Xiang Y, Ridky T W, Krishna N K, Leis J
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
J Virol. 1997 Mar;71(3):2083-91. doi: 10.1128/JVI.71.3.2083-2091.1997.
Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.
通过在多蛋白切割位点或蛋白酶（PR）编码序列中引入氨基酸替换，罗氏肉瘤病毒（RSV）Gag前体的蛋白水解加工在体内发生了改变。根据体外肽底物切割数据预测，会降低PR从Gag多蛋白释放速率的单个氨基酸替换（V(P2)S和P(P4)G），被置于NC-PR连接区的NC部分。这些替换并不影响病毒样颗粒从COS细胞的释放效率，尽管回收的颗粒除了成熟病毒蛋白外还含有大量未切割的Pr76gag。能提高PR从Gag释放速率的单个氨基酸替换（A(P3)F和S(P1)Y），同样不影响病毒样颗粒从细胞的出芽。用来自快速切割的NC-PR序列的八个氨基酸替换Gag中切割效率低下的MA-p2连接序列，导致新MA-p2连接位点的切割显著增加，但同样对出芽没有影响。然而，当MA和p2蛋白之间的NC-PR连接序列中包含A(P3)F或S(P1)Y替换时，观察到出芽减少。将含有三个氨基酸替换（R105P、G106V和S107N）的PR亚基替换Gag，该替换位于底物结合口袋中，可增加PR的催化活性，也会导致出芽缺陷。该缺陷似乎是过早蛋白水解加工的结果，通过将催化天冬氨酸残基替换为丝氨酸使PR失活可以挽救这一缺陷。NC-PR切割位点的单个氨基酸替换降低了PR从Gag的释放速率，也挽救了这种出芽缺陷。用通过四个甘氨酸残基共价连接的两个PR亚基替换Gag PR，也会导致类似的出芽缺陷。与三重取代PR导致的缺陷不同，连接的PR二聚体观察到的出芽缺陷不能通过NC-PR切割位点突变来挽救，这表明PR二聚化是成熟过程中的一个限制步骤。总体而言，这些结果与一个模型一致，即在PR亚基从Gag多蛋白释放后发生病毒蛋白成熟。
