PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.

Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.

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.

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.

p53 and RNA viruses: The tug of war.

Host factors play essential roles in viral infection, and their interactions with viral proteins are necessary for establishing effective pathogenesis. p53 is a host factor that maintains genomic integrity by controlling cell-cycle progression and cell survival. It is a well-known tumor suppressor protein that gets activated by various stress signals, thereby regulating cellular pathways. The cellular outcomes from different stresses are tightly related to p53 dynamics, including its alterations at gene, mRNA, or protein levels. p53 also contributes to immune responses leading to the abolition of viral pathogens. In turn, the viruses have evolved strategies to subvert p53-mediated host responses to improve their life cycle and pathogenesis. Some viruses attenuate wild-type p53 (WT-p53) function for successful pathogenesis, including degradation and sequestration of p53. In contrast, some others exploit the WT-p53 function through regulation at the transcriptional/translational level to spread infection. One area in which the importance of such host factors is increasingly emerging is the positive-strand RNA viruses that cause fatal viral infections. In this review, we provide insight into all the possible mechanisms of p53 modulation exploited by the positive-strand RNA viruses to establish infection. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Regulation RNA in Disease and Development > RNA in Disease.

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.

Picolinic acid is a broad-spectrum inhibitor of enveloped virus entry that restricts SARS-CoV-2 and influenza A virus in vivo.

The COVID-19 pandemic highlights an urgent need for effective antivirals. Targeting host processes co-opted by viruses is an attractive antiviral strategy with a high resistance barrier. Picolinic acid (PA) is a tryptophan metabolite endogenously produced in mammals. Here, we report the broad-spectrum antiviral activity of PA against enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), flaviviruses, herpes simplex virus, and parainfluenza virus. Mechanistic studies reveal that PA inhibits enveloped virus entry by compromising viral membrane integrity, inhibiting virus-cellular membrane fusion, and interfering with cellular endocytosis. More importantly, in pre-clinical animal models, PA exhibits promising antiviral efficacy against SARS-CoV-2 and IAV. Overall, our data establish PA as a broad-spectrum antiviral with promising pre-clinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

The "LINC" between Δ40p53-miRNA Axis in the Regulation of Cellular Homeostasis.

Previous research has shown that Δ40p53, the translational isoform of p53, can inhibit cell growth independently of p53 by regulating microRNAs. Here, we explored the role of Δ40p53 in regulating the long noncoding RNA-micro-RNA-cellular process axis, specifically focusing on LINC00176. Interestingly, LINC00176 levels were predominantly affected by the overexpression/stress-mediated induction and knockdown of Δ40p53 rather than p53 levels. Additional assays revealed that Δ40p53 transactivates LINC00176 transcriptionally and could also regulate its stability. RNA immunoprecipitation experiments revealed that LINC00176 sequesters several putative microRNA targets, which could further titrate several mRNA targets involved in different cellular processes. To understand the downstream effects of this regulation, we ectopically overexpressed and knocked down LINC00176 in HCT116 p53-/- (harboring only Δ40p53) cells, which affected their proliferation, cell viability, and expression of epithelial markers. Our results provide essential insights into the pivotal role of Δ40p53 in regulating the novel LINC00176 RNA-microRNA-mRNA axis independent of FL-p53 and in maintaining cellular homeostasis.

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.

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.

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.

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.

Contrasting Distribution of SARS-CoV-2 Lineages across Multiple Rounds of Pandemic Waves in West Bengal, the Gateway of East and North-East States of India.

The evolution of viral variants and their impact on viral transmission have been an area of considerable importance in this pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed the viral variants in different phases of the pandemic in West Bengal, a state in India that is important geographically, and compared the variants with other states like Delhi, Maharashtra, and Karnataka, located in other regions of the country. We have identified 57 pango-lineages in 3,198 SARS-CoV-2 genomes, alteration in their distribution, as well as contrasting profiles of amino acid mutational dynamics across different waves in different states. The evolving characteristics of Delta (B.1.617.2) sublineages and alterations in hydrophobicity profiles of the viral proteins caused by these mutations were also studied. Additionally, implications of predictive host miRNA binding/unbinding to emerging spike or nucleocapsid mutations were highlighted. Our results throw considerable light on interesting aspects of the viral genomic variation and provide valuable information for improved understanding of wave-defining mutations in unfolding the pandemic. IMPORTANCE Multiple waves of infection were observed in many states in India during the coronavirus disease 2019 (COVID19) pandemic. Fine-scale evolution of major SARS-CoV-2 lineages and sublineages during four wave-window categories: Pre-Wave 1, Wave 1, Pre-Wave 2, and Wave 2 in four major states of India: Delhi (North), Maharashtra (West), Karnataka (South), and West Bengal (East) was studied using large-scale virus genome sequencing data. Our comprehensive analysis reveals contrasting molecular profiles of the wave-defining mutations and their implications in host miRNA binding/unbinding of the lineages in the major states of India.

