LTR), gag, pol, vif, and the 5' end of vpr were designated "5' subclones," while the plasmids containing the 3' end of the vpr, the vpu, tat, rev, env, nef, and 3' LTR were designated "3' subclones." The 3' plasmid subclones were further manipulated to exchange separately the env and LTR regions between the two subclones (Fig. 2B). A 3.3-kilobase fragment was ex-
Both truncated
env Total gp120 gp4l
nef LTR
91.3 90.2 93.1 92.7
4017
84.0 82.5 86.7 88.1
96.4
a DNA from a recombinant HIV-lSF33 lambda clone was digested with EcoRI, and the 3' EcoRI genomic fragment was subcloned into a pUC19 plasmid vector. Restriction enzyme-digested DNA fragments of the insert DNA were isolated, cloned into M13 vectors, and used as templates for DNA sequencing by the dideoxynucleotide-chain termination method as described previously (28, 29). Homologous regions of the viral genes were identified by using the GENALIGN computer program (23).
tgp12 HIV-1 SF33 MrareTRkNYQcLWRWGTmLLGMLMICSAaEnLWVTVYYGVPVWKdATTTLFCASDAkAYDTEVHNVWATHA 111111 11111 HIM 11111111111 11111111111111 HIV-1SI2 MkvkgTRrNYQhLWRWGTlLLGMLMICSAtEkLWVTVYYGVPVWKeATTTLFCASDArAYDTEVHNVWATHA VI
73 CVPTDPNPOEVVLGNVTENFWNMRdMhEDIvSLWDOSLKPCVKLTPLCVTLNCTDvLGnATNTNnSS 6 1111111111111111111111111 11111111111111111 11 11111 11
73
CVPTDPNPQEVVLGNVTENFNMWK_NMVeQMqEDIiSLWDQSLKPCVKLTPLCVTLNCTD LGkATNTN
SS
V7 I Is.* 145 ggtveKeEIKNCSFNITTgIRDKvQKayAyFykLDVVPID ddnTN TsYRLIHCNsSVITQtCPKVSFE I 1111111111 M11 11 I I 1111111 11 1 1111111 11111 1111111 143 nwkeeiKgEIKNCSFNITTsIRDKiQKenAlFrnLDVVPIDnasttTNyTnYRLIHlCNrSVITQaCPKVSFE I
*
0
*
213 PIPIHYCaPAGFAILKCNNKkFsGKGqCTNVSTVQCTHGIkPvVSTQLLLNGSLAEEEVVIRSDNFTNNAKT 1111111 111111111111 11 11111111111111111111111 215 PIPIHYCtPAGFAILKCNNKtFnGKGpCTNVSTVQCTHGIrPiVSTQLLLNGSLAEEEVVIRSDNFTNNAKT
V3 285 IlVQLNvSVeINCTRPNNNrRrrItsGPGkvlyTTGeIIGDIRKAyCNISRAkWNkTLEQvatKLREQFGN
1 1111
II 111111111
III
III IIIIIIII IIIIII 111
11111111
287 IiVQLNeSVaINCTRPNNNtRksIyiGPGrafhTTGrIIGDIRKAhCNISRAqWNnTLEQivkKLREQFGNn 356 359 428 431
V4 KTIVFkQSSGGDPEIVMHSFNCRGEFFYCNTTkLFNsTWneNsTwnatGNDTItLPCRIKQIINMWQEVGKA 11111 11111111111111111IIIII III 1 I I H KTIVFnQSSGGDPEIVMHSFNCRGEFFYCNTTqLFNnTWrlNhTegtkGNDTIiLPCRIKQIINMWQEVGKA V5 ******************** I MYAPPIeGQIrCSSNITGLLLTRDGGgdknstTEiFRPaGGnMkDNWRSELYKYKVvKIEPLGvAPTKAKRR 111111 III IIIIIIIIIIIIIII IIIIIIIIIIII 111111 11111111 11 111 11 MYAPPIgGQIsCSSNITGLLLTRDGGtnvtndTEvFRPgGGdMrDNWRSELYKYKViKIEPLGiAPTKAKRR
500 VVQREKRAVGviGAMFLGFLGAAGSTMGAaSiTLTVQARkLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQ 0
V1111111
11111111111111111 1
1111111
11111111111111111111L11111111111I
503 WQREKRAVGivGAMFLGFLGAAGSTMGAvSlTLTVQARqLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQ
572
1111111111111111111111111111111 1111 1111111 11 11111 111111111 111111 575 LQARVLAVERYLRDQQLLGIWGCSGKLICTTaVPWNaSWSNKSLedIWdNMTWMqWEREIDNYTntIYTLLE 644
