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 c ause disease. These viruses are particularly useful in identifying viral de terminants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxx phi) in the envelop e glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This do main constitutes both a potent endocytosis signal that reduces Env expressi on on infected cells and a sorting signal that directs Env expression to th e basolateral surface of polarized cells. Rhesus macaques were inoculated w ith SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutat ion (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (Delt a GY). 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 an d disease. One of two animals that received SIVmac239Y/I and two of three a nimals that received SIVmac239 Delta GY remained healthy for up to 140 week s with low to undetectable plasma viral RNA levels and normal CD4(+) T-cell percentages. These animals exhibited ongoing viral replication as determin ed by detection of viral sequences and culturing of mutant viruses from per ipheral blood mononuclear cells and persistent anti-SN antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was as sociated with a high plasma RNA level and disease, while one animal that re ceived SIVmac239 Delta GY also developed a high viral load that was associa ted with novel and possibly compensatory mutations in the TM cytoplasmic do main. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicati ng that the mutant viruses had replicated well enough to repair this mutati on. These findings indicate that the Yxx phi signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenua te SN through mechanisms that are distinct from those caused by mutations i n nef, this Tyr-based sorting signal represents a novel target for future m odels of SIV and human immunodeficiency virus attenuation that could be use ful in new vaccine strategies.