Vana Marcy L, Chen Aiping, Boross Peter, Weber Irene, Colman Dalbinder, Barklis Eric, Leis Jonathan
Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Retrovirology. 2005 Sep 27;2:58. doi: 10.1186/1742-4690-2-58.
A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with 35S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology.
一系列氨基酸取代（M239F、M239G、P240F、V241G）被引入p10-CA蛋白酶切割位点（VVAM*PVVI），以改变连接处的切割速率。通过测量含有野生型和突变型p10-CA位点的模型肽底物的切割动力学，证实了这些取代对呼吸道合胞病毒（RSV）蛋白酶切割p10-CA的影响。通过用35S-甲硫氨酸和半胱氨酸标记转染了Gag的COS-1细胞，并从培养基和裂解物组分中免疫沉淀Gag及其切割产物，确定了这些取代对Gag多蛋白加工的影响。除M239F外，所有取代均导致可检测到的Gag加工减少以及随后从细胞中释放减少。几个突变体还导致三种CA蛋白的产生出现缺陷。将p10-CA突变亚克隆到RSV前病毒载体（RCAN）中，并引入鸡胚成纤维细胞系（DF-1）。除M239F外，所有突变均阻断了RSV复制。此外，通过电子显微镜检查了M239F和M239G取代对释放的病毒颗粒形态的影响。虽然M239F颗粒看起来与野生型颗粒相似，但M239G颗粒含有大且形状异常的核心。这些结果表明，影响p10-CA蛋白酶切割位点切割的突变会阻断RSV复制，并可能对病毒颗粒形态产生负面影响。