1111111111111111111111111~11111 11111 111111111 1111 11111111111111111111 647 ESQNQQEKNEQELLELDKWASLWNWFSITNWLWYIkIFIMIVGGLvGLRIvFAVLSIVNRVROGYSPLSFQT
716 liPaqRGPDRPeGIEEgGGERDRDRStRLVnGFLALfWdDLRSLCLFSYhRLtDLLLIvARiVElLGrRGWE
1 1 11P1 D
1
1
1 1
1
I1
1 1
1 1
1 1
11 11 l lr lDl aG1
7 1 9 DF 719 rlPvpRGPDRPdGIEEeGGERDRDRSvRLVdGFLAI iWeDLRSLCLFSYrRLrDLLLIaARtVEiLGhRGWE
788 vLKYWWnLLlYWsQELKNSAVSlLNATAIAVaEGTDRVIEVvQRvgRAILHIptRIRQGfERaLLO
11111 11 II 111111111 11111111 IIIIIIIII 11 111111 11111 11 111 aLKYWWsLLqYWiQELKNSAVSwLNATAIAVtEGTDRVIEVaQRayRAILHIhrRIRQGlERlLLO FIG. 1. Alignment of the predicted amino acid sequences of the gp160 proteins of HIV-1SF33 and HIV-1sF2 with the GENALIGN program (23). Vertical bars and capital letters indicate amino acid identity. Hypervariable regions are boxed. Potential cleavage sites are indicated by arrows. Dots show cysteine residues. The CD4-binding domain is shown by asterisks, and the fusion peptide domain is underlined. 791
J. VIROL.
NOTES
4018 A.
. .......
vif
LateV.-
ti ..rn
vpr
nef
.
vZu
cellular LTR flanking _ _ 5'
pol
F Ri
env
_
3LTR -
Rl
JI
5.1SUBCLONE _
B.
1
3' SUBCLONE -
,'
_
1
changed by using homologous EcoRI and Asp 718 restriction endonuclease sites in the two genomes. Due to restriction site limitations, as well as gene overlap, the 3' region of vpr, the vpu, tat, rev, and the initial 77 amino acids of nef were also included in the "env" exchange. Plasmid DNAs were then prepared by the boiling method (22) and banded once in cesium chloride. Recombinant viruses were generated as described in Table 3, assayed for replication in T-cell lines as well as in CD4-negative HOS cells (5, 17, 32), and evaluated for cytopathology and plaque formation (33). Input virus was quantitated both by 50% tissue culture infectious dose determination and by equal RT activity. The results, as summarized in Table 3, show that the parental isolates generated in this manner (R7 and R8) display properties identical to those of molecular clones obtained by transfection with the full-length HIV-1 genome (Tables 1 and 3). Induction of extensive cytopathic effect and plaque-forming ability were found associated with the HIV-lsF33 isolate (R8). The inter-
......tat .. rev
.-_
v2n MU env f
3' LTR
Rl1~ Asp 718
.j Rl
FIG. 2. Diagrammatic scheme for construction of recombinant DNA. An internal EcoRI (R1) site approximately halfway through the genome is present in both genomes. (A) Rl fragments containing the halves of the HIV-1 genome were generated by Rl digestion of lambda clone DNA and inserting into pUC19 plasmid vectors, creating 5' and 3' subclones. (B) Recombinant plasmid constructs of the 3' subclones were created by reciprocally exchanging the EcoRI-Asp7l8 fragments of HIV-1sF2 and HIV-lSF33.
