A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N-acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells; infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation.

IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the diversity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity.

研究了人类免疫缺陷病毒 (HIV) 和猿猴免疫缺陷病毒 (SIV) gp120 C 末端附近高度保守的苏氨酸对包膜蛋白功能和病毒粒子感染性的贡献。当这个高度保守的苏氨酸残基被丝氨酸（另一种可以接受 O-糖基化的氨基酸）以外的任何残基取代时，产生的病毒是非传染性的。我们发现该 Thr 对于 gp120 与病毒粒子的结合至关重要，并且氨基酸取代增加了细胞培养上清液中解离的 gp120 的量。当在过表达多肽N-乙酰半乳糖胺基转移酶1（GalNAcT1）的细胞中产生HIV病毒粒子时，与野生型HEK293T细胞中产生的病毒相比，病毒感染性增加了2.5倍；当使用 Thr499Ser 突变体时，感染性增加了 8 倍。当使用 GalNAcT3 时，没有观察到这些感染性增强。使用完全缺乏进行 O 连接糖基化能力的 HEK293T 敲除细胞系，我们证明了在完全不存在 O 连接碳水化合物的情况下，可以产生正常水平的病毒体和正常水平的感染性。我们的数据表明 O-糖基化对于 HIV 和 SIV 的自然复制周期不是必需的。尽管如此，理论上仍然有可能，体内HIV 和 SIV 天然靶细胞中的 GalNAc 转移酶同种型可能导致相关苏氨酸残基发生 O-糖基化，这可能会增强病毒颗粒的感染性，使其超出病毒颗粒的感染水平。不存在这种O-糖基化。

重要性HIV 和 SIV 的 gp120 包膜糖蛋白质量的大约 50% 是 N 连接碳水化合物。这种 N 连接碳水化合物的贡献之一是保护保守的肽序列不被体液免疫识别。这种 N-连接糖基化是 HIV 和 SIV 的初级分离株对抗体介导的中和具有如此严重抵抗的原因之一。 O-连接糖基化的潜在贡献则研究较少。迄今为止关于这个主题的文献有些令人困惑和模糊。我们在本报告中描述的研究明确表明，O-连接糖基化对于 HIV 和 SIV 的自然复制周期不是必需的。然而，由于启动 O-连接碳水化合物附着的众多 GalNAc 转移酶的多样性以及体内HIV 和 SIV 天然靶细胞可能完成这种 O-连接碳水化合物附着以进一步增加理论上的可能性，这扇门并没有完全关闭。传染性。