Simultaneous and systematic analysis of cellular and viral gene expression during Enterovirus 71-induced host shutoff
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Enteroviruses, including poliovirus, enterovirus 71 (EV71), enterovirus 68, coxsackievirus A16, cause millions of infections every year. The infection can lead to serious human diseases and is a significant public health problem. Among them, EV71 is an emerging pathogen that causes severe hand, foot and mouth disease (HFMD) and neurological disease, especially in young children. Currently, there is no effective treatment to EV71-caused diseases, partially blaming to a lack of understanding EV71 replication mechanism (Solomon et al., 2010). As a member of Picornaviridae, EV71 infection induces a rapid induction of host shutoff, which is marked by the inhibition of cellular protein synthesis (Holland, 1963). In the meantime, viral protein synthesis takes over the cellular translational machinery. It is believed that the cellular protein synthesis shutoff benefits viral replication by relocating cellular resources and facilitating viral escape from host cell immune responses (Cao et al., 2017). Both transcription and translation inhibition have been suggested to contribute to the host protein synthesis shutoff during picornavirus infection (Holland, 1963; Belsham, 2009). However, the relative role of transcription and translation inhibition in virus-induced host shutoff is yet elusive. Similarly, the relative role of viral mRNA production and translation in viral protein synthesis advantage is not completely clear.
Past studies on piconavirus-induced host shutoff mainly took a reductionist approach that could not systematically and simultaneously quantitate transcription and translation. Here, we employed RNA-sequencing (RNA-Seq) and ribosome profiling, two high-throughput techniques that can simultaneously access both viral and cellular gene expression in virus-infected cells (Ingolia et al., 2011), to systematically view EV71-induced host shutoff. Total RNA from RNA-Seq indicates RNA synthesis activity, while the ribosome protected fragments (RPF) serve as the indicator of active translation. In addition, the two techniques can simultaneously access both viral and cellular gene expression in virus-infected cells. We have previously examined viral and host cell transcription and translation using these techniques in vaccinia virus infected cells (Yang et al., 2015; Dai et al., 2017). We intended to choose time points that could cover active translation of viral RNA after EV71 infection. We have determined that the active EV71 gene expression was between 3 h and 7 h post infection (hpi) using rhabdomyosarcoma (RD) cells at a multiplicity of infection (MOI) of 40 through preliminary experiments (data not shown). The use of a high MOI ensured that all cells were synchronically infected based on our preliminary experiments. Based on the results, we decided to take the snapshots of gene expression in EV71-infected and mock-infected cells at 3.5 hpi and 6.25 hpi. We carried out experiments with two independent biological replicates at each time point. In one of the experiments (Set 1), we also chose an earlier time point, 1.75 hpi, to view the early event. Total mRNA and ribosome protected RNA from EV71-infected cells and mock-infected cells were sequenced. We obtained an average of 35.3-million reads from individual RNA-Seq samples and an average of 19.4-million RPF reads from individual ribosome profiling samples that were mapped to human and EV71 genomes. The high quality of ribosome profiling reads was evidenced by high mapping rates of cellular RPF reads in coding regions (CDSs) and 5′-UTRs as well as low mapping rates of 3′-UTRs and introns for both mock and infected experiments (Fig. S1).
Many unexpected transcripts and translational products have been identified from various RNA and DNA viruses (Stern-Ginossar et al., 2012; Yang et al., 2015; Irigoyen et al., 2016). However, there is no evidence of additional translational products from either 5′-UTR, 3′-UTR, or negative sense RNA of EV71 in this study, consistent with conclusions from early studies using classical molecular approaches. In addition, it has been suggested that negative sense RNA of a virus (e.g., influenza A virus) may encode a gene (Clifford et al., 2009). Only a very few reads from negative sense EV71 RNA were associated with ribosomes, indicating no translation from EV71 negative sense RNA.
In summary, the present study for the first time simultaneously analyzed viral and host cell mRNA synthesis and translation in picornavirus-infected cells using RNA-Seq and ribosome profiling. The analyses provided a systematic overview on the dynamic change of EV71 and cellular gene expression with high resolution during EV71-induced host shutoff. Host shutoff, marked by a global cellular protein synthesis inhibition, is caused by many virus infections (Walsh and Mohr, 2011). Many of them do so by eliminating host cell mRNA pools available for translation For example, vaccinia virus encodes decapping enzymes that render cellular mRNAs sensitive to degradation and also inhibits host transcription (Parrish et al., 2007). Kaposi’s sarcoma-associated herpesvirus, and influenza A virus also deplete existing or nascent host mRNAs to block host protein synthesis (Glaunsinger and Ganem, 2004; Bercovich-Kinori et al., 2016). Our study indicates that EV71 employs a distinct approach to the aforementioned viruses mainly by a suppression of cellular mRNA translation. Because the majority of the cellular mRNAs are translated through a cap-dependent mode, while the viral mRNA is translated through an IRES-mediated cap-independent mode, the inhibition of cap-dependent translation and the activation of cap-independent translation suppressed cellular mRNA translation. Our analyses also showed that viral protein is selectively synthesized in EV71-infected cells both by employing a translational advantage and by producing a large amount of viral mRNA during EV71-induced host shutoff. At the relatively earlier time of viral infection (3.5 hpi in this study) when the viral RNA amount is not high enough, EV71 RNA is translated at a higher efficiency than most of the cellular mRNAs. At a later time of infection (6.25 hpi in this study), EV71 RNA translation efficiency decreased compared to that at 3.5 hpi. However, because of the rapid increase in viral RNA, viral proteins keep accumulating. EV71’s employment of both translational advantage and large RNA production at different stage of infection differentiates it from many other viruses. This study has broad implications in understating picornavirus-induced host shutoff. It is highly likely that other picornaviruses employ the same strategy to boost their replication. An in-depth understanding of this process following the direction pointed by this study will facilitate the development of novel anti-viral strategies by targeting EV71-induced host shutoff to reduce the public health and economic burden caused by EV71 and other enterovirus pandemic.
This work was supported by the National Key Basic Research Program of China (2013CB911104 to Juan Tan); the National Institutes of Health (AI128406 to Zhilong Yang); the National Natural Science Foundation of China (31471232 to Zhi Xie); Science and Technology Planning Projects of Guangdong Province (2014B030301040 to Zhi Xie); Joint Research Fund for Overseas Natural Science of China (3030901001222 to Zhi Xie); Major Program of Science and Technology of Guangzhou (201607020001 to Zhi Xie); and the short term exchange programs for doctoral tutor from the China Scholarship Council (CSC (2015)3039 to Wentao Qiao). We thank Professor Zhiyong Lou (Tsinghua University, China) for his generous gifts of the EV71 infectious clones.
Yonqguan Lin carried out experiments and analyzed data. Yan Wang analyzed data. Hui Li and Yuhang Chen carried out experiments. Zhilong Yang, Juan Tan and Zhi Xie conceived experiments and analyzed data. Yonqguan Lin, Zhilong Yang and Wentao Qiao wrote the paper. All authors gave final approval of the submitted and published versions.
Yonqguan Lin, Yan Wang, Hui Li, Yuhang Chen, Wentao Qiao, Zhi Xie, Juan Tan and Zhilong Yang declare that they have no conflicts of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.
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