Deciphering the role of host protein HuR in RNA virus life cycle and pathogenesis: Hepatitis C Virus and SARS-CoV-2 as exemplars.

Abstract

Viruses pose a major threat to human health and the ongoing SARS-CoV-2 pandemic proves as the best evidence for that. Historically, RNA viruses have a major potential to cause such pandemics. They utilise RNA binding proteins at several stages of their life cycle. We have worked on one such RBP, ELAVL1 (also known as HuR) to study its role in life cycle and pathogenesis of two kind of RNA viruses, one which causes chronic infection, Hepatitis C Virus and the other that causes lytic infection, SARS-CoV-2. HuR is an RNA-binding protein which binds to AU-rich elements in the RNA and regulates the transport, stability and translation of associated RNAs. The function of HuR is regulated by its cellular localisation, post-translational modifications and binding partners. We have outlined the following objectives to understand the role and regulation of HuR in HCV and SARS-CoV-2 life cycle and pathogenesis. Objective 1: To study the mechanism and implications of HuR relocalisation from Nucleus to cytoplasm upon HCV infection. Hepatitis C virus is a positive sense, single-stranded RNA virus which completes its life cycle in the cytoplasm of the host cells, primarily, in hepatocytes. 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 coordinately 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 AMPK, either separately or in combination, to inhibit HCV infection.

Objective 2: HuR protein interaction with SARS-CoV-2 RNA and its consequences in viral RNA translation. SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic is a positive strand RNA virus belonging to Coronaviridae family and causes mild to severe respiratory illness. The viral genome is 30 Kb long and accumulates various mutations as it spreads. RNA-binding proteins (RBPs) play important roles in the life cycle of RNA viruses. Viral 5’UTR and 3’UTR are more than 99% conserved in the emerging mutants, suggesting the importance of sequence conservation in viral processes. We searched for sequence dependent potential RBP binding sites in the viral 5’UTR. Of the proteins obtained, we focused on HuR which is also known to influence translation and replication of other RNA viruses and has been found to be SARS-CoV-2 interacting RBP in genome-wide screens. Strong binding of HuR with viral 5’UTR was observed in in vitro, and ex vivo assays. The binding sites of HuR in the 5’UTR was mapped, and it was found that the mutations in 5’UTRs of SARS-CoV-2 Variants of Concern (VoC) altered the HuR binding affinity. While deciphering the mechanism of HuR effect on CoV-2 lifecycle, we found that it promotes the binding of another host factor PTB to the 5’UTR, promoting viral RNA translation. Interestingly, the role of HuR was found to be completely opposite on the sub-genomic (sg) RNAs of SARS-CoV-2, which code for the structural proteins. While knockout of HuR inhibited translation from genomic 5’UTR, it enhances sg5’UTR translation resulting higher ratio of structural to non-structural proteins. In conclusion, we show the role of an RNA binding protein in differential translation regulation of SARS-CoV-2 genomic and sub-genomic RNAs which opens up new avenues for the design of antivirals against the virus.

Objective 3: Role of HuR in SARS-CoV-2 virus replication and pathogenesis. We have shown that HuR binds to SARS-CoV-2 5’UTR. Therefore, we checked its effect on virus life cycle. The knock-down and knock-out of HuR reduced viral RNA levels and viral titres. The alpha variant exhibited lesser and delayed dependence on HuR as compared to the original Wuhan and Delta variant, which could be one of the mechanisms behind the longer generation time and hence lesser transmission of the alpha variant. Interestingly, Anti-sense oligo (ASO) targeting the HuR binding site was found to reduce the viral titre in WT, but not in HuR KO cell line. We further show that the knockout of HuR increased the sensitivity of the cells for Remdesivir treatment by decreasing its IC50 10 folds. Since HuR KO reduced viral RNA levels in the cells, to uncouple it from pathogenesis, we looked for a system harbouring viral proteins, but was replication deficient. For this, a non-infectious Virus like particle (VLP) system was designed. The VLPs from Baculovirus expression system were purified and characterised and its similarity studied with the kinetics of infectious virus infection. VLPs were used as a platform to study the impact of mutations in the variants and the corresponding changes in virus entry, virus pathogenesis and also explored as vaccine candidates. It appears, the knock-down of HuR did not alter the VLP entry in the cells but altered the pathogenesis. IL-8 was used as a marker for pro-inflammatory cytokines, and we found that the VLP-induced IL-8 secretion was hampered in the HuR KO cell line. Results suggest that in addition to virus life cycle, HuR regulates SARS-CoV-2 pathogenesis and could be an attractive target for possible therapeutics, possibly using ASO alone or in combination with remdesivir. Taken together, we demonstrate important roles of an RNA binding protein HuR, in two RNA viruses, HCV and SARS-CoV-2, and explored different ways to target it for tackling virus infections. The chronic virus, HCV manipulates HuR by post-translational modifications and alteration of subcellular localisation; and the lytic virus, SARS-CoV-2, utilises it to maintain a balanced ratio of structural and non-structural proteins for productive virus infection, while maintaining its subcellular localisation and exploiting it to induce inflammation.