Carnahan, K. E. | Search bioRxiv

bioRxiv2023

Amino acid substitutions in norovirus VP1 dictate cell tropism via an attachment process dependent on membrane mobility.

Viruses interact with receptors on the cell surface to initiate and co-ordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here we use murine norovirus as a model for the study of molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein had a key impact on the interaction between virus and receptor. This variation did not affect virion production or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments, and significantly improved cellular attachment when infecting immune cells in suspension. However, reducing plasma membrane mobility counteracted this phenotype, providing insight into for the role of membrane fluidity and receptor recruitment in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid variations was compared in vivo, there were significant differences in the distribution of viruses in a murine model, demonstrating a role in cellular tropism in vivo. Overall, these results highlight the importance of lipid rafts and virus-induced receptor recruitment in viral infection, as well as how capsid evolution can greatly influence cellular tropism, within-host spread and pathogenicity. ImportanceAll viruses initiate infection by utilising receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important to developing novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilise the murine norovirus model system to show that variation in a single amino acid of the major capsid protein can alone can affect viral infectivity through improved attachment to suspension cells. Reducing plasma membrane mobility reduced infectivity, providing an insight into the importance of membrane mobility for receptor recruitment. Furthermore, variation at this site was able to change viral distribution in a murine model, illustrating how in-host capsid evolution can influence viral infectivity and immune evasion.

microbiology↗

bioRxiv2019

Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Protein kinase R (PKR) plays a major role in activating host immunity during infection by sensing dsRNA produced by viruses. Once activated by dsRNA, PKR phosphorylates the translation factor eIF2, halting cellular translation. Many viruses have methods of inhibiting PKR activation or its downstream effects, circumventing protein synthesis shutdown. These include sequestering dsRNA or producing proteins that bind to and inhibit PKR activation. Here we describe our finding that in multiple cell types, PKR was depleted during mouse adenovirus type 1 (MAV-1) infection. MAV-1 did not appear to be targeting PKR at a transcriptional or translational level because total PKR mRNA levels and levels of PKR mRNA bound to polysomes were unchanged or increased during MAV-1 infection. However, inhibiting the proteasome reduced the PKR depletion seen in MAV-1-infected cells, whereas inhibiting the lysosome had no effect. This suggests that proteasomal degradation alone is responsible for PKR degradation during MAV-1 infection. Time course experiments indicate that the degradation occurs early after infection. Infecting cells with UV-inactivated virus prevented PKR degradation, whereas inhibiting viral DNA replication did not. Together these results suggest that an early viral gene is responsible. Degradation of PKR is a rare mechanism to oppose PKR activity, and it has only been described in six RNA viruses. To our knowledge, this is the first example of a DNA virus counteracting PKR by degrading it.\n\nImportanceThe first line of defense in cells during viral infection is the innate immune system, which is activated by different viral products. PKR is a part of this innate immune system and is induced by interferon and activated by dsRNA produced by DNA and RNA viruses. PKR is such an important part of the antiviral response that many viral families have gene products to counteract its activation or the resulting effects of its activity. Although a few RNA viruses degrade PKR, this method of counteracting PKR has not been reported for any DNA viruses. MAV-1 does not encode virus-associated RNAs, a human adenoviral defense against PKR activation. Instead, MAV-1 degrades PKR, and it is the first DNA virus reported to do so. The innate immune evasion by PKR degradation is a previously unidentified way for a DNA virus to circumvent the host antiviral response.

microbiology↗