Characterization of N-linked glycosylation sites on envelope proteins of simian/human immunodeficiency virus in peripheral blood of Chinese rhesus macaques during acute infection

Abstract

Objective: To investigate the number and distribution of N-linked glycosylation sites of simian/human immunodeficiency virus envelope proteins(SHIVSF162P3)and SHIV transmission. Methods: Two female adult Chinese rhesus macaques(4 years old)were intravenously inoculated with 300 TCID50 SHIVSF162P3. The macaques weighed 4 and 5 kg and were identified as Rh1 and Rh2. We collected plasma samples at days 3, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, 56, 63, 70 and 77 post-challenge. Subsequently, we monitored plasma viral load by real-time PCR after viral RNA isolation and cDNA synthesis. We amplified the full-length envelope gene by single genome amplification(SGA)at days 7, 14, 28 and 77. BioEdit, MEGA, and the HIV Databases were used to analyze envelope sequences. Sequence diversity and N-linked glycosylation sites were compared between virus stock and plasma viruses of the two macaques. Results: A total of 55 env sequences were obtained from virus stock and their average pairwise distances were(0.166 6± 0.096 3)%. Viral loads peaked at 7.68 and 7.49 log10 copies/ml at day 10 and reached the set point at day 42(4.27 and 4.81 log10 copies/ml). The percentages of envelope sequences containing 25 potential N-linked glycosylation sites(PNGSs)were 83%(20/24)and 94%(29/31)in Rh1 and Rh2, respectively, at day 7; these were significantly higher than the proportion in SHIVSF162P3 stock(49%(27/55)). Viral diversity after infection increased with time whereas the proportion of sequences containing 25 PNGSs decreased and sequences containing 27 PNGSs gradually increased. In Rh1, the percentage of sequences containing 27 PNGSs increased to 29% at day 28 and reached 35% at day 77 in Rh2. By analyzing the number of PNGSs in the V1-V5 regions, we found that PNGS variation mainly occurred in the V4 loop. Compared with sequences containing 27 PNGSs, a seven amino acid(TWNNTIG)deletion was found in the V4 loop, which resulted in a loss of two PNGSs at positions 392 and 396. Conclusion: Low glycosylation of the SHIVSF162P3 V4 loop may facilitate spread of the SHIV virus whereas viruses with highly glycosylated V4 loops showed replication advantages after infection.