Background:  The progression to disease in simian immunodeficiency virus (SIV) infection has been associated with the emergence of neutralization-resistant viruses that contain mutations in the envelope gene, resulting in the addition or relocation of potential N-linked glycosylation sites, or in the substitution or deletion of amino acids that alter the size of variable loops. The goal of this study was to address the role of variation in the V4 loop on neutralization of SIV/17E-CL (an attenuated, neutralization-sensitive clone). We hypothesized that introduction of mutations in the V4 loop (i.e., introduction of potential N-linked glycosylation sites or deletions) would result in viruses that were resistant to neutralization by a panel of rhesus monoclonal antibodies.

Methods:  Mutations were introduced into the SIV/17E-CL envelope gene using overlapping polymerase chain reaction (PCR). These included:  the addition of single potential N-linked glycosylation sites (N415S or A417T); the deletion of 5 amino acids (∆421-425); or the replacement of the SIV V4 with the antigenically “silent” HIV-1 V4 to create a chimeric envelope protein. Recombinant viruses were generated by transfection of 293T cells, and neutralizing antibody titers were determined in TZM-bl cells.

Results:  Introduction of point mutations that created the addition of single potential N-linked glycosylation sites in the V4 region of SIV gp120 resulted in replication competent viruses with no difference in replication compared to the parental SIV/17E-CL virus. Monoclonal antibody neutralization of the parental and glycosylation mutant viruses was also similar. In contrast, while partial deletion of the SIV V4 loop resulted in a replication competent virus, a marked reduction in neutralization sensitivity of monoclonal antibodies that recognize discontinuous epitopes in the C-terminal half of SIV gp120 was observed. In addition, partial deletion of V4 resulted in enhanced neutralization sensitivity by monoclonal antibodies that recognize the V1/V2 regions of SIV gp120.

Conlcusions:  Partial deletion of the V4 region likely resulted in abrogation of neutralizing monoclonal antibody binding by causing conformational changes in Env that resulted in disruption of the discontinuous antibody binding site. Furthermore, these results suggest the existence of potential interactions between the V1/V2 and V4 regions of Env in its trimeric form on the virus particle, and that these interactions may involve conformational epitopes important for recognition of neutralizing antibodies.