A truncated form of Nef selected during pathogenic reversion of simian immunodeficiency virus SIVmac239Δnef increases viral replication
A truncated form of Nef selected during pathogenic reversion of simian immunodeficiency virus SIVmac239Δnef increases viral replication
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DOI：
10.1128/jvi.77.2.1245-1256.2003
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发表时间：
2003-01-01
影响因子：
5.4
通讯作者：
Cheng-Mayer, C
中科院分区：
文献类型：
--
作者：
Chakrabarti, LA;Metzner, KJ;Cheng-Mayer, C
猴免疫缺陷病毒SIVmac 239 Deltanef减毒活疫苗可有效保护恒河猴免受野生型SIVmac感染，但偶尔会导致CD 4（+）T细胞耗竭并进展为猴艾滋病（SAIDS）。从进展为SAIDS的接种疫苗的猕猴（Rh 1490）中回收的病毒在nef基因中获得了额外的缺失，导致了恢复原始nef开放阅读框的移码（R. I.康纳，D. C. Montefiori，J. M. J. P. Binley，摩尔，S. Bonhoeffer，A. Gettie，E. A.费纳莫尔湾E.谢里丹，D. D. Ho，P.J. Dailey和P.A.马克思，J. Virol. 72：7501-7509，1998）。静脉接种Rh 1490病毒分离株到4只未感染的恒河猴中，2年内4只动物中有3只出现CD 4（+）T细胞耗竭和疾病，表明毒力恢复。将包含从Rh 1490分离株扩增的截短nef基因的DNA片段插入SIVmac 239的遗传骨架中。所得克隆SIVmac 239-Delta 2nef表达约23 kDa的Nef蛋白，而原始SIVmac 239 Deltanef克隆表达8 kDa的较短蛋白。Nef的回复突变体不会引起CD 4、CD 3或主要组织相容性复合物I类的下调。在使用指示细胞系的单循环测定中，SIVmac 239-Delta 2nef的感染性与SIVmac 239 Deltanef的感染性相似。相比之下，在未刺激条件下感染的外周血单核细胞（PBMC）培养物中，SIVmac 239-Delta 2nef比SIVmac 239 Deltanef更有效地复制。在SIVmac 239-Delta 2nef感染的培养物中p27 Gag抗原水平仍然低于用野生型SIVmac 239获得的那些，与Nef功能的部分恢复一致。含有nef缺失的长末端重复序列（LTR）-荧光素酶构建体的转录活性与野生型LTR没有显著差异。在Nef-Delta 2内引入提前终止密码子消除了PBMC中的复制优势，表明Nef-Delta 2蛋白而不是LTR的U3区的结构负责病毒复制的增加。总之，这些结果表明，SIV与nef基因的缺失可以恢复到毒力和表达的nef与多个缺失的形式可能有助于通过增加病毒复制这一过程。
The live, attenuated vaccine simian immunodeficiency virus SIVmac239Deltanef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4(+) T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4(+) T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Delta2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Deltanef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Delta2nef was similar to that of SIVmac239Deltanef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Delta2nef replicated more efficiently than SIVmac239Deltanef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Delta2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Delta2 abolished the replicative advantage in PBMCs, demonstrating that the Nef-Delta2 protein, rather than the structure of the U3 region of the LTR, was responsible for the increase in viral replication. Taken together, these results show that SIV with a deletion in the nef gene can revert to virulence and that expression of a form of nef with multiple deletions may contribute to this process by increasing viral replication.