A Viral Nuclear Noncoding RNA Binds Re-localized Poly(A) Binding Protein and Is Required for Late KSHV Gene Expression

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

在感染裂解阶段，γ疱疹病毒卡波西肉瘤相关疱疹病毒（Kaposi's Sarcoma-Associated Herpesvirus, KSHV）会表达一种丰度极高、长度为1.1 kb的功能未知的核非编码RNA。我们观测到，这种聚腺苷酸化核（polyadenylated nuclear, PAN）RNA会与宿主聚腺苷酸结合蛋白C1（poly(A)-binding protein C1, PABPC1）紧密结合；该蛋白通常定位于细胞质，通过结合mRNA的聚腺苷酸尾调控mRNA的稳定性与翻译效率。在KSHV感染的裂解阶段，病毒关闭核酸外切酶（viral shutoff exonuclease, SOX）的表达会导致PABPC1重新定位至细胞核；同时SOX还介导了宿主关闭效应：在此过程中宿主mRNA被下调，而病毒mRNA得以选择性表达。我们的研究显示，尽管PAN RNA对于宿主关闭效应或PABPC1的核定位重分布并非必需，但在瞬时转染实验中，SOX可显著上调PAN RNA的表达水平。这种上调作用会被两种方式阻断：一是使宿主关闭效应和PABPC1核重定位功能丧失的SOX突变体，二是移除PAN RNA的聚腺苷酸尾。在被诱导进入裂解期的KSHV感染细胞中，利用核糖核酸酶H（RNase H）靶向反义寡核苷酸敲低PAN RNA后发现，该RNA对于带聚腺苷酸尾的晚期病毒mRNA的蛋白质合成是必需的。本研究拓展了长链非编码RNA的功能范畴，并揭示了核非编码RNA在γ疱疹病毒感染中的作用机制。