Biology subjects

Khaperskyy, D.

Publications and source records attributed to Khaperskyy, D..

4 recordsLinked to original sources

bioRxiv2025

Impaired host shutoff is a fitness cost associated with baloxavir marboxil resistance mutations in influenza A virus PA/PA-X nuclease domain.

The polymerase acidic (PA) protein is a subunit of the trimeric influenza A virus (IAV) RNA-dependent RNA polymerase and the target of the anti-influenza drug baloxavir marboxil (BXM). As with other direct-acting antivirals, treatment with BXM can lead to selection of viruses carrying resistance mutations. If these mutations have negligible fitness costs, resistant viruses can spread widely and render existing treatments obsolete. Multiple BXM resistance mutations in the nuclease domain of PA have been identified, with I38T and I38M amino acid substitutions occurring frequently. These mutations have minimal to no effects on viral polymerase activity, virus replication, or transmission. However, for reasons that are not well understood, viruses with BXM resistance substitutions have not been able to compete with parental wild-type strains. The IAV genome segment encoding PA also encodes the host shutoff nuclease PA-X, which shares the endonuclease domain with PA but has a unique C-terminal domain generated by ribosomal frameshifting during translation. Unlike their effects on PA activity, the effects of BXM or the I38T/M substitutions on PA-X function remain uncharacterized. In our work, for the first time, we directly examine the effects of baloxavir and the I38T/M substitutions on PA-X activity and show that baloxavir inhibits PA-X activity in a dose dependent manner. Most importantly, we also demonstrate that the I38T/M mutations significantly impair the host shutoff activity of PA-X proteins from different IAV strains of H1N1, H3N2, and H5N1 subtypes. Our work reveals that the deleterious effects of I38T/M on PA-X function may represent an important barrier to the spread of BXM-resistant viruses. AUTHOR SUMMARYA general shut off of the host cells ability to produce new proteins, including those that are needed for antiviral defence, is an important feature of the influenza A virus infection. It enables the virus to effectively suppress intrinsic immune responses. Furthermore, influenza viruses continuously evolve and cause seasonal epidemics by escaping adaptive immunity resulting from previous infections or vaccinations. Young children, elderly, and immunocompromised individuals are at increased risk of severe disease and hospitalization following influenza infection. Antiviral drugs are an important option in treating influenza and limiting disease severity. Baloxavir marboxil is a single dose oral anti-influenza drug effective in treatment of influenza A and B. However, viruses frequently evolve resistance to this antiviral, and if resistance spreads, it can render this treatment obsolete. How likely is the emergence of widespread baloxavir resistance remains an open question and in our work we tested if these mutations weaken the virus, and specifically its general host shutoff ability. We discovered that the viral host shutoff is decreased by baloxavir resistance mutations and propose that this could serve as an important barrier to a successful spread of baloxavir-resistant viruses.

microbiology↗

bioRxiv2023

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their own protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine if NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X effects on host gene expression are not simply additive and that these factors functionally interact in mediating host shutoff. IMPORTANCERespiratory viruses including influenza A virus continue to cause annual epidemics with high morbidity and mortality due to limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff - a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the non-structural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterised some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs development of improved live attenuated vaccines.

microbiology↗

bioRxiv2022

Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation

Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus- infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the highly pathogenic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2 (eIF2) phosphorylation and SG formation induced by exogenous stress (e.g. sodium arsenite treatment). Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV-2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2 phosphorylation, and the Nsp1 of SARS- CoV2 alone was sufficient to cause decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear retention of the SG- nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in infection compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication. Author SummaryHost cells possess many mechanisms that can detect viral infections and trigger defense programs to suppress viral replication and spread. One of such antiviral mechanisms is the formation of stress granules - large aggregates of RNA and proteins that sequester viral components and cellular factors needed by the virus to replicate. Because of this threat, viruses evolved specific mechanisms that prevent stress granule formation. Understanding these mechanisms can reveal potential targets for therapies that would disable viral inhibition of stress granules and render cells resistant to infection. In this study we analyzed inhibition of stress granules by two human coronaviruses: the common cold coronavirus OC43 and the pandemic SARS-CoV2. We have demonstrated that these viruses employ at least two proteins - nucleocapsid protein (N) and the non-structural protein 1 (Nsp1) to suppress stress granules. These proteins act through distinct complementary mechanisms to ensure successful virus replication. Because both OC43 and SARS-CoV2 each dedicate more than one gene product to inhibit stress granule formation, our work suggests that viral disarming of stress granule responses is central for a productive infection.

microbiology↗

bioRxiv2020

UV damage induces G3BP1-dependent stress granule formation that is not driven by translation arrest via mTOR inhibition

Translation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not cause dissociation of the 48S preinitiation complexes. The catalytic activity of the general control non-derepressible 2 (GCN2) kinase contributes to UV-induced SG formation, which is independent of the GCN2-mediated phosphorylation of the eukaryotic translation initiation factor 2. Like many other types of SGs, condensation of UV-induced granules specifically requires the Ras-GTPase-Activating Protein SH3-Domain-Binding Protein 1 (G3BP1). Our work reveals that in UV-treated cells the mechanisms of translation arrest and SG formation may be unlinked, resulting in condensation of ribonucleoproteins that do not represent the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.

cell biology↗