Parsing the functions of the tumor suppressor tumor protein p53 (TP53) is complex due to the multiple isoforms it encodes. ∆40p53, an N-terminally truncated p53 isoform and the only translational isoform, modulates full-length p53 (FLp53) activity and independently regulates targets such as the miR-186-5p/transcriptional repressor protein YY1 axis. To identify additional miRNAs regulated by ∆40p53, we performed small RNA sequencing. We found that ectopic overexpression of ∆40p53, but not FLp53, significantly downregulated miR-4671-5p. Expression of both isoforms at varying ratios revealed that miR-4671-5p may be modulated by FLp53 in a ∆40p53-dependent manner. In silico analysis identified N-sulfoglucosamine sulfohydrolase (SGSH) as a potential miR-4671-5p target. SGSH expression showed an inverse correlation with miR-4671-5p in cancer datasets and had prognostic significance. SGSH mRNA and protein levels were reduced upon miR-4671-5p overexpression or si∆40p53 treatment, confirming regulatory linkage. Functionally, miR-4671-5p overexpression induced intra-S-phase cell cycle arrest, implicating SGSH in cell cycle regulation. These results reveal a previously unknown ∆40p53/miR-4671-5p/SGSH axis that, when dysregulated, induces intra-S-phase cell cycle arrest and may contribute to cancer outcomes. Our findings highlight the distinct regulatory role of ∆40p53, independent of FLp53, in maintaining cellular and metabolic homeostasis via miRNA-mediated mechanisms.

Abstract Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (Δ40p53, Δ133p53, and Δ160p53) participate in tetramer formation, they are important regulators of cancer fate. Although Δ40p53- and Δ133p53-mediated regulation of cancer is well reported, the mechanism underlying Δ160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of Δ160p53 and its role in cancer regulation. As differential synthesis of Δ160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by Δ160p53. Furthermore, Δ160p53 did not induce p53-responsive promoters. RNA sequencing analysis of Δ160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of Δ160p53, which can be considered a novel target for cancer treatment.

Dengue virus is a global health concern resulting in approximately 100 million infections and 21 000 deaths annually. The disease progresses from dengue without warning signs (PLAN A) to dengue with warning signs (PLAN B) and then to severe dengue (PLAN C). However, the molecular mechanisms underlying this transition are not known. In the absence of effective antivirals and vaccines, predicting disease severity early is the only option for better disease management. Extracellular vesicles (EVs) are membrane-bound vesicles secreted from cells and contain miRNA, proteins, and mRNA, which play a crucial role in cell-to-cell communication. Our work involves identifying and characterizing the mRNAs in the EVs isolated from patients in different stages of dengue severity, i.e., PLAN A, PLAN B, and PLAN C. The pathway analysis of these mRNAs enriched in EVs revealed that they play crucial roles in cytokine and chemokine signaling and platelet degranulation, all of which are known to be dysregulated as dengue progresses to severe disease. Further validations using patient samples showed that the selective secretion of these mRNAs from platelets may affect platelet functions. Overall, our study is the first to show the full secretory mRNA profile across different stages of dengue disease severity and reveals a novel mechanism by which the selective secretion of specific mRNAs upon infection may dysregulate host immune responses and contribute to thrombocytopenia, thereby exacerbating disease severity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA associates with different RNA-binding host proteins at each stage of its life cycle. We found sequence-dependent binding of one such important protein, human antigen R (HuR), to the SARS-CoV-2 5'-UTR and studied its potential role in the viral life cycle. The knockdown and knockout studies revealed the importance of such binding in viral translation. We identified 5'-UTR mutations in SARS-CoV-2 variants of concern that altered the HuR-binding affinity. Interestingly, HuR enhanced non-structural protein translation through the genomic 5'-UTR by promoting polypyrimidine tract-binding protein binding to the 5'-UTR. HuR knockout increased the sensitivity to remdesivir treatment by decreasing its half-maximal inhibitory concentration. An antisense oligonucleotide (whose binding site overlapped the HuR-binding site) reduced viral RNA levels in wild-type cells but not HuR-knockout cells. Our results indicate that HuR regulates the balance between SARS-CoV-2 structural and non-structural proteins and guides the infection of viral variants, implying that HuR can potentially be explored as an antiviral target.IMPORTANCEViruses interact with various host proteins throughout their life cycle. A key protein is HuR, an RNA-binding protein regulating RNA stability and translation. HuR binds to viral RNAs at the 5'-UTR or 3'-UTR, influencing translation and replication. We identified conserved HuR binding sites in the SARS-CoV-2 5'-UTR across beta coronaviruses. This binding enhances translation initiation from the genomic 5'-UTR, increasing non-structural protein production essential for replication. Additionally, we discovered that another host protein, PTB, is recruited by HuR to the viral 5'-UTR, aiding ribosome loading. This regulation shows that the virus exploits HuR for its benefit. Targeting HuR may help control the SARS-CoV-2 life cycle. HuR knockout increased sensitivity to remdesivir, an antiviral drug. Using an antisense oligonucleotide to block HuR binding effectively reduced viral RNA levels. Our findings highlight HuR's critical role in viral protein production regulation and its potential as a therapeutic target against SARS-CoV-2.

