Kaposi’s sarcoma-associated herpesvirus ORF57 protein protects viral transcripts from specific nuclear RNA decay pathways by preventing hMTR4 recruitment

Nuclear RNAs are subject to a number of RNA decay pathways that serve quality control and regulatory functions. As a result, any virus that expresses its genes in the nucleus must have evolved mechanisms that avoid these pathways, but the how viruses evade nuclear RNA decay remains largely unknown. The multifunctional Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 (Mta) protein is required for the nuclear stability of viral transcripts. In the absence of ORF57, we show that viral transcripts are subject to degradation by two specific nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) in which decay factors are recruited through poly(A) tails, and an ARS2-mediated RNA decay pathway dependent on the 5ʹ RNA cap. In transcription pulse chase assays, ORF57 appears to act primarily by inhibiting the ARS2-mediated RNA decay pathway. In the context of viral infection in cultured cells, inactivation of both decay pathways by RNAi is necessary for the restoration of ORF57-dependent viral genes produced from an ORF57-null bacmid. Mechanistically, we demonstrate that ORF57 protects viral transcripts by preventing the recruitment of the exosome co-factor hMTR4. In addition, our data suggest that ORF57 recruitment of ALYREF inhibits hMTR4 association with some viral RNAs, whereas other KSHV transcripts are stabilized by ORF57 in an ALYREF-independent fashion. In conclusion, our studies show that KSHV RNAs are subject to nuclear degradation by two specific host pathways, PPD and ARS2-mediated decay, and ORF57 protects viral transcripts from decay by inhibiting hMTR4 recruitment.

细胞核内RNA存在多种兼具质量控制与调控功能的RNA降解通路。据此，任何在细胞核内表达基因的病毒都必须演化出规避这些降解通路的机制，但目前学界对病毒如何逃逸细胞核内RNA降解的认知仍较为有限。多功能的卡波西肉瘤相关疱疹病毒（Kaposi’s sarcoma-associated herpesvirus, KSHV）开放阅读框57（ORF57，又称Mta）蛋白，对病毒转录本的核稳定性至关重要。研究发现，在缺失ORF57的情况下，病毒转录本会通过两种特异性核RNA降解通路被降解：其一为多聚腺苷酸结合核蛋白1（PABPN1）与PAPα/γ介导的RNA降解通路（PPD），该通路的降解因子通过多聚腺苷酸尾完成招募；其二为依赖5'端RNA帽结构的ARS2介导的RNA降解通路。在转录脉冲追踪实验中，ORF57主要通过抑制ARS2介导的RNA降解通路发挥功能。在培养细胞的病毒感染模型中，仅当通过RNA干扰同时灭活这两种降解通路时，才能恢复由ORF57缺失型杆状病毒穿梭载体（bacmid）所表达的、依赖ORF57的病毒基因产物。从机制层面而言，本研究证实ORF57可通过阻止外切体辅助因子hMTR4的招募，从而保护病毒转录本免受降解。此外，研究数据表明，ORF57招募ALYREF可抑制hMTR4与部分卡波西肉瘤相关疱疹病毒RNA的结合，而其余病毒转录本则通过不依赖ALYREF的途径被ORF57稳定。综上，本研究表明卡波西肉瘤相关疱疹病毒的RNA可通过两种特异性宿主通路发生核降解，即PPD通路与ARS2介导的降解通路；而ORF57通过抑制hMTR4的招募，保护病毒转录本免受降解。