Apr 20, 1989 - Joseph Sodroski is a Scholar of the Leukemia ... Fisher, A. G., B. Ensoli, L. Ivanoff, M. Chamberlain, S. Pette- way, L. Rattner, R. C. Gallo, and F.
Vol. 63, No. 9
JOURNAL OF VIROLOGY, Sept. 1989, p. 4085-4087 0022-538X/89/094085-03$02.00/0
Copyright ©3 1989, American Society for Microbiology
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 retrovirus that is the etiologic agent of acquired immunodeficiency syndrome (AIDS) (2, 6). HIV-1 shares many features with other retroviruses, including the ability of the gag, pro, pol, and env genes to encode core proteins, a protease, reverse transcriptase, and envelope glycoproteins, respectively (8). HIV-1 also possesses additional genes modulating viral replication. The HIV-1 genome encodes vif, vpr, tat, rev, vpu, and nef proteins (8). As in other retroviruses, the long terminal repeats (LTRs) of HIV-1 contain cis-acting sequences important for integration, transcription, and polyadenylation. Additional cis-acting signals allow regulation of HIV-1 sequences by some of the novel HIV-1 gene products (8). In avian and murine retroviruses, cis-acting sequences located between the 5' LTR and the gag gene initiation codon are necessary for the efficient packaging of viral RNA into virions (3, 9, 13, 15, 19). Recently, these sequences, in addition to sequences overlapping the gag gene, were demonstrated to be sufficient for viral RNA encapsidation by Moloney murine leukemia virus (1). The signals important for packaging HIV-1 RNA into virion particles have not been identified. The region between the 5' major splice donor and the gag gene initiation codon is highly conserved in different HIV-1 strains sequenced to date (14). We investigated the phenotype produced by a deletion in this region. The region between the HIV-1 5' LTR and the gag gene is 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 origin of replication to allow efficient gene expression in COS-1 cells. The mutation was produced by site-directed 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 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 transfection were precipitated (11) with AIDS serum no. *
19501. The overall levels of viral protein detected in cell lysates were comparable for the wild-type HXBc2 and mutant HXBAP1 viruses (Fig. 2A). The levels of viral proteins precipitated from the supernatants of COS-1 cells were slightly lower for the HXBAP1 mutant than for the wild-type virus (Fig. 2A). The amount of reverse transcriptase activity (7) measured in the supernatants of COS-1
cells transfected with the pHXBAP1 plasmid was 60% of that measured in cells transfected with the pHXBc2 plasmid (data not shown). COS-1 cells transfected with the pHXBAP1 plasmid were fixed 48 h following transfection and examined by electron microscopy. Viral particles, including budding forms, with normal HIV-1 morphology were observed (Fig. 2B). To evaluate the effect of the HXBAP1 mutation on HIV-1 replication, supernatants from COS-1 cells transfected with pHXBc2 and pHXBAP1 plasmids were filtered (0.2-,umpore-size filter), and the amount of reverse transcriptase was measured. Supernatants containing equal amounts of reverse transcriptase activity for mutant and wild-type viruses were added to Jurkat human T lymphocytes. The Jurkat cultures and a mock-infected culture were maintained with changes of medium every 3 days. At intervals, samples of Jurkat cells were labeled and assessed for expression of HIV-1 proteins by immunoprecipitation with AIDS serum no. 19501 (Fig. 3). Jurkat cultures exposed to the HXBAP1 virus exhibited marked delays in and lower levels of viral SD
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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 19-base-pair (bp) deletion in pHXBAP1.
Corresponding author. 4085
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