Ri
3' RECOMBINANT - PLASMID -' s Ri
cc-o
TABLE 3. Biologic properties of HIV-lSF2 and HIV-1SF33 recombinant virusesa
ytatologyb Cytopath
Virus construct
Plaque formation
PMC
Replication in: CEM
HOS
772.6
1.7
2.1
5'SF2 + 3'SF2
Rl
RI
Rl
+
R8 5'SF33 + 3'SF33
RI
Rl
RI
++
+
858.2
333.3
353.0
R9 5'SF2 + 3'SF33
Rl
Rl
RI
++
+
1079.9
200.2
618.8
R10 5'SF33 + 3'SF2
RI
RI
RI
+
_
887.1
2.6
0.9
Rll SF2 + SF33 env
Rl
RI
Rl
++
+
402.9
291.3
188.0
R12 SF33 + SF2 env
R1
R
AXRi1+
169.9
1.3
41.4
SF2 + SF33 LTR
R
Ri
Asp{11
+
-
496.3
1.5
59.7
SF33 + SF2 LTR
Rin1
A41
++
+
1047.2
225.6
194.0
a The original isolates (R7 and R8) were regenerated by cotransfecting their 5' and 3' subclones into the RD-4 cell line by the calcium phosphate precipitation method (14). The plasmid DNAs were linearized by EcoRI digestion before transfection. Three days after transfection, virus was rescued by overlaying the RD-4 monolayer with 106 HUT 78 cells or 3 x 106 PMC from seronegative individuals as described previously (14). The presence of virus was monitored by visual inspection for ballooning and syncytial formation, by RT activity, and by enzyme-linked immunosorbent assay for p25 core protein in the supernatant of cocultivated cells. No difference in the time of recovery of parental viruses (R7 and R8) was observed by using this cotransfection method as compared with transfection with the full-length viral genome. To generate interisolate recombinant viruses exchanging 5' and 3' halves of the genome (R9 and R10), 10 F±g of the 5' subclone of one isolate and 10 ,ug of the 3' subclone of the other isolate were used for the cotransfection. For the generation of recombinant viruses containing the env regions exchanged (Rll and R12), the 5' subclone of the opposite isolate was cotransfected with the 3' env recombinant subclone. For the 3' LTR viral recombinants, the 5' subclone was cotransfected with the 3' recombinant subclone containing the env region of the same isolate. b Cytopathicity, plaque formation, and replication in the different cell types were determined as described in the footnotes of Table 1.
NOTES
VOL. 64, 1990
isolate hybrid viruses exhibited this same prominent cytopathic property when the 3' half of the HIV-lsF33 genome was included (R9). Furthermore, this property was maintained in recombinant viruses containing only a portion of the 3' half of the HIV-1sF33 genome (Rll and R14). The ability to replicate in specific cell lines or types also segregated with the 3' end of the HIV-lsF33 genome. Viruses R9, Rll, and R14 readily infected the CEM cell line, whereas RT activity similar to that of the parental HIV-lsF2 isolate (R7) was detected in CEM cells with the R10, R12, and R13 viruses after cocultivation with PMC. The env region also appears to be a major determinant in the infection of the CD4-negative HOS cell line. Similar to the HIV-lsF33 virus (R8), high levels of RT activity were found in PMC cocultivated with the R9-, Rll-, and R14-infected HOS cells. Nevertheless, the detection of low-level virus replication in the HOS cells with the R12 and R13 viruses, unlike that of R7 and R10, also suggests that the HIV-lsFs3 LTR contributes to efficient replication in HOS cells. These studies with recombinant viruses generated by taking advantage of intracellular ligation processes indicate that a region from nucleotide 5750 to 9032 is responsible for the cytopathic and replicative characteristics of an HIV-1 isolate. This sequence includes part of the vpr, tat, rev, vpu, env, and part of the nef genes. tat and rev act as positive regulators of virus production (3, 12, 21, 24), and the nef gene appears to be a negative regulator of viral replication (1, 19, 25, 35). vpu has been shown to control the efficient release of virions (31), while the vpr protein increases the rate of replication and accelerates the cytopathic effect of the virus in T cells (7). However, the function of vpr appears to be dispensable for viral replication (8). It is noteworthy that both isolates used in this study contain truncated vpu genes but replicate efficiently and to high titers in PMC and established T-cell lines. The env gpl20 has been shown to induce cell fusion (18, 30), with resulting syncytial formation and cell death. Antibodies against a 24-amino-acid sequence of the viral env gpl20 inhibit this fusion process (26). Our results with these recombinant viruses and those obtained with other HIV recombinants (10) support the findings that the env gene is the major determinant of cytopathicity. The recombinant viruses also demonstrated an important role of the env region in determining replicative properties of HIV-1. The host range of a virus is determined by three features: infectivity, rate of replication, and