Assembly of HIV GAG-B-galactosidase fusion proteins into virus particles.
Assembly of HIV GAG-B-galactosidase fusion proteins into virus particles.
复制标题
将 HIV GAG-B-半乳糖苷酶融合蛋白组装成病毒颗粒。
作者：
C. T. Wang;J. Stegeman;Y. Zhang;E. Barklis
我们研究了在HIV Gag蛋白存在下，人免疫缺陷病毒（HIV-1）Gag-B-半乳糖苷酶（Gag-B-gal; GBG）融合蛋白向HIV颗粒中的组装。通过测定培养基与细胞B-gal活性来测量融合蛋白从细胞的释放，并且该释放依赖于未融合Gag蛋白的共表达。通过去污剂处理和密度梯度分级分离研究证明了Gag-B-gal掺入病毒颗粒，并且取决于需要HIV CA结构域C末端三分之二的蛋白质-蛋白质相互作用。中央MA域融合蛋白的掺入似乎不重要，一个nonmyristylated GBG蛋白被纳入，但在一个相对降低的水平，而NC和p6域略有影响融合蛋白组装成颗粒。亚细胞分级分离研究表明，所有的融合蛋白，包括nonmyristylated一个丰富的细胞质颗粒级分。然而，组装成颗粒与亚细胞分馏模式无关。同样，Gag-B-gal蛋白的病毒体掺入水平与其免疫荧光定位模式无关。然而，我们观察到，虽然大多数融合蛋白显示核周环与整个细胞的异质性染色，短的融合蛋白出现丰富的细胞内膜上，和完整的MA，但删除NC结构域的融合蛋白表现出增强的表面染色没有一个明确的核周环。总之，我们的数据表明，CA结构域是HIV融合蛋白组装成病毒颗粒的主要决定因素。
We have studied the assembly of human immunodeficiency virus (HIV-1) Gag-B-galactosidase (Gag-B-gal; GBG) fusion proteins into HIV particles in the presence of HIV Gag proteins. Release of fusion proteins from cells was measured by assay of media versus cellular B-gal activities and was dependent on co-expression of unfused Gag proteins. Gag-B-gal incorporation into virus particles was demonstrated by detergent treatment and density gradient fractionation studies and was dependent on protein-protein interactions requiring the C-terminal two-thirds of the HIV CA domain. The central MA domain appeared unimportant for fusion protein incorporation; a nonmyristylated GBG protein was incorporated but at a relatively reduced level, while the NC and p6 domains slightly affected the assembly of fusion proteins into particles. Subcellular fractionation studies showed that all fusion proteins including the nonmyristylated one were enriched in the cytoplasmic pellet fraction. However, assembly into particles did not correlate with subcellular fractionation patterns. Similarly, virion incorporation levels of Gag-B-gal proteins did not correlate with their immunofluorescence localization patterns. However, we observed that while most fusion proteins displayed a perinuclear ring with heterogeneous staining throughout cells, short fusion proteins appeared enriched on the intracellular membranes, and fusion proteins with intact MA but deleted NC domains showed an enhanced surface staining without a clear perinuclear ring. Altogether, our data suggest that the CA domain is the primary determinant for assembly of HIV fusion proteins into virus particles.