Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit.

胞囊噬菌体科（Cystoviridae）包含若干拥有双链RNA（double-stranded RNA，dsRNA）基因组的噬菌体。我们此前已纯化得到该科成员之一——噬菌体φ6的催化性聚合酶亚基（Pol），并证实该蛋白可在体外催化RNA合成。该反应中，噬菌体特异性RNA与异源RNA均可充当模板，但带有φ6负链3′末端序列的模板更受青睐。这为“体内仅能由φ6的dsRNA片段转录产生正链而非负链”这一观测结果提供了分子基础。为验证此类调控机制是否也存在于其他dsRNA病毒中，我们纯化得到了与φ6亲缘关系较近的噬菌体φ8和φ13的重组聚合酶亚基，并在体外对它们的聚合酶活性进行了检测。这些酶均可利用单链RNA（single-stranded RNA，ssRNA）与双链RNA（dsRNA）作为模板，催化依赖模板的RNA合成。但它们彼此之间，以及与φ6的聚合酶之间，在部分生化特性上存在差异。值得注意的是，每种聚合酶均对带有病毒特异性负链短3′末端序列的单链RNA展现出独特的偏好性。这表明，除其他因素外，胞囊噬菌体科的RNA转录过程可通过聚合酶亚基的模板特异性进行调控。