miRNA-based regulation of Coxsackievirus B3 replication and pathogenesis

Abstract

Coxsackievirus B3 (CVB3) is a single-stranded, positive-sense RNA virus known to cause acute myocarditis, pancreatitis, and aseptic meningitis. In many cases, the long-term impact of CVB3 infection-induced cell death causes dilated cardiomyopathy (DCM) and type I diabetes. In fact, CVB3 is one of the main viruses that cause viral myocarditis. CVB3 genome is approximately 7.4 kb single-stranded uncapped RNA with the open reading frame (ORF) flanked by the 5' and 3' untranslated regions (UTRs). The 5'UTR contains a cloverleaf region, essential for the virus replication, and an Internal Ribosome Entry Site (IRES) element for its translation. Upon encountering the host cell, the virus enters it through receptor-mediated endocytosis and releases its genome into the cytoplasm. The genome undergoes the initial rounds of IRES-mediated translation and starts synthesizing viral proteins. These viral proteins hijack the host translation machinery for its benefit. The viral protease 2A cleaves translation initiation factor eIF4GI such that all cap-dependent translations are inhibited in the cell; however, cap-independent translations can occur. The non-structural viral proteins are further utilized by the CVB3 RNA for its replication to form the negative strand of the viral RNA, which is further utilized to produce more positive-strand RNAs. These positive-strand RNAs are then packaged in the viral structural proteins to form complete virion particles, which are released by cell lysis. From CVB3 RNA release into the cytoplasm to virus release after the infection cycle, CVB3 requires host factors at various steps. Our laboratory has extensively studied host proteins such as La, PTB, PSF, hnRNP C, DAP5, and HuR, which regulate the CVB3 RNA translation and replication. In addition to host proteins, host non-coding RNAs also impact virus infection in various ways. In this study, we were interested in exploring how miRNAs might impact CVB3 infection. microRNAs are 22-nucleotide long non-coding RNAs that bind to their target mRNAs, leading to their degradation and/or translation inhibition. Here, we wanted to elucidate their role during CVB3 infection at a molecular level and in disease progression. To study the role of microRNA-22 during Coxsackievirus B3 infection In the literature, several miRNAs have been reported that regulate CVB3 infection through the modulation of their host targets. However, the CVB3 genome, being an RNA, can also be targeted by the miRNAs. We were particularly interested in miRNAs that could bind to the UTR regions of CVB3 RNA and regulate its translation. To start with, we shortlisted miRNAs with a potential binding site in the CVB3 genomic RNA using the online software ViTa. We shortlisted 14 miRNAs that have a high probability of binding to CVB3 UTRs. We found one miRNA, miR-22, whose binding site was predicted in a crucial region in the 5'UTR between stem-loop V and VI. There are many ITAFs that bind in this region, and also, it is near the Shine-Dalgarno-like sequence where ribosome binds to start scanning and initiate translation. So, we picked miR-22 for further investigation. First of all, since it was a predicted binding site, we confirmed the binding of miR-22 on the 5'UTR using mutational analysis and pull down assays. This binding negatively regulated the translation of CVB3 RNA. Moreover, cells from which miR-22 was knocked out showed a higher level of CVB3 infection than the wild type. Since the binding site of miR-22 overlapped several ITAFs, we investigated their crosstalk with miR-22. We found that while miR-22 binding had no effect on the binding of another ITAF (HuR), which binds to stem-loop I, it specifically inhibited the binding of several ITAFs (La, PSF, and PTB) on viral mRNA. This altered the spatial structure necessary for ribosome recruitment on the CVB3 RNA, ultimately inhibiting its translation. However, viral RNA translation is an essential event in establishing infection. So, we were interested in checking the dynamics of miR-22 over the time of CVB3 infection. We found that miR-22 levels were upregulated post 4 hours of infection, allowing enough time to synthesize viral proteins. This increase was found to be induced by viral protease 2A, possibly to benefit the virus by balancing the translation and replication of the CVB3 genomic RNA. Along with the direct effect on viral RNA, the altered level of miR-22 could also affect the level of its