MicroRNA-22-3p displaces critical host factors from the 5' UTR and inhibits the translation of Coxsackievirus B3 RNA.

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication.

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.

Helical Domain Changes between hGBP3 and hGBP3ΔC Result in Distinct Oligomers and Anti-HCV Activity.

Human guanylate binding proteins (hGBPs), which are large GTPases, are crucial for cell-autonomous immunity, including antiviral activity. hGBPs contain two domains: an N-terminal catalytic domain and a C-terminal helical domain. hGBP3 and its splice variant hGBP3ΔC have been shown to possess anti-influenza activity in lung epithelial cells. These two proteins have identical catalytic domains but different helical domains. It is unclear whether this difference affects GTPase activity or protein oligomerization. Using combined approaches, we show that both proteins hydrolyze GTP to GDP and further to GMP. However, they form different oligomers. hGBP3 exists as a hexamer in the free form, whereas hGBP3ΔC forms large oligomers, indicating that helical domain modifications of the splice variant result in distinct oligomers. Furthermore, unlike other homologues, neither protein changes its oligomeric state upon substrate binding or hydrolysis. Deleting the helical domain of hGBP3 (hGBP31-309) yields a monomer, suggesting that the helical domain promotes the hexamerization of hGBP3. We overexpressed hGBP3 and hGBP3ΔC to test their efficacy against HCV growth and found that hGBP3 inhibits HCV multiplication, while the splice variant has little effect. Our mutational studies on hGBP3 show that substrate hydrolysis, rather than substrate binding, is required for inhibiting HCV growth. This suggests that substrate hydrolysis generates a protein conformation essential for anti-HCV activity. Additionally, truncated hGBP31-309 does not exhibit anti-HCV activity. Altogether, these findings suggest that the helical domain of hGBP3 is crucial for reducing HCV growth through hexamer formation and that its variations result in different oligomers and antiviral activities.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

Exosome-associated microRNA-375 induces cell proliferation by regulating IGFBP4 upon hepatitis C virus infection.

Hepatitis C virus (HCV) infection is one of the most common causes of liver cancer. HCV infection causes chronic disease followed by cirrhosis, which often leads to hepatocellular carcinoma (HCC). In this study, we investigated the roles of exosome-associated miRNAs in HCV-induced disease pathology. Small RNA sequencing was performed to identify miRNAs that are differentially regulated in exosomes isolated from patient sera at two different stages of HCV infection: cirrhosis and hepatocellular carcinoma. Among the differentially expressed miRNAs, miR-375 was found to be significantly upregulated in exosomes isolated from patients with cirrhosis and HCC. A similar upregulation was observed in intracellular and extracellular/exosomal levels of miR-375 in HCV-JFH1 infected Huh7.5 cells. The depletion of miR-375 in infected cells inhibited HCV-induced cell migration and proliferation, suggesting a supportive role for miR-375 in HCV pathogenesis. miR-375, secreted through exosomes derived from HCV-infected cells, could also be transferred to naïve Huh7.5 cells, resulting in an increase in cell proliferation and migration in the recipient cells. Furthermore, we identified Insulin growth factor binding protein 4 (IGFBP4), a gene involved in cell growth and malignancy, as a novel target of miR-375. Our results demonstrate the critical involvement of exosome-associated miR-375 in HCV-induced disease progression.

Emergence of a unique SARS-CoV-2 Delta sub-cluster harboring a constellation of co-appearing non-Spike mutations.

Accumulation of diverse mutations across the structural and nonstructural genes is leading to rapid evolution of SARS-CoV-2, altering its pathogenicity. We performed whole genome sequencing of 239 SARS-CoV-2 RNA samples collected from both adult and pediatric patients across eastern India (West Bengal), during the second pandemic wave in India (April-May 2021). In addition to several common spike mutations within the Delta variant, a unique constellation of eight co-appearing non-Spike mutations was identified, which revealed a high degree of positive mutual correlation. Our results also demonstrated the dynamics of SARS-CoV-2 variants among unvaccinated pediatric patients. 41.4% of our studied Delta strains harbored this signature set of eight co-appearing non-Spike mutations and phylogenetically out-clustered other Delta sub-lineages like 21J, 21A, or 21I. This is the first report from eastern India that portrayed a landscape of co-appearing mutations in the non-Spike proteins, which might have led to the evolution of a distinct Delta subcluster. Accumulation of such mutations in SARS-CoV-2 may lead to the emergence of "vaccine-evading variants." Hence, monitoring of such non-Spike mutations will be significant in the formulation of any future vaccines against those SARS-CoV-2 variants that might evade the current vaccine-induced immunity, among both the pediatric and adult populations.

