Yamshchikov G V, Ritter G D, Vey M, Compans R W
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Virology. 1995 Dec 1;214(1):50-8. doi: 10.1006/viro.1995.9955.
The requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gag precursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envt protein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP.
利用杆状病毒表达系统研究了猴免疫缺陷病毒（SIV）颗粒组装和包膜整合的要求。在多角体蛋白启动子（pPolh）控制下表达的Pr56gag前体蛋白在Sf9昆虫细胞中表达时产生了高水平的未成熟逆转录病毒样颗粒（VLP）。为了确定包膜蛋白（Env）整合到VLP中的最佳条件，比较了两种重组杆状病毒，一种在极晚期pPolh或晚期/极晚期衣壳/多角体蛋白（Pcap/polh）杂交启动子控制下表达SIV包膜蛋白，另一种在杂交Pcap/polh启动子控制下表达截短形式的SIV包膜蛋白（Envt）。我们观察到，利用较早的杂交启动子可导致细胞表面Env表达水平更高，并使其整合到出芽病毒颗粒中。我们还发现，与全长Env相比，Envt蛋白更有效地转运到昆虫细胞表面并整合到VLP中。此外，我们研究了蛋白酶弗林蛋白酶共表达的影响，该蛋白酶与哺乳动物细胞中Env前体gp160的蛋白水解切割有关。在昆虫细胞中共表达弗林蛋白酶可导致更有效地蛋白水解切割成gp120和gp41, 并且切割后的蛋白被整合到VLP中。
