Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity.
The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry.
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Department of Microbiology and Immunology, University of California, San Francisco 94143-0414.
Stearns
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Littman
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D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D015686:Gene Products, env / Q000235:genetics* / Q000378:metabolism; D015726:Giant Cells / Q000166:cytology; D015699:HIV Envelope Protein gp120 / Q000235:genetics* / Q000378:metabolism; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism / Q000382:microbiology; D014162:Transfection
D015704:CD4 Antigens; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D011498:Protein Precursors