Spence S L, Pipas J M
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
J Virol. 1994 Jul;68(7):4227-40. doi: 10.1128/JVI.68.7.4227-4240.1994.
The simian virus 40 (SV40) T antigen host range mutants dl1066 and dl1140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants dl1066 and dl1140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with dl1066 and dl1140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40% sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants dl1066 and dl1140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests that the host range blockage may result from a failure of such mutants to add VP1 to 75S assembly intermediates.
猿猴病毒40（SV40）T抗原宿主范围突变体dl1066和dl1140在噬斑形成上表现出复制后阻滞，这表明T抗原在病毒生命周期后期具有新的作用。宿主范围突变体dl1066和dl1140能够在BSC细胞中生长并形成噬斑，但不能在CV1猴肾细胞（一种通常允许病毒生长的宿主细胞）上生长。先前的研究表明，在感染了dl1066和dl1140的CV1细胞中，病毒DNA复制水平和晚期衣壳蛋白积累水平仅略有降低，而agnoprotein未能积累不太可能是导致突变体生长缺陷的主要因素。在这里，我们表明宿主范围突变体在病毒颗粒组装方面存在缺陷。SV40组装过程是75S病毒染色质复合物逐步转化为200S - 240S组装病毒体的过程。当病毒感染细胞提取物在5%至40%的蔗糖梯度上分离时，野生型提取物在对应于组装病毒峰的200S - 240S组分中显示出病毒晚期蛋白的最大积累，而在对应于未成熟组装中间体的75S - 150S组分中积累较少。然而，在非允许性CV1细胞中生长的宿主范围突变体dl1066和dl1140未能组装任何可观数量的成熟200S - 240S病毒体，并积累了75S中间体，而在允许性BSC细胞中，组装水平比野生型略有降低。对梯度组分蛋白质组成的分析表明，SV40组装的机制与多瘤病毒提出的机制相似，并表明宿主范围阻滞可能是由于此类突变体未能将VP1添加到75S组装中间体所致。
