Dinant S, Janda M, Kroner P A, Ahlquist P
Institute for Molecular Virology, University of Wisconsin, Madison 53706.
J Virol. 1993 Dec;67(12):7181-9. doi: 10.1128/JVI.67.12.7181-7189.1993.
The positive-strand RNA bromoviruses encode two nonstructural proteins, 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase, and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interaction is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independently of viral replication by using RNA- or DNA-based transient expression, and their in vivo RNA replication activities were tested in protoplasts with BMV and CCMV RNA3 templates. RNA-based transient expression confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a expression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologous combinations exhibited RNA synthesis defects. The combination of CCMV 1a and BMV 2a did not support detectable synthesis of negative-strand, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and subgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis, which was only slightly affected. These results indicate that at least some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA synthesis are differentiated in part by features of such interactions.
正义链RNA雀麦花叶病毒编码两种参与RNA依赖性RNA复制的非结构蛋白，即1a和2a。这些蛋白与其他植物和动物病毒的甲基转移酶、解旋酶和聚合酶蛋白具有广泛的序列相似性。1a和2a在体外也能形成复合物。为了探究1a - 2a相互作用在体内RNA复制过程中是否必需，我们对来自两种不同雀麦花叶病毒，即雀麦花叶病毒（BMV）和豇豆花叶病毒（CCMV）的1a和2a基因进行了重配。通过基于RNA或DNA的瞬时表达，使1a和2a独立于病毒复制进行表达，并在原生质体中使用BMV和CCMV RNA3模板测试它们的体内RNA复制活性。基于RNA的瞬时表达证实了之前的研究结果，即雀麦花叶病毒RNA复制对1a表达降低的敏感性高于对2a表达降低的敏感性。1a和2a同源组合的基于DNA的表达支持高水平的RNA合成，但两种1a - 2a异源组合均表现出RNA合成缺陷。CCMV 1a和BMV 2a的组合不支持检测到负链、正链或亚基因组RNA的合成。BMV 1a和CCMV 2a的相反组合在正链和亚基因组RNA积累方面存在优先缺陷，表明1a - 2a相互作用以不同于负链RNA合成的方式参与这些过程，负链RNA合成仅受到轻微影响。这些结果表明，1a和2a的至少一些功能在体内以相互依赖的方式发挥作用，并且正链和负链RNA合成的机制部分由这种相互作用的特征区分。