magnitude of virus production. Infectivity relates to the ability of a virus to gain entry into target cells. After entry, infection is further characterized by the replication kinetics and magnitude or levels of virus production. In the infection of CEM cells, the slower rate and lower levels of virus production observed in HIV-1SF2-infected cultures could be the result of inefficient viral entry or regulation at a postentry level. Replication in the CEM cells segregated with the env-exchanged region, which also contains the regulatory genes tat and rev and part of the vpr gene. Further fine-structure mapping with recombinant viruses segregating the env and these regulatory and accessory genes will be required to specifically identify the major determinants of HIV-1 replication in CEM cells. Infection of the CD4-negative HOS cell line also segregated with the env-exchanged region. A fusion process, independent of CD4 binding, could determine viral entry into these cells (6, 32). A region in the N terminus of gp4l has been shown to share sequence homology with fusion peptides of paramyxo viruses (11), and a 24-amino-acid sequence in the third hypervariable region of gpl20 (RP 135) has been shown to be important for envelope-induced fusion (26). The con-
4019
sensus amino acid sequence GPGR was not conserved in the RP 135 region of HIV-1SF33, and amino acid differences were found in the gp4l regions of HIV-lSF2 and HIV-lSF33 (Fig. 1). Whether these changes are determinants of viral entry into CD4-negative cell types requires further investigation. Finally, the observation that recombinant viruses with HIV1SF33 LTR (R12 and R13) replicated to low levels in the non-CD4-expressing HOS cells suggests that the LTR may play a role in determining levels of HIV-1 replication after viral entry into target cells. We thank Shenbei Tang for performing the MT-4 plaque assays. This work was supported in part by funds from the National Cooperative Vaccine Development Group (AI22778) of the National Institutes of Health and in part by funds provided by the California Department of Health Services. Additional funding was provided by Public Health Service grants from the National Institutes of Health to C.C.-M. and J.A.L. (ROl A125284 and AI24499).
1.
2. 3. 4.
5. 6. 7.
8.
9.
10.
11.
12.
13.
14. 15.
LITERATURE CITED Ahmad, N., and S. Venkatesan. 1988. nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:14811485. Alizon, M., S. Wain-Hobson, L. Montagnier, and P. Sonigo. 1986. Genetic variability of the AIDS virus: nucleotide sequence analysis of two isolates from African patients. Cell 46:63-74. Arya, S. K., C. Guo, S. F. Joseph, and F. Wong-Staal. 1985. trans-Activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73. Benn, S., R. Rutledge, T. Folks, J. Gold, L. Baker, J. McCormick, P. Feorino, P. Piot, T. Quinn, and M. Martin. 1985. Genomic heterogeneity of AIDS retroviral isolates from North America and Zaire. Science 230:949-951. Cheng-Mayer, C., D. Seto, M. Tateno, and J. A. Levy. 1988. Biologic features of HIV-1 that correlate with virulence in the host. Science 240:80-82. Chesebro, B., R. Buller, J. Portis, and K. Wehrly. 1990. Failure of human immunodeficiency virus entry and infection in CD4positive human brain and skin cells. J. Virol. 64:215-221. Cohen, E. A., E. F. TerwilHiger, Y. Jalinoos, J. Pronix, J. G. Sodroski, and W. A. Haseltine. 1990. Identification of HIV-1 vpr product and function. J. Acquired Immune Defic. Syndr. 3: 11-18. Dedera, D., W. Hu, N. Vander Heyden, and L. Ratner. 1989. Viral protein R of human immunodeficiency virus types 1 and 2 is dispensable for replication and cytopathogenicity in lymphoid cells. J. Virol. 63:3205-3208. Fenyo, E. M., L. Morfeldt-Manson, F. Chiodi, B. Lind, A. vonGegerfelt, J. Albert, E. Olausson, and B. Asjo. 1988. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates. J. Virol. 62:4414- 419. Fisher, A. G., B. Ensoli, D. Looney, A. Rose, R. C. Gallo, M. S. Saag, G. M. Shaw, B. H. Hahn, and F. Wong-Staal. 1988. Biologically diverse molecular variants within a single HIV-1 isolate. Nature (London) 334:444 447. Gallaher, W. R. 1987. Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus. Cell 50:327-328. Hadzopoulou-Cladaras, M., B. K. Felber, C. Cladaras, A. Athanassopoulos, A. Tse, and G. N. Pavlakis. 1989. The rev (trslart) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J. Virol. 63:1265-1274. Hoffman, A. D., B. Banapour, and J. A. Levy. 1985. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology 147:326-335. Levy, J. A., C. Cheng-Mayer, D. Dina, and P. A. Luciw. 1986. AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts. Science 232:998-1001. Levy, J. A., A. D. Hoffman, S. M. Kramer, J. A. Landis, J. M.
