Kempf Brian J, Peersen Olve B, Barton David J
Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
Department of Biochemistry, Colorado State University, Fort Collins, Colorado, USA.
J Virol. 2016 Sep 12;90(19):8410-21. doi: 10.1128/JVI.00078-16. Print 2016 Oct 1.
RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3D(pol), as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3D(pol) may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3D(pol)-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3D(pol) decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3D(pol) increased the frequency of recombination. The 3D(pol) Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3D(pol) Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance.
Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.
RNA重组在小RNA病毒种属群的形成以及种属群内病毒的持续进化中起着重要作用。在本研究中，我们研究了脊髓灰质炎病毒聚合酶3D(pol)与RNA重组相关的结构和功能。当新生RNA产物在RNA复制过程中用一个病毒RNA模板替换另一个模板时，就会发生重组。由于重组是小RNA病毒复制的一个自然方面，我们推测3D(pol)的某些特征可能部分存在以促进RNA重组。此外，我们推断破坏聚合酶延伸复合物内3D(pol)-RNA相互作用的丙氨酸替代突变可能会增加和/或降低重组的幅度。我们发现3D(pol)中的L420A突变降低了RNA重组的频率，而3D(pol)中其他位点的丙氨酸替代增加了重组的频率。3D(pol)的亮氨酸420侧链与聚合酶活性位点3个核苷酸处新生RNA产物中的一个核糖相互作用。值得注意的是，降低重组的L420A突变也使病毒更容易受到利巴韦林的抑制，这与利巴韦林诱导的病毒RNA中G→A和C→U突变的积累一致。我们得出结论，3D(pol)亮氨酸420对RNA重组至关重要，并且RNA重组有助于利巴韦林抗性。
重组有助于小RNA病毒种属群的形成以及循环疫苗衍生脊髓灰质炎病毒(cVDPV)的出现。自然界中出现的重组病毒偶尔比任何一个亲本毒株更具适应性，特别是当重组中的两个伙伴密切相关时，即特征种属群的成员，如肠道病毒A至H种属群或鼻病毒A至C种属群。我们的研究表明RNA重组需要病毒聚合酶的保守特征。此外，使重组失活的聚合酶突变使病毒更容易受到抗病毒药物利巴韦林的影响，这表明重组有助于利巴韦林抗性。阐明RNA复制和重组的分子机制可能有助于人类实现并维持脊髓灰质炎病毒的根除。
