Identification of Residues of the Moloney Murine Leukemia Virus Nucleocapsid Critical for Viral DNA Synthesis In Vivo

The nucleocapsid (NC) protein of retroviruses is a small nucleic acid-binding protein important in virion assembly and in the encapsidation of the viral RNA genome into the virion particle. Multiple single-amino-acid substitutions were introduced into the NC of Moloney murine leukemia virus to examine further its role in viral replication. Two residues were shown to play important roles in the early events of replication. Unlike viruses with previously characterized NC mutations, these viruses showed no impairment in the late events of replication. Viruses containing the substitutions L21A and K30A expressed the normal complement of properly processed viral Gag proteins. Analysis of the RNA content of mutant virions revealed normal levels of unspliced and spliced viral RNA, and the tRNA(Pro) primer was properly annealed to the primer binding site on the viral genome. The virions demonstrated no defect in initiation of reverse transcription using the endogenous tRNA primer or in the synthesis of long viral DNA products in vitro. Nonetheless, viruses possessing these NC mutations demonstrated significant defects in the synthesis and accumulation of viral DNA products in vivo.

逆转录病毒的核衣壳（nucleocapsid, NC）蛋白是一类小型核酸结合蛋白，在病毒颗粒组装以及病毒RNA基因组包装进入病毒颗粒的过程中发挥关键作用。研究人员在莫洛尼鼠白血病病毒（Moloney murine leukemia virus）的NC蛋白中引入多种单氨基酸替换突变，以进一步探究其在病毒复制中的作用。研究发现，其中两个残基在病毒复制的早期事件中发挥重要功能。与此前已被表征的携带NC突变的病毒不同，这些突变病毒并未出现复制晚期事件的功能缺损。携带L21A与K30A替换突变的病毒，可表达正常丰度且经正确剪切加工的病毒Gag蛋白。对突变病毒颗粒的RNA含量分析显示，其未剪接与剪接后的病毒RNA水平正常，且tRNA(Pro)（脯氨酸tRNA）引物已正确退火至病毒基因组的引物结合位点。该类病毒颗粒在使用内源性tRNA引物启动逆转录，或是体外合成长链病毒DNA产物的过程中，均未表现出任何缺陷。尽管如此，携带上述NC突变的病毒在体内的病毒DNA产物合成与积累过程中，却出现了显著的功能缺陷。