| dc.description.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. | en_US |
