Virus Entry, Tropism, and Attachment
Session Day and Time: Thursday 1:30 - 3:30 pm
Room: Hall D
Background: Neutralizing antibody responses contribute significantly to the containment of many viral illnesses. Although HIV infected individuals generate high titers of anti-env antibodies, neutralizing titers remain weak and appear late after seroconversion. Extensive glycosylation of SIV and HIV env is thought to play a role in masking neutralization epitopes. Our laboratory has previously shown that monkeys infected with a mutated and highly neutralization sensitive virus missing 5 N-linked glycosylation sites in the V1-V2 region, demonstrated initial viral peaks similar to WT but then controlled viremia at low or undetectable levels for more than 2 yrs. The role of glycosylation in masking CD4 binding site epitopes of neutralization has not been previously examined. Exposure of epitopes that contain well-conserved residues for CD4 binding could theoretically elicit strong, broadly neutralizing antibody responses.
Methods: Using PCR mutagenesis, we created 4 SIVmac239 strains by substituting the N within the N-linked glycosylation site of NXT/S with Q at sites proximal to the ridge of the predicted CD4 binding pocket of SIV env. Viral replication was evaluated by measurement of p27gag production in the supernatant of transfected cells by ELISA.
Results: Changes were made at env positions 278, 284, 371, and 377 of SIVmac239. Three (3) mutant strains, gp278, gp371 and gp377, replicated well in transfected CEMX174 cells while gp284 did not. A second, independently made, mutant strain with N to Q changes at both positions of 278 and 284 also did not replicate. To determine whether loss of the glycan at gp284 vs substitution of N alone caused the replication defect, we created another mutant with disruption of the glycosylation site by substitution of T of NXT with A. This mutant demonstrated delayed growth kinetics compared to WT. The concentration of soluble CD4 (sCD4) necessary to inhibit viral replication by 50% (IC50)was similar for gp371, gp377 and wild-type virus; the IC50 for gp278 was twice that of the other strains.
Conclusions: One (1) of 3 glycosylation mutants studied showed a modest change in sensitivity to sCD4 inhibition. The identification of 3 glycosylation sites that are predicted to lie on or near the ridge of the CD4 binding pocket that are dispensable for native structure and function raises the hope that this may be a promising approach to improving the neutralization response of env-containing vaccines.