In Vivo Attenuation of Simian Immunodeficiency Virus by Disruption of a Tyrosine-Dependent Sorting Signal in the Envelope Glycoprotein Cytoplasmic Tail

Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxφ) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (ΔGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239ΔGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239ΔGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxφ signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.

已有研究报道可产生低水平血浆病毒粒子RNA、致病能力减弱的减毒猴免疫缺陷病毒（simian immunodeficiency viruses, SIVs）。此类病毒在鉴定致病相关病毒决定簇方面具有重要应用价值。本研究表明，包膜糖蛋白（envelope glycoprotein, Env）胞质尾区第721至724位氨基酸YRPV处的高度保守酪氨酸（tyrosine, Tyr）基序（Yxxφ）发生突变，可显著降低SIVmac239的体内致病能力。该结构域同时具备两种功能：一是作为强效内吞信号，降低感染细胞表面的Env表达水平；二是作为分选信号，指导Env定向表达于极化细胞的基底外侧膜表面。研究人员将SIVmac239对照组病毒，或携带第721位酪氨酸突变为异亮氨酸的SIVmac239（SIVmac239Y/I），或缺失第721位酪氨酸及其上游甘氨酸的SIVmac239（ΔGY）接种至恒河猴。为评估病毒的体内复制能力，所有病毒的nef基因均引入了终止密码子——已有研究证实，该终止密码子可在体内复制过程中发生回复突变，但在体外复制时不会出现。所有3只对照组动物均出现高病毒载量及相关病症。在接种SIVmac239Y/I的2只恒河猴中，有1只；接种SIVmac239ΔGY的3只恒河猴中，有2只，在长达140周的观测期内保持健康状态，血浆病毒RNA水平低至无法检出，CD4(+) T细胞占比维持正常。通过对外周血单个核细胞中的病毒序列进行检测、突变病毒分离培养，以及持续监测抗SIV抗体滴度，可证实这些动物体内存在持续的病毒复制。在1只接种SIVmac239Y/I的恒河猴体内，异亮氨酸回复突变为酪氨酸，伴随血浆RNA水平升高及病症发作；而1只接种SIVmac239ΔGY的恒河猴也出现了高病毒载量，该现象与跨膜蛋白（transmembrane protein, TM）胞质区出现的新型且可能具有代偿功能的突变相关。在所有对照组及实验组动物体内，nef基因的终止密码子均在接种后前2个月内回复为可读框，这表明突变病毒已具备足够的复制能力以修复该突变。上述研究结果表明，Yxxφ信号基序在SIV致病过程中发挥关键作用。此外，由于该基序的突变可通过不同于nef基因突变的途径实现SIV减毒，因此这种基于酪氨酸的分选信号基序，可为未来构建SIV及人类免疫缺陷病毒（human immunodeficiency virus, HIV）减毒模型提供全新靶点，有望应用于新型疫苗研发策略。