Identification of a Sequence Required for Efficient Packaging of Human

Identification of a Sequence Required for Efficient Packaging of Human Immunodeficiency Virus Type 1 RNA into Virions ANDREW LEVER, HEINRICH GOTTLINGER, WILLIAM HASELTINE, AND JOSEPH SODROSKI* Laboratory of Human Retrovirology, Dana-Farber Cancer Institute, Department of Pathology, Harvard Medical School, and Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115 Received 16 December 1988/Accepted 20 April 1989

Sequences required for efficient packaging of human immunodeficiency virus type 1 (HIV-1) genome RNA into virus particles were identified. Deletion of 19 base pairs between the 5' long terminal repeat and the gag gene initiation codon of HIV-1 resulted in a virus markedly attenuated for replication in human T lymphocytes. The mutant virus was characterized by nearly wild-type ability to encode viral proteins and to produce virion particles. The mutant virions exhibited a significant reduction in the content of HIV-1-specific RNA. These results identify an important component of the HIV-1 packaging signal.

Human immunodeficiency virus type 1 (HIV-1) is a retro- 19501. The overall levels of viral protein detected in cell virus that is the etiologic agent of acquired immunodefi- lysates were comparable for the wild-type HXBc2 and ciency syndrome (AIDS) (2, 6). HIV-1 shares many features mutant HXBAP1 viruses (Fig. 2A). The levels of viral with other retroviruses, including the ability of the gag, pro, proteins precipitated from the supernatants of COS-1 cells pol, and env genes to encode core proteins, a protease, were slightly lower for the HXBAP1 mutant than for the reverse transcriptase, and envelope glycoproteins, respec- wild-type virus (Fig. 2A). The amount of reverse tran- tively (8). HIV-1 also possesses additional genes modulating scriptase activity (7) measured in the supernatants of COS-1 viral replication. The HIV-1 genome encodes vif, vpr, tat, cells transfected with the pHXBAP1 plasmid was 60% of that rev, vpu, and nef proteins (8). As in other retroviruses, the measured in cells transfected with the pHXBc2 plasmid long terminal repeats (LTRs) of HIV-1 contain cis-acting (data not shown). COS-1 cells transfected with the sequences important for integration, transcription, and poly- pHXBAP1 plasmid were fixed 48 h following transfection adenylation. Additional cis-acting signals allow regulation of and examined by electron microscopy. Viral particles, in- HIV-1 sequences by some of the novel HIV-1 gene products cluding budding forms, with normal HIV-1 morphology were (8). observed (Fig. 2B). In avian and murine retroviruses, cis-acting sequences To evaluate the effect of the HXBAP1 mutation on HIV-1 located between the 5' LTR and the gag gene initiation replication, supernatants from COS-1 cells transfected with codon are necessary for the efficient packaging of viral RNA pHXBc2 and pHXBAP1 plasmids were filtered (0.2-,um- into virions (3, 9, 13, 15, 19). Recently, these sequences, in pore-size filter), and the amount of reverse transcriptase was addition to sequences overlapping the gag gene, were dem- measured. Supernatants containing equal amounts of re- onstrated to be sufficient for viral RNA encapsidation by verse transcriptase activity for mutant and wild-type viruses Moloney murine leukemia virus (1). The signals important were added to Jurkat human T lymphocytes. The Jurkat for packaging HIV-1 RNA into virion particles have not been cultures and a mock-infected culture were maintained with identified. The region between the 5' major splice donor and changes of medium every 3 days. At intervals, samples of the gag gene initiation codon is highly conserved in different Jurkat cells were labeled and assessed for expression of HIV-1 strains sequenced to date (14). We investigated the HIV-1 proteins by immunoprecipitation with AIDS serum phenotype produced by a deletion in this region. no. 19501 (Fig. 3). Jurkat cultures exposed to the HXBAP1 The region between the HIV-1 5' LTR and the gag gene is virus exhibited marked delays in and lower levels of viral shown in Fig. 1. A 19-base-pair deletion in this region was created in an infectious HIV-1 proviral clone on plasmid pHXBc2 (4). This plasmid also contains a simian virus 40 SD -I of to gene in origin replication allow efficient expression 5' LTR COS-1 cells. The mutation was produced by site-directed _Lng- mutagenesis (10), and the sequence was confirmed by DNA sequencing (16). The mutated plasmid was designated pHXBAP1. To evaluate the effect of the mutation on viral protein loRXBCE2 expression and virion production, COS-1 cells were transfected with the pHXBc2 and pHXBAP1 plasmids by the DEAE-dextran procedure (17). COS-1 cell lysates and supernatants radiolabeled with [35S]cysteine (17) 48 h after pHXBL5P1 GTGAGTACGCC 9bp -. GGCTA= transfection were precipitated (11) with AIDS serum no. D*letion FIG. 1. HIV-1 genome from the 5' LTR to the gag initiation codon showing the major splice donor (SD) and the site of a * Corresponding author. 19-base-pair (bp) deletion in pHXBAP1.

