Sabino Cunha Marcela, Lima Sampaio Thatiane, Peterlin B Matija, Jesus da Costa Luciana
Departamento de Virologia-Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho 373-CCS-Bloco I, Rio de Janeiro 21941-902, Brazil.
Departments of Medicine, Microbiology and Immunology, University of California, San Francisco, 533 Parnassus Avenue, San Francisco, CA 94143, USA.
Viruses. 2016 Jul 7;8(7):189. doi: 10.3390/v8070189.
Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity.
来自所有灵长类慢病毒的Nef蛋白，包括黑猩猩猿猴免疫缺陷病毒（SIVcpz），均可提高病毒子代的感染性。然而，Nef在病毒感染性增加中所涉及的功能仍未完全明确。尽管如此，到目前为止，对SIVcpz的Nef功能的研究都是在HIV-1前病毒的背景下进行的。为了探究Nef在SIVcpz复制周期中所起的作用，通过在nef序列内的一个独特限制位点引入移码突变，从SIVcpzWTGab2克隆中获得了一个Nef缺陷衍生物。这个缺失nef的克隆表达了Nef的N端74个氨基酸的截短肽，并被命名为SIVcpz-tNef。我们发现，SIVcpz-tNef的行为与经典的缺失nef的HIV-1或猕猴猿猴免疫缺陷病毒SIVmac不同。值得注意的是，来自Hek-293T和Molt生产细胞的SIVcpz-tNef子代完全没有感染性。此外，SIVcpz-tNef感染性的丧失与Gag和GagPol加工的抑制相关。在病毒裂解物中检测到Gag的明显积累和极低水平的逆转录酶。此外，一旦在表达SIVcpzΔNef和HIV-1WT的细胞中反式表达tNef肽，这些观察结果就会重现，表明截短肽对SIVcpz和HIV-1的病毒加工和感染性具有显性负作用。我们证明，截短的Nef肽在蛋白酶区域之外与GagPol结合，这样做可能在非常早期阶段就阻断了GagPol和Gag前体的加工。这项研究首次证明，天然存在的Nef肽可以有效地阻断慢病毒的加工和感染性。