Association of exosomal miR-96-5p and miR-146a-5p with the disease severity in dengue virus infection.

Exosomes are small extracellular vesicles secreted by cells and have a major role in cell-to-cell signaling. As dengue infection progresses from a mild to a severe form of infection, the exosome's microRNA (miRNA) composition might change, which may contribute to pathogenesis. In this study, a comprehensive analysis of serum exosomal miRNAs was performed and their involvement in dengue virus-induced disease progression in an Indian cohort was assessed. Small RNA-seq showed 50 differentially expressed exosomal miRNAs that were significantly dysregulated during dengue infection. After extensive validation, miR-96-5p was found to be significantly upregulated, whereas miR-146a-5p was significantly downregulated with the progression of disease to severe form. Interestingly, a strong positive correlation was found between the expression levels of miR-96-5p and miR-146a-5p and the platelet levels of the patients. Further, study of miR-146a-5p showed that it regulates the expression of the proteins which are involved in the immune responses. These results suggest that miR-96-5p and miR-146a-5p could be used as diagnostic and prognostic markers for dengue disease progression, in addition to the already available biochemical and pathological parameters.

LncRNA NEAT1 regulates HCV-induced Hepatocellular carcinoma by modulating the miR-9-BGH3 axis.

Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver diseases, such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Several cellular entities, including paraspeckles and their related components, are involved in viral pathogenesis and cancer progression. NEAT1 lncRNA is a major component of paraspeckles that has been linked to several malignancies. In this study, analysis of the Cancer Genome Atlas (TCGA) database and validation in HCV-induced HCC tissue and serum samples showed significantly high expression of NEAT1 in patients with liver cancer. Moreover, we found that NEAT1 levels increased upon HCV infection. To further understand the mechanism of NEAT1-induced HCC progression, we selected one of its targets, miR-9-5 p, which regulates BGH3 mRNA levels. Interestingly, miR-9-5 p levels were downregulated upon HCV infection, whereas BGH3 levels were upregulated. Additionally, partial NEAT1 knockdown increased miR-9-5 p levels and decreased BGH3 levels, corroborating our initial results. BGH3 levels were also upregulated in HCV-induced HCC and TCGA tissue samples, which could be directly correlated with NEAT1 levels. As a known oncogene, BGH3 is directly linked to HCC progression mediated by NEAT1. We also found that NEAT1 levels remained upregulated in serum samples from patients treated with direct-acting antivirals (DAA), indicating that NEAT1 might be a molecular trigger that promotes HCC development. Collectively, these findings provide molecular insights into HCV-induced HCC progression via the NEAT1-miR-9-BGH3 axis.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3).

The 5'UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5'UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

Responsive polymer-assisted 3D cryogel supports Huh7.5 as in vitro hepatitis C virus model and ectopic human hepatic tissue in athymic mice.

The three-dimensional (3D) cell culture models serve as the interface between conventional two-dimensional (2D) monolayer culture and animal models. 3D culture offers the best possible model system to understand the pathophysiology of human pathogens such as hepatitis C virus (HCV), which lacks a small animal model, due to narrow host tropism and non-permissiveness of murine hepatocytes. In this study, functionally robust spheroids of HCV permissive Huh7.5 cells were generated, assisted by the temperature or pH-responsive polymers PNIPAAm and Eudragit respectively, followed by the long-term growth of the multilayered 3D aggregates in poly(ethylene glycol) (PEG)-alginate-gelatin (PAG) cryogel. The human serum albumin (HSA), marker of hepatic viability was detected up to 600 ng/ml on 24th day of culture. The 3D spheroid culture exhibited a distinct morphology and transcript levels with the upregulation of hepato-specific transcripts, nuclear factor 4α (HNF4α), transthyretin (TTr), albumin (Alb), phase I and phase II drug-metabolizing genes. The two most important phase I enzymes CYP3A4 and CYP2D6, together responsible for 90% metabolism of drugs exhibited up to 9- and 12-fold increment, respectively in transcripts. The 3D culture was highly permissive to HCV infection and supported higher multiplicity of infection compared to monolayer Huh7.5 culture. Quantitation of high levels of HSA (500-200 ng/ml) in circulation in mice for 32 days asserted integration with host vasculature and in vivo establishment of 3D culture implants as an ectopic human hepatic tissue in mice. The study demonstrates the 3D spheroid Huh7.5 culture as a model for HCV studies and screening potential for anti-HCV drug candidates.

SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA.

During enteroviral infections, the canonical translation factor eukaryotic translation initiation factor 4 γ I (eIF4GI) is cleaved by viral protease 2A. The resulting C-terminal fragment is recruited by the viral internal ribosome entry site (IRES) for efficient translation of the viral RNA. However, the 2A protease is not present in the viral capsid and is synthesized only after the initial round of translation. This presents the conundrum of how the initial round of translation occurs in the absence of the C-terminal eIF4GI fragment. Interestingly, the host protein DAP5 (also known as p97, eIF4GIII, and eIF4G2), an isoform of eIF4GI, closely resembles the eIF4GI C-terminal fragment produced after 2A protease-mediated cleavage. Using the Coxsackievirus B3 (CVB3) IRES as a model system, here we demonstrate that DAP5, but not the full-length eIF4GI, is required for CVB3 IRES activity for translation of input viral RNA. Additionally, we show that DAP5 is specifically required by type I IRES but not by type II or type III IRES, in which cleavage of eIF4GI has not been observed. We observed that both DAP5 and C-terminal eIF4GI interact with CVB3 IRES in the same region, but DAP5 exhibits a lower affinity for CVB3 IRES compared with the C-terminal eIF4GI fragment. It appears that DAP5 is required for the initial round of viral RNA translation by sustaining a basal level of CVB3 IRES activity. This activity leads to expression of 2A protease and consequent robust CVB3 IRES-mediated translation by the C-terminal eIF4GI fragment.

lncRNA HULC facilitates efficient loading of HCV-core protein onto lipid droplets and subsequent virus-particle release.

The cellular lipid pool plays a central role in hepatitis C virus (HCV) life cycle, from establishing infection to virus propagation. Here, we show that a liver abundant long noncoding RNA, highly upregulated in liver carcinoma (HULC), is upregulated during HCV infection and manipulates the lipid pool to favour virus life cycle. Interestingly, HULC was found to be crucial for the increase in number of lipid droplets in infected cells. This effect was attributed to the role of HULC in lipid biogenesis. Further, we demonstrated that HULC knockdown decreases the association of HCV-core protein with lipid droplets. This exhibited a direct consequence on the release of HCV particles. The role of HULC in HCV-particle release was further substantiated by additional knockdown and mutation experiments. Additionally, we found that increased level of HULC in HCV-infected cells was a result of Retinoid X Receptor Alpha (RXRA)-mediated transcription, which seemed to be aided by HCV-core protein. Taken together, the results identify a distinct role of long noncoding RNA HULC in lipid dynamics during HCV infection, which provides new insights into the complex process of HCV propagation and pathogenesis.

Kinesin-dependent mechanism for controlling triglyceride secretion from the liver.

Despite massive fluctuations in its internal triglyceride content, the liver secretes triglyceride under tight homeostatic control. This buffering function is most visible after fasting, when liver triglyceride increases manyfold but circulating serum triglyceride barely fluctuates. How the liver controls triglyceride secretion is unknown, but is fundamentally important for lipid and energy homeostasis in animals. Here we find an unexpected cellular and molecular mechanism behind such control. We show that kinesin motors are recruited to triglyceride-rich lipid droplets (LDs) in the liver by the GTPase ARF1, which is a key activator of lipolysis. This recruitment is activated by an insulin-dependent pathway and therefore responds to fed/fasted states of the animal. In fed state, ARF1 and kinesin appear on LDs, consequently transporting LDs to the periphery of hepatocytes where the smooth endoplasmic reticulum (sER) is present. Because the lipases that catabolize LDs in hepatocytes reside on the sER, LDs can now be catabolized efficiently to provide triglyceride for lipoprotein assembly and secretion from the sER. Upon fasting, insulin is lowered to remove ARF1 and kinesin from LDs, thus down-regulating LD transport and sER-LD contacts. This tempers triglyceride availabiity for very low density lipoprotein assembly and allows homeostatic control of serum triglyceride in a fasted state. We further show that kinesin knockdown inhibits hepatitis-C virus replication in hepatocytes, likely because translated viral proteins are unable to transfer from the ER to LDs.

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication.

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of ß-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.

Strand-specific affinity of host factor hnRNP C1/C2 guides positive to negative-strand ratio in Coxsackievirus B3 infection.

