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Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization.

ABSTRACT: Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable cis-acting element named 5'-UAR-flanking stem (UFS) in the flavivirus genomic 5' terminus. We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses. Interestingly, stabilization of the DENV UFS impaired both genome cyclization and vRNA replication. Moreover, the UFS unwound in response to genome cyclization, leading to the decreased affinity of NS5 for the viral 5' end. Thus, we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication. This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment, highlighting the presence of switch-like mechanisms among RNA viruses.

SUBMITTER: Liu ZY

PROVIDER: S-EPMC5101012 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization.

Liu Zhong-Yu ZY Li Xiao-Feng XF Jiang Tao T Deng Yong-Qiang YQ Ye Qing Q Zhao Hui H Yu Jiu-Yang JY Qin Cheng-Feng CF

eLife 20161001

Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable <i>cis</i>-acting element named 5'-UAR-flanking stem (UFS) in the flavivirus genomic 5' terminus. We demonstrated that the UFS was critical for efficient NS5 recruitment and viral ...[more]

PMID: 27692070

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Project description:In previous studies, we showed that the cellular RNA-binding protein AUF1 supports the replication process of the flavivirus West Nile virus. Here we demonstrate that the protein also enables effective proliferation of dengue virus and Zika virus, indicating that AUF1 is a general flavivirus host factor. Further studies demonstrated that the AUF1 isoform p45 significantly stimulates the initiation of viral RNA replication and that the protein's RNA chaperone activity enhances the interactions of the viral 5'UAR and 3'UAR genome cyclization sequences. Most interestingly, we observed that AUF1 p45 destabilizes not only the 3'-terminal stem-loop (3'SL) but also 5'-terminal stem-loop B (SLB) of the viral genome. RNA structure analyses revealed that AUF1 p45 increases the accessibility of defined nucleotides within the 3'SL and SLB and, in this way, exposes both UAR cyclization elements. Conversely, AUF1 p45 does not modulate the fold of stem-loop A (SLA) at the immediate genomic 5' end, which is proposed to function as a promoter of the viral RNA-dependent RNA polymerase (RdRp). These findings suggest that AUF1 p45, by destabilizing specific stem-loop structures within the 5' and 3' ends of the flaviviral genome, assists genome cyclization and concurrently enables the RdRp to initiate RNA synthesis. Our study thus highlights the role of a cellular RNA-binding protein inducing a flaviviral RNA switch that is crucial for viral replication.IMPORTANCE The genus Flavivirus within the Flaviviridae family includes important human pathogens, such as dengue, West Nile, and Zika viruses. The initiation of replication of the flaviviral RNA genome requires a transformation from a linear to a cyclized form. This involves considerable structural reorganization of several RNA motifs at the genomic 5' and 3' ends. Specifically, it needs a melting of stem structures to expose complementary 5' and 3' cyclization elements to enable their annealing during cyclization. Here we show that a cellular RNA chaperone, AUF1 p45, which supports the replication of all three aforementioned flaviviruses, specifically rearranges stem structures at both ends of the viral genome and in this way permits 5'-3' interactions of cyclization elements. Thus, AUF1 p45 triggers the RNA switch in the flaviviral genome that is crucial for viral replication. These findings represent an important example of how cellular (host) factors promote the propagation of RNA viruses.

Project description:A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

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Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization.

Publications

Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization.

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