Rhesus monkeys were infected with mutant forms of simian immunodeficiency virus lacking dual combinations of the 4th, 5th and 6th sites for N-linked glycosylation in the external envelope glycoprotein of the virus. When compared with sera from monkeys infected with the parental virus, sera from monkeys infected with the mutant viruses exhibited markedly increased antibody binding to specific peptides from this region and markedly increased neutralizing activity. These results demonstrate a role for N-linked glycosylation in limiting the neutralizing antibody response to SIV and in shielding the virus from immune recognition.

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

Coding sequences for the first two variable loops of the gp120 envelope glycoprotein were removed from simian immunodeficiency virus (SIV) strain 239 (SIVmac239). This deletion encompassed 100 amino acids. The resulting virus replicated poorly after transfection into immortalized T-cell lines, with peak replication occurring only after 25 to 30 days. Limited passaging of SIVmac239⌬V1V2 in cultures gave rise to a variant which had significantly improved replication kinetics but which retained the original 100-amino-acid deletion in gp120. Cloning and sequencing revealed 11 changes in the envelope, including amino acid substitutions in both gp120 (5 substitutions) and gp41(6 substitutions). Four of the five changes in gp120 are predicted to lie within and around the putative coreceptor binding domain, a region which is believed to be covered by the V1 and V2 loops in the native envelope complex. Analysis of recombinant clones surprisingly revealed that the changes in gp41 were sufficient to overcome the replication deficiency created by deletion of the V1 and V2 loops from gp120. The SIVmac239⌬V1V2 envelope displayed a significant reduction in its ability to mediate cell-cell fusion, and the infectious titer of SIVmac239⌬V1V2 was approximately four-to eightfold lower than that of parental SIVmac239. Although SIVmac239 is strongly dependent on both CD4 and a coreceptor for entry, envelope protein lacking the V1 and V2 loops was able to mediate fusion with CD4 ؊ CCR5 ؉ cells at 60% the level observed with CD4 ؉ CCR5 ؉ cells. Plasma from SIVmac239-infected monkeys was at least 100 to 1,000 times more effective at neutralizing SIVmac239⌬V1V2 than SIVmac239. These results demonstrate the dispensability of the V1-V2 sequences of SIVmac239 for viral replication, a role for V1 and V2 in shielding the coreceptor binding region of the envelope, and the extreme sensitivity of a SIV lacking these sequences to antibody-mediated neutralization.