Selection for neutralization resistance of the simian/human immunodeficiency virus SHIVSF33A variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify N-linked glycosylation

Abstract

We previously reported on the in vivo adaptation of an infectious molecular simian/human immunodeficiency virus (SHIV) clone, SHIVSF33, into a pathogenic biologic viral variant, designated SHIVSF33A. In the present study, we show that SHIVSF33A is resistant to neutralization by human immunodeficiency virus (HIV) and SHIV antisera. Multiple amino acid substitutions accumulated over time throughout the env gene of SHIVSF33A; some of them coincided with the acquisition of the neutralization resistance of the virus. Of interest are changes that resulted in the removal, repositioning, and addition of potential glycosylation sites within the V1, V2, and V3 regions of envelope gp120. To determine whether potential glycosylation changes within these principal neutralization domains of HIV type 1 formed the basis for the resistance to serum neutralization of SHIVSF33A, mutant viruses were generated on the backbone of parental SHIVSF33 and tested for their neutralization sensitivity. The mutations generated did not alter the in vitro replication kinetics or cytopathicity of the mutant viruses in T-cell lines. However, the removal of a potential glycosylation site in the V1 domain or the creation of such a site in the V3 domain did allow the virus to escape serum neutralization antibodies that recognized parental SHIVSF33. The combination of the V1 and V3 mutations conferred an additive effect on neutralization resistance over that of the single mutations. Taken together, these data suggest that (i) SHIV variants with changes in the Env SU can be selected in vivo primarily by virtue of their ability to escape neutralizing antibody recognition and (ii) carbohydrates play an important role in conferring neutralization escape, possibly by altering the structure of envelope gp120 or by shielding principal neutralization sites.

FIG. 1

Amino acid alignment of the V1, V2, and V3 domains of SHIV_SF33, SHIV_SF33A, and sequential isolates obtained from Mnu25814. Viral DNA sequences encoding the V1 to V5 regions of the HIV-1 env gene were amplified by nested PCR. The amplified products were cloned, and the predicted amino acid sequences of the V1, V2, and V3 domains of the variants were determined and compared to the corresponding sequences of the reference SHIV_SF33 clone (33WT). The numbers in parentheses represent the numbers of clones displaying the indicated sequence divided by the total number of clones sequenced; the consensus sequence of the SHIV_SF33A isolate (33A) is provided for comparison. Dots are used to indicate identity, and overlining denotes the positions of glycosylation changes within the various domains. 52wk, 72wk, 91wk, and 96wk represent the variants present in Mnu25814 at 52, 72, 91, and 96 weeks postinfection, respectively.

FIG. 2

Replication of SHIV_SF33 (WT), SHIV_SF33A (33A), and glycosylation mutants in cells CEMX174 (A) and RhPBMC (B). CEMX174 cells (10⁵) or RhPBMC (2 × 10⁶) were infected with 100 TCID₅₀ of each virus for 3 h at 37°C. The viral inocula were then removed, and the infected cells were maintained in culture media. At the times indicated, p27 antigen production in culture supernatants was determined.

FIG. 3

Neutralization of SHIV_SF33 (WT), SHIV_SF33A (33A), and single (A) or double and triple (B) glycosylation mutants by serum collected from Mnu25814 at 96 weeks postinfection. Serum neutralization was performed as described in Materials and Methods. The percent neutralization of each virus was determined and plotted against the reciprocal of serum dilutions used. Data represent one of three independent neutralization experiments, and similar findings were obtained with serum collected from Mnu25814 at 104 weeks postinfection.

FIG. 4

Neutralization of V1, V2, and V3 glycosylation mutants by antisera to SHIV and HIV-1. Serum neutralization was performed with serum collected from Mnu25814 at 96 weeks postinfection (A) and with a pool of sera collected from HIV-1-infected individuals (B) as described in Materials and Methods. The percent neutralization was determined and plotted against the reciprocal of serum dilutions used. Data represent one of three independent neutralization experiments.