Isolation of enzymatically active replication complexes from feline calicivirus-infected cells
Isolation of enzymatically active replication complexes from feline calicivirus-infected cells
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DOI：
10.1128/jvi.76.17.8582-8595.2002
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发表时间：
2002-09-01
影响因子：
5.4
通讯作者：
Sosnovtsev, SV
中科院分区：
文献类型：
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作者：
Green, KY;Mory, A;Sosnovtsev, SV
从猫杯状病毒（FCV）感染的细胞中分离出一种在镁盐、核糖核苷酸和atp再生系统存在下体外合成病毒RNA的膜状部分。该部分的酶活性成分被命名为FCV复制复合体（RCs），类似于其他正链RNA病毒。新合成的RNA通过Northern blot分析进行了表征，结果表明其全长（8.0 kb）和亚基因组长度（2.5 kb）的RNA分子与fcv感染细胞中合成的RNA分子相似。研究了与该部分相关的病毒蛋白的特性。60 kda的VP1主要衣壳蛋白是检测到的最丰富的病毒蛋白。VP2是一个由开放阅读框3 （ORF3）编码的次要结构蛋白。与该片段相关的非结构蛋白包括前体多肽Pro-Pol （76 kDa）和p30- vpg (43 kDa)，以及成熟的非结构蛋白p32（来自ORF1多蛋白的n端区域）、p30（推测的“3a样”蛋白）和p39（推测的核苷三磷酸酶）。分离含有病毒蛋白和细胞蛋白的酶活性RCs有助于解剖病毒和宿主对FCV RNA复制的贡献。
A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.