4085 4086 NOTES J. VIROL.

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FIG. 2. (A) Immunoprecipitation of 135S-labeled viral proteins from COS-1 cell lysates (lanes 1 through 3) or from supernatants (lanes 4 through 6) with AIDS serum no. 19501 after transfection with no DNA (lanes 1 and 4). 10 kg of pHXBc2 (lanes 2 and 5), or 10 kg of pHXBAP1 (lanes 3 and 6). kD, Kilodaltons. (B) Electron micrographs of COS-1 cells transfected with pHXBAP1 showing virus particles with normal HIV-1 morphology. protein production relative to results with cultures exposed Jurkat cells infected with the HXBAP1 virus produced to the wild-type virus. The replication of the HXBzXP1 virus detectable levels of reverse transcriptase activity and viral in human T lymphocytes appears to be significantly attenu- antigens in the supernatant 3 to 4 weeks following infection. ated compared with replication of the HXBc2 virus. To investigate the basis for the replication defect observed in the HXBAP1 virus in Jurkat cells, supernatants from these cultures were filtered (0.2-gm-pore-size filter) and equivalent amounts of reverse transcriptase activity were pelleted by centrifugation at 12,000 x g for 1 h at 20°C. Viral pellets kD were lysed by Nonidet P-40 in the presence of vanadyl -- 160/120 ribonucleotides, and dilutions of virus were dot blotted onto ..p.. nitrocellulose filters. Some samples were treated with so- ~ _g . dium hydroxide (5 M, 60°C for 15 min) prior to dot blotting. _a . Filters were hybridized with a DNA probe consisting of 55 HIV-1 gag and eur gene sequences, washed, and autoradiographed as previously described (12). For the wild-type HXBc2 virus, a signal specific for RNA could be detected after 1,000 reverse transcriptase units of filtered supernatant were blotted (not shown). For the HXBAP1 mutant, even 5

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FIG. 3. Immunoprecipitation of labeled viral proteins from Jur- kat T-cell lysates (lanes 1 through 3, 7 through 9. and 13 through 15) or supernatants (lanes 4 through 6, 10 through 12, and 16 through 18) exposed to supernatants from COS-1 cells that were mock trains- FIG. 4. RNA dot blot without (column 1) and with (column 2) fected (lanes 1, 4, 7, 10. 13. and 16), transfected with pHXBc2 (lanes sodium hydroxide treatment following blotting of filtered superna- 2, 5, 8, 11, 14, and 17), or transfected with pHXBAPI (laines 3. 6. 9. tants from Jurkat cultures. The supernatants contained reverse 12, 15, and 18). The Jurkat cells were examined at day 7 (lanes transcriptase activities of 5 x 10i cpm of HXBAP1 virus (row A), 5 through 6), day 14 (lanes 7 through 12). and day 21 (lanes 13 through x 10' cpm of HXBc' virus (row B). and 1 x 105 cpm of HXBc2 virus 18) following infection. Kilodaltons. (row C). VOL. 63, 1989 NOTES 4087

