Alin K, Goff S P
Department of Biochemistry, Columbia University P & S, New York 10032, USA.
Virology. 1996 Aug 15;222(2):339-51. doi: 10.1006/viro.1996.0431.
The capsid domain (CA) of the retroviral Gag protein is a major constituent of the virion core. To examine the role of this protein in M-MuLV morphogenesis and replication, a series of substitution mutations affecting the central region of CA were introduced into an infectious proviral DNA. The altered DNAs were introduced into cells, and the resulting lines were analyzed for production of infectious virions. Only one of the replication defective mutants analyzed was blocked in virion assembly. The remaining mutant DNAs induced the formation and release of particles containing genomic RNA and polymerase protein. The reverse transcriptase associated with these mutant virions was capable of transcribing both minus strand strong stop and extended DNA products using the endogenous genomic RNA as template in vitro. Upon infection of fresh cells, however, no viral DNA synthesis could be detected either by Southern analysis or by an RNase protection assay developed specifically to detect intermediate products of reverse transcription. The results indicate that the bulk of the CA mutants are blocked before reverse transcription of the viral genome and suggest an important role for the capsid protein in an early stage of viral replication.
逆转录病毒Gag蛋白的衣壳结构域(CA)是病毒粒子核心的主要成分。为了研究该蛋白在莫洛尼鼠白血病病毒(M-MuLV)形态发生和复制中的作用,一系列影响CA中心区域的替代突变被引入到一个感染性前病毒DNA中。将改变后的DNA导入细胞,并对所得细胞系进行分析,以检测感染性病毒粒子的产生情况。在分析的复制缺陷型突变体中,只有一个在病毒粒子组装过程中受阻。其余的突变DNA诱导了含有基因组RNA和聚合酶蛋白的颗粒的形成与释放。与这些突变病毒粒子相关的逆转录酶能够以细胞内源性基因组RNA为模板,在体外转录负链强终止DNA产物和延伸的DNA产物。然而,在感染新鲜细胞后,无论是通过Southern分析还是通过专门开发用于检测逆转录中间产物的核糖核酸酶保护试验,均未检测到病毒DNA合成。结果表明,大多数CA突变体在病毒基因组逆转录之前就被阻断,这提示衣壳蛋白在病毒复制的早期阶段具有重要作用。