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首页> 美国卫生研究院文献>The Journal of Experimental Medicine >The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages
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The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages

机译：人类免疫缺陷病毒-1 nef基因产物：原发性淋巴细胞和巨噬细胞中病毒感染和复制的积极因素

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Considerable controversy and uncertainty have surrounded the biological function of the Human Immunodeficiency Virus (HIV)-1 nef gene product. Initial studies suggested that this early, nonstructural viral protein functioned as a negative regulatory factor; thus, it was proposed to play a role in establishing or maintaining viral latency. In contrast, studies in Simian Immunodeficiency Virus (SIV)mac-infected rhesus monkeys have suggested that Nef is not a negative factor but rather plays a central role in promoting high-level viral replication and is required for viral pathogenesis in vivo. We sought to define a tissue culture system that would approximate the in vivo setting for virus infection in order to assess the role of HIV-1 Nef in viral replication. We show that infection of mitogen-activated peripheral blood mononuclear cells (PBMC) with Nef+ HIV results in enhanced replication as evidenced by earlier gag p24 expression when compared with infections performed with nef mutant viruses. Moreover, when unstimulated freshly isolated PBMC are infected with Nef+ and Nef- viruses and then subsequently activated with mitogen, the Nef-induced difference in viral replication kinetics is even more pronounced, with the Nef- viruses requiring much more time in culture for appreciable growth. A positive effect of Nef on viral replication was also observed in primary macrophages infected with a recombinant of YU-2, a patient- derived molecular clone with macrophage tropism. These positive effects of Nef on viral replication are dependent on the initial multiplicity of infection (MOI), in that infections of unstimulated PBMC at low MOI are most dependent upon intact nef for subsequent viral growth. We now provide evidence that the Nef+ HIV is more infectious than Nef- HIV from both a tissue culture infectious dose analysis, and a single-cell HIV infection assay. In the latter case, we demonstrate that infection with equivalent doses of HIV based on virion-associated gag p24 yields five- to sixfold more infected cells if Nef+ viral stocks were used. Furthermore, we find that the differential infectivity is not dependent on CD4 down-regulation as Nef+ virus produced from transfected COS cells lacking CD4 is also more infectious. However, normalization of PBMC infections to equivalent infectivity between that of the Nef+ and Nef- viruses continues to reveal delayed viral replication in the absence of Nef, suggesting that secondary viral spread in PBMC is also enhanced in Nef+ infections. We demonstrate this directly by showing a 13-15-fold increase in infectivity of PBMC-derived Nef+ HIC.(ABSTRACT TRUNCATED AT 400 WORDS)
机译：人类免疫缺陷病毒（HIV）-1 nef基因产物的生物学功能已引起相当大的争议和不确定性。初步研究表明，这种早期的非结构性病毒蛋白起着负调节因子的作用。因此，建议在建立或维持病毒潜伏期中发挥作用。相反，对猿猴免疫缺陷病毒（SIV）mac感染的恒河猴的研究表明，Nef不是负面因素，而是在促进高水平病毒复制中起核心作用，是体内病毒发病机制所必需的。我们试图定义一种组织培养系统，该系统应近似于病毒感染的体内环境，以便评估HIV-1 Nef在病毒复制中的作用。我们显示，与用nef突变病毒进行的感染相比，用Nef + HIV感染有丝分裂原活化的外周血单个核细胞（PBMC）会导致复制增强，这是由较早的gag p24表达所证明的。此外，当未刺激的新鲜分离的PBMC感染Nef +和Nef-病毒，然后被有丝分裂原激活时，Nef诱导的病毒复制动力学差异更加明显，Nef病毒需要更多的培养时间才能明显生长。 。在用YU-2重组体感染的原代巨噬细胞中也观察到Nef对病毒复制的积极作用，这是一种由患者衍生的具有巨噬细胞嗜性的分子克隆。 Nef对病毒复制的这些积极影响取决于感染的最初多重性（MOI），因为在低MOI时未刺激的PBMC的感染最依赖于完整的nef来进行随后的病毒生长。我们现在提供证据，从组织培养物感染剂量分析和单细胞HIV感染分析来看，Nef + HIV比Nef-HIV更具感染力。在后一种情况下，我们证明，如果使用Nef +病毒原种，用基于病毒体相关的gag p24的等量HIV感染，感染细胞的数量会增加5至6倍。此外，我们发现差异感染力不依赖于CD4下调，因为从缺少CD4的转染COS细胞产生的Nef +病毒也更具感染力。然而，将PBMC感染归一化为与Nef +和Nef-病毒之间相等的感染力继续显示出在没有Nef的情况下病毒复制被延迟，这表明在Nef +感染中PBMC中的二次病毒传播也得到了增强。我们通过显示PBMC衍生的Nef + HIC的感染性增加13-15倍来直接证明这一点（摘要截短了400字）

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