The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds the viral RNA to form a helical nucleocapsid and enables genome packaging and its release into host cells. Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), one of the most abundant RNA-binding proteins in eukaryotes, regulates key aspects of RNA metabolism, including splicing, transcription, localisation, and transport. Here, we report a direct physical interaction between the SARS-CoV-2 N protein and hnRNPA1, primarily mediated through their intrinsically disordered regions. Furthermore, we found that these proteins co-phase separate in vitro and colocalise within stress granules in cells. In vivo studies reveal that hnRNPA1 suppresses viral replication, suggesting that the N protein-hnRNPA1 interaction plays an important role in modulating the viral life cycle.

Host RNA-binding proteins (RBPs) play a pivotal role in regulating dengue virus (DENV) translation and replication through interactions with untranslated regions (UTRs) of viral RNA. We investigated host proteins associated with detergent-resistant membranes (DRMs) of the DENV replication complex and identified Human antigen R (HuR) as a key RBP enriched in the DRM. HuR was found to negatively regulate DENV replication by binding the DENV-3′UTR and impeding the association of polypyrimidine tract-binding protein (PTB), a known RNA stabilizer. Additionally, infection-induced modulation of HuR stabilized host mRNAs involved in innate immunity. Interestingly, preliminary in vivo validation in the AG129 mouse model reveals an inverse correlation between HuR expression and viral load, implicating HuR in cytokine dysregulation. Notably, HuR promoted cap-independent translation of viral RNA during later stages of infection, when cap-dependent translation is suppressed. These findings reveal a dual role for HuR: restricting viral RNA replication while enhancing translation, highlighting its critical, phase-specific function in the DENV life cycle.

Retinoblastoma gene (RB1) mutation has been reported in lung cancer, retinoblastoma and cervical cancer etc. Here, we elucidate the involvement of RB1 mutations and their regulation in endometrial cancer. Analysis of mutation data of 547 endometrial cancer samples revealed that 12% of samples harboured RB1 mutations. However, 26% of the patients aged between 30 and 50 years harboured RB1 mutations, compared to only 10% in the older age group. Further, in silico interaction studies and structural analysis of these novel RB1 mutations and E2F revealed possible physiological relevance. We found 14,117 genes were significantly mutated, and 5,770 genes were differentially expressed in RB1-altered patients. Pathway analysis showed a significant correlation between RB1 mutations and estrogen receptor mutations. Different cancer-related pathways were also altered in RB1-mutated conditions. More importantly, a positive link between RB1 mutations and HPV infection pathways was observed, indicating HPV infections might induce RB1 mutations in endometrial cancer, which could be prevented by early vaccination. Our findings suggest RB1 mutations as a potential contributor to endometrial cancer in younger women, highlighting the importance of viral co-factors in risk assessments, advocating HPV vaccination in individuals with RB1 mutations. ### Competing Interest Statement The authors have declared no competing interest.