RG203KR Mutations in SARS-CoV-2 Nucleocapsid: Assessing the Impact Using a Virus-Like Particle Model System.

The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of mutations at the virion level, recombinant baculovirus expression system-based virus-like particles (VLPs) were generated for the prototype Wuhan sequence along with spike protein mutants like D614G and G1124V and the significant RG203KR mutation in nucleocapsid. All four structural proteins were assembled in a particle for which the morphology and size, confirmed by transmission electron microscopy, closely resembled that of the native virion. The VLP harboring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by immunized mouse sera. Results demonstrate a noninfectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant. IMPORTANCE Since its origin in late 2019, the SARS-CoV-2 virus has been constantly mutating and evolving. Current studies mostly employ spike protein (S) pseudovirus systems to determine the effects of mutations on the infectivity and immunogenicity of variants. Despite its functional importance and emergence as a mutational hot spot, the nucleocapsid (N) protein has not been widely studied. The generation of SARS-CoV-2 VLPs in a baculoviral system in this study, with mutations in the S and N proteins, allowed examination of the involvement of all the structural proteins involved in viral entry and eliciting an immune response. This approach provides a platform to study the effect of mutations in structural proteins of SARS-CoV-2 that potentially contribute to cell infectivity, immune response, and immune evasion, bypassing the use of infectious virus for the same analyses.

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.

Regulation of miR-186-YY1 axis by the p53 translational isoform ∆40p53: implications in cell proliferation.

We have earlier shown that p53-FL and its translational isoform ∆40p53 are differentially regulated. In this study, we have investigated the cellular effect of ∆40p53 regulation on downstream gene expression, specifically miRNAs. Interestingly, ∆40p53 showed antagonistic regulation of miR-186-5p as compared to either p53 alone or a combination of both the isoforms. We have elucidated the miR-186-5p mediated effect of ∆40p53 in cell proliferation. Upon expression of ∆40p53, we observed a significant decrease in YY1 levels, an established target of miR-186-5p, which is involved in cell proliferation. Further assays with anti-miR-186 established the interdependence of ∆40p53- miR-186-5p-YY1- cell proliferation. The results unravel a new dimension toward the understanding of ∆40p53 functions, which seems to regulate cellular fate independent of p53FL.

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.

Stimulation of GMP formation in hGBP1 is mediated by W79 and its effect on the antiviral activity.

Interferon-inducible large GTPases are critical for innate immunity. The distinctive feature of a large GTPase, human guanylate binding protein-1 (hGBP1), is the sequential hydrolysis of GTP into GMP via GDP. Despite several structural and biochemical studies, the underlying mechanism of assembly-stimulated GMP formation by hGBP1 and its role in immunity are not fully clarified. Using a series of biochemical, biophysical, and in silico experiments, we studied four tryptophan residues, located near switch I-II (in and around the active site) to understand the conformational changes near these regions and also to investigate their effect on enhanced GMP formation. The W79A mutation showed significantly reduced GMP formation, whereas the W81A and W180A substitutions exhibited only a marginal defect. The W114A mutation showed a long-range effect of further enhanced GMP formation, which was mediated through W79. We also observed that after first phosphate cleavage, the W79-containing region undergoes a conformational change, which is essential for stimulated GMP formation. We suggest that this conformational change helps to reposition the active site for the next cleavage step, which occurs through a stable contact between the indole moiety of W79 and the main chain carbonyl of K76. We also showed that stimulated GMP formation is crucial for antiviral activity against hepatitis C. Thus, the present study not only provides new insight for the stimulation of GMP formation in hGBP1, but also highlights the importance of the enhanced second phosphate cleavage product in the antiviral activity.

Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility.

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in SD domain) and G1124V (in S2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

Potent HCV NS3 Protease Inhibition by a Water-Soluble Phyllanthin Congener.

NS3/4A protease of hepatitis C virus (HCV) plays an important role in viral RNA replication. A 1,4-diphenylbutanedicarboxylic acid derivative, namely, phyllanthin, extracted from the leaf of a herbal plant, Phyllanthus amarus, inhibits HCV NS3/4A protease and replication activities. However, the reduced aqueous solubility, high toxicity, and poor oral bioavailability are major impediments with phyllanthin. We herein present a design approach to generate phyllanthin congeners in order to potentiate inhibition activity against protease. The phyllanthin congeners were synthesized by chemical methods and subjected to systematic biological studies. One of the congeners, annotated as D8, is identified as a novel and potent inhibitor of the HCV-NS3/4Aprotease activity in vitro and the viral RNA replication in cell culture. Structural analysis using the computational-based docking approach demonstrated important noncovalent interactions between D8 and the catalytic residues of the viral protease. Furthermore, D8 was found to be significantly nontoxic in cell culture. More importantly, oral administration of D8 in BALB/c mice proved its better tolerability and bioavailability, as compared to native phyllanthin. Taken together, this study reveals a promising candidate for developing anti-HCV therapeutics to control HCV-induced liver diseases.