Okolovitch Evan M, Govindarajan Vishnu, Robles-Sikisaka Refugio, Campagnola Grace, Kempf Brian J, Routh Andrew L, Peersen Olve B, Barton David J
Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA.
J Virol. 2025 Jun 24:e0043425. doi: 10.1128/jvi.00434-25.
Enteroviruses frequently recombine with one another in nature; however, it is unclear how viral replication machinery can distinguish between related and unrelated partners during recombination. We hypothesize that viral RNA recombination involves two parental RNA templates, nascent RNA products, and their dynamic interactions with the viral polymerase-a sexual replication strategy. When nascent RNA products move from one parental RNA template to another, RNA sequence similarity may be an important factor underpinning the mechanism and efficiency of recombination. To test this hypothesis, we focused on recombination between two related group C enteroviruses, poliovirus and Coxsackievirus A21 (CVA21), using bioinformatic, biological, and biochemical approaches. Bioinformatic analyses comparing 22 prototypical group C enteroviruses delineated four recombination groups where viruses in each group exhibit high RNA sequence and amino acid similarity in their polymerase genes. ClickSeq and ViReMa methods detect recombinant forms of poliovirus with P3 genes from CVA21, analogous to recombinant circulating vaccine-derived polioviruses (cVDPV). Biochemical assays show that poliovirus and CVA21 polymerases can detect mismatched base pairs as they traverse an extended primer grip surface adjacent to the active site. Mismatched base pairs in the -2 and -3 positions destabilize polymerase elongation complexes, consistent with the predicted role of RNA sequence similarity in recombination. Two subgroup-specific genetic elements, upstream open-reading frames (uORFs) and RNase L competitive inhibitor RNAs (RNase L ciRNAs), reinforce the existence and biological relevance of enterovirus C recombination groups. Altogether, our observations suggest that enterovirus RNA replication machinery can distinguish between related and unrelated partners during recombination.
Viral RNA recombination transforms live-attenuated polioviruses into neurovirulent circulating vaccine-derived polioviruses, complicating the planned eradication of poliovirus. When humans are co-infected with poliovirus and related non-polio enteroviruses, viral replication machinery can produce recombinant viruses. However, who recombines with whom? What factors determine whether two distinct viruses can produce recombinant progeny that are fit for transmission from person to person? In this study, we clarify which viruses recombine with one another in nature and further elucidate the mechanisms by which the viral polymerase distinguishes between related and unrelated RNA templates-a sexual form of replication. Understanding these mechanisms could lead to better strategies for virus control and/or eradication.
肠道病毒在自然界中经常相互重组；然而，尚不清楚病毒复制机制在重组过程中如何区分相关和不相关的伙伴。我们假设病毒RNA重组涉及两个亲本RNA模板、新生RNA产物以及它们与病毒聚合酶的动态相互作用——一种有性复制策略。当新生RNA产物从一个亲本RNA模板转移到另一个模板时，RNA序列相似性可能是支撑重组机制和效率的一个重要因素。为了验证这一假设，我们使用生物信息学、生物学和生化方法，重点研究了两种相关的C组肠道病毒——脊髓灰质炎病毒和柯萨奇病毒A21（CVA21）之间的重组。生物信息学分析比较了22种典型的C组肠道病毒，划分出四个重组组，每组中的病毒在其聚合酶基因中表现出高度的RNA序列和氨基酸相似性。ClickSeq和ViReMa方法检测到带有来自CVA21的P3基因的脊髓灰质炎病毒重组形式，类似于重组循环疫苗衍生脊髓灰质炎病毒（cVDPV）。生化分析表明，脊髓灰质炎病毒和CVA21聚合酶在穿过与活性位点相邻的延伸引物握持表面时能够检测到错配碱基对。-2和-3位置的错配碱基对会使聚合酶延伸复合物不稳定，这与RNA序列相似性在重组中的预测作用一致。两个亚组特异性遗传元件，上游开放阅读框（uORF）和核糖核酸酶L竞争性抑制剂RNA（核糖核酸酶L ciRNA），强化了C组肠道病毒重组组的存在及其生物学相关性。总之，我们的观察结果表明，肠道病毒RNA复制机制在重组过程中能够区分相关和不相关的伙伴。
病毒RNA重组将减毒活脊髓灰质炎病毒转变为具有神经毒性的循环疫苗衍生脊髓灰质炎病毒，使脊髓灰质炎病毒的计划根除复杂化。当人类同时感染脊髓灰质炎病毒和相关的非脊髓灰质炎肠道病毒时，病毒复制机制可产生重组病毒。然而，谁与谁重组？哪些因素决定两种不同的病毒能否产生适合人际传播的重组后代？在本研究中，我们阐明了自然界中哪些病毒相互重组，并进一步阐明了病毒聚合酶区分相关和不相关RNA模板的机制——一种有性复制形式。了解这些机制可能会带来更好的病毒控制和/或根除策略。
