Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity

Abstract

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 gpl60 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 gpl60 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.

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV‐1) envelope glycoprotein gp120

“…The GPG apex of the V3 loop, important for binding of most mAbs, did not seem to be involved in MTCPP binding, but portions of t 0th flanking sequences adjoining GPG appeared esseiitial. Interestingly, introduction of amino acid substitutions into the GPG sequence diminished but did not abrogate virus infectivity and fusogenic activity (Page et al, 1992).…”

Molecular determinants of human immunodeficiency virus type I phenotype variability

“…These observations may at least in part explain the increased virus load in late asymptomatic HIV infection in individuals, regardless of SI capacity of the variants present in the infected individuals [13,140,141]. Since V3 together with other variable domains determine both SI capacity and tropism of HIV-1, evolution from NSI macrophage-tropic towards SI and/or T-cell-tropic isolates may result from consecutive changes in these variable domains [65,66,68,70,71,[92][93][94]100,104]. Knowledge of the exact sequence of amino acid substitutions would enable prediction of phenotype evolution, and may thus be useful as a prognostic marker for disease progression and for prescription of antiviral therapy.…”

“…The variable domains V1, V2 and V3 of gp120 have been suggested to be involved in HIV-1 fusion and infectivity at a stage subsequent to CD4 binding. Amino acid substitutions in and binding of mAbs directed against these domains affect syncytium formation and infectivity without affecting CD4 binding [65][66][67][68][69][70][71]. Gp120-CD4 interaction induces conformational changes in both CD4 and gp120 [72][73][74][75][76] but the significance of these conformational alterations for the subsequent fusion event remains undefined.…”

“…Construction of HIV-1 proviruses and vaccinia vectors expressing chimeric envelope proteins have clearly defined the V2 and V3 domains as important determinants of SI capacity of HIV-1 [65,66,68,70,71,93,94]. The almost perfect correlation between SI capacity of HIV-1 isolates with positively charged amino acid residues at positions 306 and 320 in V3 indicate that V3 is the major determinant of SI phenotype [95,96].…”

Human Immunodeficiency Virus Type 1-Associated Cd4 Downmodulation

“…Antibodies against V3 block infectivity and inhibit cell fusion but do not prevent gp120 binding to CD4 (Javaherian et al, 1989;Skinner et al, 1988). Mutations that altered either the Gly-Pro-Gly sequence or certain adjacent residues, including Arg-285 (depending on viral isolate), abolished or greatly reduced syncytium formation and infectiousness (Freed et al, 1990;Grimaila et al, 1992;Page et al, 1992). However, such mutants were not impaired in either synthesis, processing, or transport of gp160, or in its ability to bind CD4, demonstrating the involvement of V3 in fusion.…”