Host RNA-binding proteins (RBPs) play a pivotal role in regulating dengue virus (DENV) translation and replication through interactions with untranslated regions (UTRs) of viral RNA. We investigated host proteins associated with detergent-resistant membranes (DRMs) of the DENV replication complex and identified Human antigen R (HuR) as a key RBP enriched in the DRM. HuR was found to negatively regulate DENV replication by binding the DENV-3′UTR and impeding the association of polypyrimidine tract-binding protein (PTB), a known RNA stabilizer. Additionally, infection-induced modulation of HuR stabilized host mRNAs involved in innate immunity. Interestingly, in vivo validation in AG129 mice model highlights an inverse correlation between HuR expression and viral load and implicates HuR in cytokine dysregulation. Notably, HuR promoted cap-independent translation of viral RNA during later stages of infection, when cap-dependent translation is suppressed. These findings reveal a dual role for HuR: restricting viral RNA replication while enhancing translation, highlighting its critical, phase-specific function in the DENV life cycle. Author summary Understanding how Dengue virus interacts with human cells is key to finding better treatments. Our study looked at a human protein called HuR, which normally control the stability and the use of RNA inside our cells. We discovered that HuR has two important but opposite roles during dengue infection. Early on, it slows down the virus’s ability to make copies of its genetic material, helping to reduce infection. But later, when the virus struggles to use the normal method of making proteins, HuR steps in to help the virus make its proteins in a different way. This double-edged role of HuR shows how the virus cleverly uses host cell machinery at different stages of infection. Overall, this study uncovers the novel role of a specific RNA-binding protein of the host in orchestrating dengue virus infection and pathogenesis, highlighting its potential as a target for antiviral therapies. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

CVB3 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 (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.

Brazil, Russia, India, China, and South Africa (BRICS) are a group of developing countries with shared economic, healthcare, and scientific interests. These countries navigate multiple syndemics, and the COVID-19 pandemic placed severe strain on already burdened BRICS’ healthcare systems, hampering effective pandemic interventions. Genomic surveillance and molecular epidemiology remain indispensable tools for facilitating informed pandemic intervention. To evaluate the combined manner in which the pandemic unfolded in BRICS countries, we reviewed the BRICS pandemic epidemiological and genomic milestones, which included the first reported cases and deaths, and pharmaceutical and non-pharmaceutical interventions implemented in these countries. To assess the development of genomic surveillance capacity and efficiency over the pandemic, we analyzed the turnaround time from sample collection to data availability and the technologies used for genomic analysis. This data provided information on the laboratory capacities that enable the detection of emerging SARS-CoV-2 variants and highlight their potential for monitoring other pathogens in ongoing public health efforts. Our analyses indicated that BRICS suffered >105.6M COVID-19 infections, resulting in >1.7M deaths. BRICS countries detected intricate genetic combinations of SARS-CoV-2 variants that fueled country-specific pandemic waves. BRICS’ genomic surveillance programs enabled the identification and characterization of the majority of globally circulating Variants of Concern (VOCs) and their descending lineages. Pandemic intervention strategies first implemented by BRICS countries included non-pharmaceutical interventions during the onset of the pandemic, such as nationwide lockdowns, quarantine procedures, the establishment of fever clinics, and mask mandates- which were emulated internationally. Vaccination rollout strategies complemented this, some representing the first of their kind. Improvements in BRICS sequencing and data generation turnaround time facilitated quicker detection of circulating and emerging variants, supported by investments in sequencing and bioinformatic infrastructure. Intra-BRICS cooperation contributed to the ongoing intervention in COVID-19 and other pandemics, enhancing collective capabilities in addressing these health challenges. The data generated continues to inform BRICS-centric pandemic intervention strategies and influences global health matters. The increased laboratory and bioinformatic capacity post-COVID-19 will support the detection of emerging pathogens.

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.

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 hGBP3 Delta C 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 hGBP3 Delta C 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 (hGBP3(1-309)) yields a monomer, suggesting that the helical domain promotes the hexamerization of hGBP3. We overexpressed hGBP3 and hGBP3 Delta C 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 hGBP3(1-309) 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.

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.

ABSTRACTPrevious 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 non-coding RNA-microRNA-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.

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis. The use of neutralizing antibodies could be a more effective therapeutic option. Previously we reported the discovery of a novel epitope at the C terminus of HCV-E2 protein, that induced potent neutralizing antibodies in the infected patients. Furthermore, monoclonal antibodies generated against this epitope could also significantly reduce virus replication in a cell culture system. In this study, we have focused on the generation of single chain variable fragments of this unique neutralizing monoclonal antibody A8A11 raised against the conserved epitope. The nucleotide sequence of the neutralizing monoclonal antibody A8A11 was determined and the scFv gene was constructed followed by cloning into the expression plasmid for recombinant protein expression. The scFv mimicked the antibody in binding to the hepatitis C virus like particles (HCV-LP). As expected, the scFv inhibited HCV-LP binding to hepatocytes and could effectively reduce viral replication in the cell culture system. More importantly, scFv A8A11 could restrict serum HCV RNA levels in HCV-infected chimeric mice harboring human hepatocytes. Results provide a basis for developing a promising scFv-based entry inhibitor, which could be more effective against viruses refractory to drugs targeting viral enzymes.

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.

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.