25 LITERATURE CITED 1. Adam, M. A., and A. D. Miller. 1988. Identification of a signal in 20 a murine retrovirus that is sufficient for packaging of nonretro- viral RNA into virions. J. Virol. 62:3802-3806. 15 2. Barre-Sinoussi, F., J.-C. Chermann, F. Rey, M. T. Nugeyre, S. RNA Chamaret, J. Gruest, C. Dauguet, C. Axier-Blin, F. Vezinet-Grun, 10 C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868-871. 5 3. Bender, M. A., T. D. Palmer, R. E. Gelinas, and A. D. Miller. 1987. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J. Virol. 61: 5 10 15 20 1639-1646. p24 (ng) 4. Fisher, A. G., E. Collati, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-1l1 with FIG. 5. Quantitation of virion RNA and p24 antigen in HXBc2 biolog- (A) and HXBAP1 (0) viruses. RNA amounts are expressed in ical activity. Nature (London) 316:262-265. arbitrary densitometric units. The specificity of the signal for RNA 5. Fisher, A. G., B. Ensoli, L. Ivanoff, M. Chamberlain, S. Pette- was confirmed by sodium hydroxide treatment of portions of RNA way, L. Rattner, R. C. Gallo, and F. Wong-Staal. 1987. The sor prepared in parallel. gene of HIV-1 is required for efficient virus transmission in vitro. Science 237:888-892. 6. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G.-M. Shearer, M. Kaplan, B. F. Haynes, T. Palker, R. Redfield, J. Oleske, B. Safai, x 104 reverse transcriptase units of supernatant gave no G. White, P. Foster, and P. D. Markham. 1984. Frequent detectable signal (Fig. 4). detection and isolation of cytopathic retroviruses (HTLV-III) In an independent experiment, the ratios of virus-specific from patients with AIDS and at risk for AIDS. Science 224: RNA to p24 capsid protein in the HXBc2 and HXBAP1 500-503. virions were determined. 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Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and scribed and the was densito- above, signal quantitated by efficient site-specific mutagenesis without phenotypic selection. metric analysis (Fig. 5). For the HXBc2 virus, a detectable Methods Enzymol. 154:367-382. signal was observed at a p24 antigen level of 1 ng and above. 11. Lee, T. H., J. E. Coligan, J. S. Allan, M. F. McLane, J. E. By contrast, no signal was detected for the HXBAP1 virus Groopman, and M. Essex. 1986. A new HTLV/LAV protein even at a p24 antigen level of 20 ng. encoded by a gene found in cytopathic retroviruses. Science The results suggest that a region between the 5' LTR and 231:1546-1549. the gag gene of HIV-1 is important for packaging viral RNA 12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular into virions. A mutation in this region exhibits minimal cloning: a laboratory manual. Cold Spring Harbor Laboratory, effects on the ability of the provirus to produce proteins and Cold Spring Harbor, N.Y. virion particles following transfection but markedly de- 13. Mann, R., R. C. Mulligan, and D. Baltimore. 1983. Construction creases the level of virion RNA and attenuates virus repli- of a retrovirus packaging mutant and its use to produce helper- cation in a human CD4-positive lymphocyte line. These free defective retrovirus. Cell 33:153-159. results suggest that the efficiency of the HXBzAP1 mutant in 14. Myers, G., A. B. Rabson, S. F. Josephs, T. F. Smith, and F. packaging virus-specific RNA into virions is less than 2% of Wong-Staal. 1988. Human retroviruses and AIDS. Theoretical that of the biology and biophysics. Los Alamos National Laboratory, Los wild-type virus. HIV-1 replicates in cultured Alamos, N. Mex. CD4-positive cells via cell-free transmission and cell-to-cell 15. Pugatsch, T., and D. W. Stacey. 1983. Identification of a transmission, with the latter involving the contact of infected sequence likely to be required for avian retroviral packaging. and uninfected cells (5, 17, 18). HIV-1 replication in Jurkat T Virology 128:505-511. lymphocytes involves both modes of transmission. The 16. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- marked attenuation in replication of the HXBAP1 mutant ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. virus in Jurkat lymphocytes suggests that packaging of viral USA 74:5463-5467. RNA into viral capsids is important for both cell-free and 17. Sodroski, J., W. C. Goh, C. Rosen, A. Tartare, D. Portetelle, A. cell-to-cell spread of the virus. Burny, and W. A. Haseltine. 1986. Replicative and cytopathic potential of HTLV-lII/LAV with sor deletions. Science 231: 1549-1553. We thank Robert Gallo and Flossie Wong-Staal for the gift of 18. Strebel, K., D. Daugherty, K. Clouse, D. Cohen, T. Folks, and plasmid pHXBc2 and Bruce Walker for the gift of serum no. 19501. M. A. Martin. 1987. The HIV 'A'" (sor) gene product is Heinrich Gottlinger was supported by a fellowship from the essential for virus infectivity. Nature (London) 328:728-730. Deutsche Forschungsgemeinschaft, Bonn, Federal Republic of Ger- 19. W'atanabe, S., and H. M. Temin. 1983. Encapsidation sequences many. This work was supported by Public Health Service grant for spleen virus and avian retrovirus are between the 5' long A127702 from the National Institutes of Health. Joseph Sodroski is terminal repeat and the start of the gaig gene. Proc. Natl. Acad. a Scholar of the Leukemia Society of America. Sci. USA 79:5986-5990.