Coxsackievirus B3 is an enterovirus, with positive-sense single-stranded RNA genome containing 'Internal Ribosome Entry Site' (IRES) in the 5'UTR. Once sufficient viral proteins are synthesized in the cell from the input RNA, viral template switches from translation to replication to synthesize negative-strand RNA. Inhibition of translation is a key step in regulating this switch as the positive-strand RNA template should be free of ribosomes to enable polymerase movement. In this study, we show how a host protein hnRNP C1/C2 inhibits viral RNA translation. hnRNP C1/C2 interacts with stem-loop V in the IRES and displaces poly-pyrimidine tract binding protein, a positive regulator of translation. We further demonstrate that hnRNP C1/C2 induces translation to replication switch, independently from the already known role of the ternary complex (PCBP2-3CD-cloverleaf RNA). These results suggest a novel function of hnRNP C1/C2 in template switching of positive-strand from translation to replication by a new mechanism. Using mathematical modelling, we show that the differential affinity of hnRNP C1/C2 for positive and negative-strand RNAs guides the final ± RNA ratio, providing first insight in the regulation of the positive to negative-strand RNA ratio in enteroviruses.

Polypyrimidine tract-binding protein (PTB) and PTB-associated splicing factor in CVB3 infection: an ITAF for an ITAF.

The 5' UTR of Coxsackievirus B3 (CVB3) contains internal ribosome entry site (IRES), which allows cap-independent translation of the viral RNA and a 5'-terminal cloverleaf structure that regulates viral replication, translation and stability. Here, we demonstrate that host protein PSF (PTB associated splicing factor) interacts with the cloverleaf RNA as well as the IRES element. PSF was found to be an important IRES trans acting factor (ITAF) for efficient translation of CVB3 RNA. Interestingly, cytoplasmic abundance of PSF protein increased during CVB3 infection and this is regulated by phosphorylation status at two different amino acid positions. Further, PSF protein was up-regulated in CVB3 infection. The expression of CVB3-2A protease alone could also induce increased PSF protein levels. Furthermore, we observed the presence of an IRES element in the 5'UTR of PSF mRNA, which is activated during CVB3 infection and might contribute to the elevated levels of PSF. It appears that PSF IRES is also positively regulated by PTB, which is known to regulate CVB3 IRES. Taken together, the results suggest for the first time a novel mechanism of regulations of ITAFs during viral infection, where an ITAF undergoes IRES mediated translation, sustaining its protein levels under condition of translation shut-off.

Interplay between PTB and miR-1285 at the p53 3'UTR modulates the levels of p53 and its isoform Δ40p53α.

p53 and its translational isoform Δ40p53 are involved in many important cellular functions like cell cycle, cell proliferation, differentiation and metabolism. Expression of both the isoforms can be regulated at different steps. In this study, we explored the role of 3'UTR in regulating the expression of these two translational isoforms. We report that the trans acting factor, Polypyrimidine Tract Binding protein (PTB), also interacts specifically with 3'UTR of p53 mRNA and positively regulates expression of p53 isoforms. Our results suggest that there is interplay between miRNAs and PTB at the 3'UTR under normal and stress conditions like DNA damage. Interestingly, PTB showed some overlapping binding regions in the p53 3'UTR with miR-1285. In fact, knockdown of miR-1285 as well as expression of p53 3'UTR with mutated miR-1285 binding sites resulted in enhanced association of PTB with the 3'UTR, which provides mechanistic insights of this interplay. Taken together, the results provide a plausible molecular basis of how the interplay between miRNAs and the PTB protein at the 3'UTR can play pivotal role in fine tuning the expression of the two p53 isoforms.

Hepatitis C virus: Enslavement of host factors.

Hepatitis C virus (HCV) has infected over 170 million people world-wide. This infection causes severe liver damage that can progress to hepatocellular carcinoma leading to death of the infected patients. Development of a cell culture model system for the study of HCV infection in the recent past has helped the researchers world-wide to understand the biology of this virus. Studies over the past decade have revealed the tricks played by the virus to sustain itself, for as long as 40 years, in the host setup without being eliminated by the immune system. Today we understand that the host organelles and different cellular proteins are affected during HCV infection. This cytoplasmic virus has all the cellular organelles at its disposal to successfully replicate, from ribosomes and intracellular membranous structures to the nucleus. It modulates these organelles at both the structural and the functional levels. The vast knowledge about the viral genome and viral proteins has also helped in the development of drugs against the virus. Despite the achieved success rate to cure the infected patients, we struggle to eliminate the cases of recurrence and the non-responders. Such cases might emerge owing to the property of the viral genome to accumulate mutations during its succeeding replication cycles which favours its survival. The current situation calls an urgent need for alternate therapeutic strategies to counter this major problem of human health. © 2017 IUBMB Life, 70(1):41-49, 2018.