cellular targets, which might contribute to CVB3 infection. We obtained a list of miR-22 targets to identify the possible players and performed pathway analysis. Several targets were shortlisted among the top hits, and their levels were checked upon CVB3 infection. As miR-22 level increases post-infection, we shortlisted four targets whose levels decreased, showing a reciprocal correlation. To further investigate whether miR-22 regulated their levels, we checked them in the miR-22 knockout cell line. We found that two target mRNAs (PCDH1 and GNB4) out of four had higher levels in the knockout cells as compared to wild-type cells. However, upon infection in the miR-22 knockout cell line, GNB4 levels were again downregulated, indicating that other mechanisms could also be involved in regulating GNB4 levels during CVB3 infection. The levels of PCDH1, i.e., Protocadherin-1, showed the expected trend. Upon infection in miR-22 knockout cells, PCDH-1 levels increased, indicating that miR-22 was keeping it under control during CVB3 infection. PCDH1 is a single-pass transmembrane protein present in the cell-cell junctions. While it is a relatively new protein, there is a report of its involvement in inducing the NF-κB signaling in the case of pancreatic cancer and another report suggesting that it helps in the entry of New World hantaviruses into the host cells. To further explore the role of PCDH1 during CVB3 infection, PCDH1 expression was partially silenced in miR-22 KO cells and checked its impact on CVB3 infection. We found that partial silencing of PCDH1 inhibited CVB3 infection at a later phase, indicating that PCDH1 might be helping the virus during infection. We concluded that while CVB3 increases the level of miR-22 for its own benefit, the increased miR-22, in turn, helps the host by acting as an antiviral molecule by keeping the pro-viral molecule, PCDH1, under control. Dysregulation of miRNAs and its possible role in CVB3-induced cardiac pathogenesis CVB3 is one of the major viral pathogens responsible for causing virus-induced myocarditis that further leads to dilated cardiomyopathy (DCM), an end-stage heart disease. Many other factors could cause myocarditis, and symptomatic treatment is given for it; hence, the underlying cause mostly remains undetected. Consequently, not many studies decipher the molecular mechanism by which CVB3 infection eventually causes DCM. We were interested in the miRNAs, which are dysregulated during CVB3 infection and might play a role in cardiac pathology. So, we performed small RNA sequencing from CVB3-infected mice heart tissue. From available literature and our sequencing data, we shortlisted approximately 50 dysregulated miRNAs and performed network analysis of these miRNAs. Further, we wanted to correlate our findings with human samples. However, we couldn’t manage to get CVB3- infected human heart samples. So, we collected tissue from hearts having DCM (mimicking CVB3-induced pathogenesis) and performed small RNA sequencing. We found 150 dysregulated miRNAs in DCM heart tissues and again performed network analysis. We made two lists of the shared genes targeted by human and mouse's upregulated and downregulated miRNAs. We got 4093 target genes of upregulated miRNAs and 2309 target genes of downregulated miRNAs. To further shortlist, we wanted to check the level of these genes in DCM condition and pick the genes that showed inverse correlation. For the same, we performed total RNA sequencing from the DCM hearts and found 384 genes out of 4093 were downregulated. Similarly, out of 2309, 120 genes were upregulated, showing that these genes might be regulated by the changing levels of miRNAs during CVB3-induced cardiac pathogenesis. When we performed pathway analysis for these genes, we found that many of the pathways are similar to studies on dilated cardiomyopathy. However, some pathways which came up in our study were underexplored with respect to it. Also, two of the pathways, Ferroptosis and Hippo signaling pathways, showed opposite trends in our study in contrast to available literature, implying that these pathways might be specific to CVB-induced pathogenesis. Further validations and detailed study will give us a proper insight into the molecular mechanism that creates the basis for CVB3-induced DCM. Taken together, we have deciphered the direct and indirect role of microRNAs during CVB3 infection by binding to CVB3 genomic RNA and by modulating host factors involved in various pathways, leading to disease progression.