Welker R, Kottler H, Kalbitzer H R, Kräusslich H G
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Virology. 1996 May 1;219(1):228-36. doi: 10.1006/viro.1996.0240.
The Nef protein of primate immunodeficiency viruses is essential for establishing a highly productive pathogenic infection in vivo. In tissue culture, Nef is not required for infection but enhances viral infectivity. This effect is most pronounced in unstimulated primary lymphocytes and occurs in the early phase of infection prior to viral gene expression. Since Nef expression does not lead to obvious changes in virus composition, it was of interest to analyze whether Nef is incorporated into virus particles. Here, we show that Nef is specifically immunoprecipitated from radioactively labeled human immunodeficiency virus type 1 (HIV-1)-infected cells and virus particle preparations. Quantitative analysis revealed Nef to be incorporated on the order of 10% of reverse transcriptase incorporation, which corresponds to 5 to 10 molecules of Nef per virion. In infected cells, Nef was detected as a full-length 27-kDa protein. In contrast, approximately 50% of particle-associated Nef corresponded to an 18-kDa species which comigrated with the larger product after in vitro cleavage of purified HIV-1 Nef by the viral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Nef is cleaved concomitantly with viral structural proteins on maturation of virus particles. This cleavage is likely to be functionally significant because it dissociates the conserved core domain from the N-terminal membrane attachment region. Our results suggest that the profound influence of Nef on establishing infection of unstimulated cells in tissue culture and in vivo is mediated by virion-associated Nef which functions in early infection before viral gene expression.
灵长类免疫缺陷病毒的Nef蛋白对于在体内建立高效的致病性感染至关重要。在组织培养中，感染并不需要Nef蛋白，但它可增强病毒的感染性。这种效应在未受刺激的原代淋巴细胞中最为明显，且发生在病毒基因表达之前的感染早期阶段。由于Nef蛋白的表达不会导致病毒组成发生明显变化，因此分析Nef蛋白是否被整合到病毒颗粒中很有意义。在此，我们表明，可从放射性标记的1型人类免疫缺陷病毒（HIV-1）感染的细胞和病毒颗粒制剂中特异性免疫沉淀出Nef蛋白。定量分析显示，Nef蛋白的整合量约为逆转录酶整合量的10%，这相当于每个病毒体中有5到10个Nef分子。在感染的细胞中，Nef蛋白被检测为全长27 kDa的蛋白质。相比之下，与颗粒相关的Nef蛋白中约50%对应于一种18 kDa的蛋白，该蛋白在体外经病毒蛋白酶切割纯化的HIV-1 Nef蛋白后，与较大的产物迁移位置相同。HIV-1蛋白酶的特异性抑制剂可完全消除颗粒制剂中Nef蛋白的切割。很可能，Nef蛋白在病毒颗粒成熟时与病毒结构蛋白同时被切割。这种切割可能在功能上具有重要意义，因为它使保守的核心结构域与N端膜附着区域分离。我们的结果表明，Nef蛋白对组织培养和体内未受刺激细胞感染的深远影响是由与病毒体相关的Nef蛋白介导的，该蛋白在病毒基因表达之前的早期感染中发挥作用。
