Cicala C, Avantaggiati M L, Graessmann A, Rundell K, Levine A S, Carbone M
Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
J Virol. 1994 May;68(5):3138-44. doi: 10.1128/JVI.68.5.3138-3144.1994.
The simian virus 40 (SV40) large-T antigen is essential for SV40 DNA replication and for late viral gene expression, but the role of the SV40 small-t antigen in these processes is still unclear. We have previously demonstrated that small t inhibits SV40 DNA replication in vitro. In this study, we investigated the effect of small t on SV40 replication in cultured cells. CV1 monkey cell infection experiments indicated that mutant viruses that lack small t replicate less efficiently than the wild-type virus. We next microinjected CV1 cells with SV40 DNA with and without purified small-t protein and analyzed viral DNA replication efficiency by Southern blotting. Replication of either wild-type SV40 or small-t deletion mutant DNA was increased three- to fivefold in cells coinjected with purified small t. Thus, in contrast to our in vitro observation, small t stimulated viral DNA replication in vivo. This result suggests that small t has cellular effects that are not detectable in a reconstituted in vitro replication system. We also found that small t stimulated progression of permissive monkey cells--but not of nonpermissive rodent cells--from G0-G1 to the S phase of the cell cycle, possibly leading to an optimal intracellular environment for viral replication.
猿猴病毒40（SV40）大T抗原对于SV40 DNA复制和病毒晚期基因表达至关重要，但SV40小t抗原在这些过程中的作用仍不清楚。我们之前已证明小t在体外可抑制SV40 DNA复制。在本研究中，我们调查了小t对培养细胞中SV40复制的影响。CV1猴细胞感染实验表明，缺乏小t的突变病毒复制效率低于野生型病毒。接下来，我们将纯化的小t蛋白与SV40 DNA一起或不与SV40 DNA一起显微注射到CV1细胞中，并通过Southern印迹分析病毒DNA复制效率。在与纯化的小t共同注射的细胞中，野生型SV40或小t缺失突变体DNA的复制增加了三到五倍。因此，与我们的体外观察结果相反，小t在体内刺激了病毒DNA复制。这一结果表明，小t具有在体外重组复制系统中无法检测到的细胞效应。我们还发现，小t刺激了允许性猴细胞（而非非允许性啮齿动物细胞）从细胞周期的G0-G1期进入S期，这可能导致了有利于病毒复制的最佳细胞内环境。
