Lin Yuan, Horvath Frank, Aligo Jason A, Wilson Rebecca, He Biao
Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802, USA.
Virology. 2005 Aug 1;338(2):270-80. doi: 10.1016/j.virol.2005.05.014.
The paramyxovirus simian virus 5 (SV5) has seven genes but encodes eight known viral proteins. The V/P gene is transcribed into two mRNA species: V mRNA from a faithful transcription of the gene and P mRNA from transcription with addition of two G residues at a specific site of the gene. V, a 222-amino acid (AA) residue protein, and P, a 392 AA residue protein, share an identical N-terminus domain of 164 amino acid residues. P is essential for SV5 RNA replication and transcription. Whereas it is known that V plays important roles in virus pathogenesis, the role of V in SV5 replication and transcription is not clear. A mini-genome system, free of vaccinia virus gene expression system, consisting of plasmids expressing NP, P, and L, as well as a plasmid encoding a reporter gene, chloramphenicol acetyltransferase (CAT) flanked by SV5 trailer and leader sequences under control of a bacteriophage T7 RNA polymerase promoter, has been established to examine the role of V in SV5 RNA transcription and replication. Addition of V-expressing plasmid in the mini-genome system caused inhibition of the reporter gene expression, suggesting that V plays a role in regulating SV5 gene expression. By examining the amount of encapsidated viral RNA genome using reverse transcription with primer annealing to viral anti-genome RNA and PCR, it was found that expression of V reduced the amount of viral RNA genome in the mini-genome system, suggesting that V inhibits viral RNA replication. To examine whether the V protein inhibits viral RNA transcription as well, a mini-genome system with a defective anti-genome promoter (AGP) such that a reporter gene (luciferase, Luc) expression is only derived from transcription of newly produced mini-genome and not from de novo replicated viral genome due to the defect in replication element has been utilized. The V protein inhibited luciferase expression from the mini-genome with a defective AGP, suggesting V inhibits SV5 transcription. Thus, SV5 V inhibits both SV5 RNA replication and transcription.
副粘病毒猴病毒5（SV5）有七个基因，但编码八种已知的病毒蛋白。V/P基因转录成两种mRNA：一种是基因忠实转录产生的V mRNA，另一种是在基因特定位点添加两个G残基后转录产生的P mRNA。V蛋白有222个氨基酸残基，P蛋白有392个氨基酸残基，它们共享一个由164个氨基酸残基组成的相同N端结构域。P蛋白对SV5 RNA复制和转录至关重要。虽然已知V蛋白在病毒发病机制中起重要作用，但V蛋白在SV5复制和转录中的作用尚不清楚。已建立了一个不含痘苗病毒基因表达系统的微型基因组系统，该系统由表达NP、P和L的质粒以及一个编码报告基因氯霉素乙酰转移酶（CAT）的质粒组成，CAT基因两侧是SV5拖尾序列和前导序列，受噬菌体T7 RNA聚合酶启动子控制，用于研究V蛋白在SV5 RNA转录和复制中的作用。在微型基因组系统中添加表达V的质粒会导致报告基因表达受到抑制，这表明V蛋白在调节SV5基因表达中发挥作用。通过使用与病毒反基因组RNA退火的引物进行逆转录和PCR来检测包装的病毒RNA基因组的量，发现V蛋白的表达减少了微型基因组系统中病毒RNA基因组的量，这表明V蛋白抑制病毒RNA复制。为了检测V蛋白是否也抑制病毒RNA转录，使用了一个具有缺陷反基因组启动子（AGP）的微型基因组系统，使得报告基因（荧光素酶，Luc）的表达仅来自新产生的微型基因组的转录，而不是由于复制元件缺陷而从头复制的病毒基因组。V蛋白抑制了具有缺陷AGP的微型基因组的荧光素酶表达，这表明V蛋白抑制SV5转录。因此，SV5的V蛋白同时抑制SV5 RNA复制和转录。