4020
16.
17.
18.
19.
20. 21.
22. 23. 24. 25.
26.
J. VIROL.
NOTES
Shimabukuro, and L. S. Oshiro. 1984. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225:840-842. Levy, J. A., and J. M. Shimabukuro. 1985. Recovery of AIDSassociated retroviruses from patients with AIDS or AIDSrelated conditions and from clinically healthy individuals. J. Infect. Dis. 152:734-738. Levy, J. A., J. Shimabukuro, T. McHugh, C. Casavant, D. Stites, and L. Oshiro. 1985. AIDS-associated retrovirus (ARV) can productively infect other cells besides human T helper cells. Virology 147:441-448. Lifson, J. D., M. B. Feinberg, G. R. Reyes, L. Rabin, B. Banapour, S. Chakrabarti, B. Moss, F. Wong-Staal, K. S. Steimer, and E. G. Engleman. 1986. Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein. Nature (London) 323:725-728. Luciw, P. A., C. Cheng-Mayer, and J. A. Levy. 1987. Mutational analysis of the human immunodeficiency virus: the orf-B region downregulates virus replication. Proc. Natl. Acad. Sci. USA 84:1434-1438. Luciw, P. A., S. J. Potter, K. Steimer, D. Dina, and J. A. Levy. 1984. Molecular cloning of AIDS-associated retrovirus. Nature (London) 312:760-763. Malim, M. H., J. Hauber, S. Y. Le, J. V. Maizel, and B. R. Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature (London) 338:254-257. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Martinez, H. M. 1988. A flexible multiple sequence alignment program. Nucleic Acids Res. 16:1683-1691. Muesing, M. A., D. H. Smith, and D. J. Capon. 1987. Regulation of mRNA accumulation by an HIV trans-activator protein. Cell 48:691-701. Niederman, T. M. J., B. J. Thielan, and L. Ratner. 1989. Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proc. Natl. Acad. Sci. USA 86:1128-1132. Rusche, J. R., K. Javaherian; C. McDanal, J. Petro, D. L. Lynn, R. Grimaila, A. Langlois, R. C. Galo, L. 0. Arthur, P. J.
27.
28.
29. 30.
31. 32.
33. 34.
35.
Fischenger, D. P. Bolognesi, S. D. Putney, and T. J. Matthews. 1988. Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120. Proc. Natl. Acad. Sci. USA 85:3198-3202. Sakai, K., S. Dewhurst, X. Ma, and D. J. Volsky. 1988. Differences in cytopathogenicity and host cell range among infectious molecular clones of human immunodeficiency virus type 1 simultaneously isolated from an individual. J. Virol. 62:40784085. Sanchez-Pescador, R., M. D. Power, P. J. Barr, K. S. Steimer, M. M. Stempien, S. L. Brown-Shimer, W. W. Gee, A. Renard, A. Randolph, J. A. Levy, D. Dina, and P. A. Luciw. 1984. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227:484-492. Sanchez-Pescador, R., and M. S. Urdea. 1984. Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing. DNA 3:339-343. Sodroski, J., W. C. Goh, C. Rosen, K. Campbell, and W. A. Haseltine. 1986. Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature (London) 322:470474. Strebel, K., T. Klimkait, and M. A. Martin. 1989. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science 241: 1221-1223. Tateno, M., F. Gonzalez-Scarano, and J. A. Levy. 1989. Human immunodeficiency virus can infect CD4-negative human fibroblastoid cells. Proc. Natl. Acad. Sci. USA 86:4287-4290. Tateno, M., and J. A. Levy. 1988. MT-4 plaque formation can distinguish cytopathic subtypes of the human immunodeficiency virus (HIV). Virology 176:299-301. Tersmette, M., R. A. Gruters, F. deWolf, R. E. Y. deGoede, J. M. A. Lange, P. T. A. Schellekens, J. Goudsmit, H. G. Huisnian, and F. Miedema. 1989. Evidence for a role of virulent human immunodeficiency virus (HIV) variants in the pathogenesis of acquired immunodeficiency syndrome: studies on sequential HIV isolates. J. Virol. 63:2118-2125. Terwilliger, E., J. G. Sodroski, C. A. Rosen, and W. A. Haseltine. 1986. Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. J. Virol. 60:754-760.
