Chakrabarti Lisa A, Metzner Karin J, Ivanovic Tijana, Cheng Hua, Louis-Virelizier Jean, Connor Ruth I, Cheng-Mayer Cecilia
Viral Immunology Unit, Pasteur Institute, Paris, France.
J Virol. 2003 Jan;77(2):1245-56. doi: 10.1128/jvi.77.2.1245-1256.2003.
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.
减毒活疫苗猿猴免疫缺陷病毒SIVmac239Deltanef能有效保护恒河猴免受野生型SIVmac感染,但偶尔会导致CD4(+) T细胞耗竭并发展为猿猴艾滋病(SAIDS)。从一只发展为SAIDS的接种疫苗的恒河猴(Rh1490)中分离出的病毒,其nef基因又发生了一次额外缺失,导致移码,恢复了原来的nef开放阅读框(R.I.康纳、D.C.蒙特菲奥里、J.M.宾利、J.P.摩尔、S.博恩霍费尔、A.格蒂、E.A.费纳莫尔、K.E.谢里丹、D.D.何、P.J.戴利和P.A.马克思,《病毒学杂志》72:7501 - 7509,1998年)。将Rh1490病毒分离株静脉接种到四只未感染的恒河猴体内,在两年内四只动物中有三只出现了CD4(+) T细胞耗竭和疾病,表明毒力恢复。从Rh1490分离株中扩增出的包含截短nef基因的DNA片段被插入到SIVmac239的基因骨架中。得到的克隆SIVmac239 - Delta2nef表达一种约23 kDa的Nef蛋白,而原始的SIVmac239Deltanef克隆表达一种8 kDa的较短蛋白。Nef的回复形式不会导致CD4、CD3或主要组织相容性复合体I类分子的下调。在使用指示细胞系的单循环试验中,SIVmac239 - Delta2nef的感染性与SIVmac239Deltanef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,SIVmac239 - Delta2nef比SIVmac239Deltanef复制得更有效。SIVmac239 - Delta2nef感染的培养物中的p27 Gag抗原水平仍低于野生型SIVmac239,这与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没有明显差异。在Nef - Delta2内引入一个提前终止密码子消除了在PBMC中的复制优势,表明是Nef - Delta2蛋白而不是LTR的U3区域结构导致了病毒复制增加。综上所述,这些结果表明,nef基因缺失的SIV可以恢复毒力,并且一种具有多个缺失的nef形式的表达可能通过增加病毒复制促进这一过程。