Biology subjects

Mulloy, R. P.

Publications and source records attributed to Mulloy, R. P..

6 recordsLinked to original sources

bioRxiv2026

Human coronavirus nucleocapsid proteins have disparate innate immune evasion abilities

During infection, coronaviruses produce abundant double-stranded RNA (dsRNA) which can induce antiviral innate immune responses such as the interferon, 2-5-oligoadenylate synthetase (OAS)/RNase L, and protein kinase R (PKR) pathways. Coronaviruses must antagonize these dsRNA responses for successful replication. The SARS-CoV-2 nucleocapsid (N) protein plays a central role in evasion of dsRNA responses, interacting with dsRNA to block interferon-{beta} production and the activation of OAS/RNase L and PKR. Despite intensive study of SARS-CoV-2 N, our understanding of the innate immune evasion abilities of N proteins produced by other human coronaviruses (HCoVs) remains incomplete. Here, we provide a comprehensive comparison of HCoV N proteins expressed in a human lung cell line and show that their abilities to block dsRNA-induced innate immune responses differ. Highly pathogenic HCoV N proteins inhibited the production of interferon-{beta} mRNA and activation of OAS/RNase L, while common cold HCoV N proteins did not. While most HCoV N proteins inhibited PKR phosphorylation, HCoV-OC43 N did not, an observation that correlated with high levels of PKR activation observed during HCoV-OC43 infection. The ability of HCoV N proteins to antagonize PKR required colocalization with dsRNA, yet the overall decrease in PKR phosphorylation mediated by N was not due to sequestration of dsRNA away from PKR. Rather, N colocalized with dsRNA and PKR at dsRNA-induced foci (dRIFs) and inhibited PKR phosphorylation within dRIFs. Sarbecovirus N proteins also relocalized PKR to G3BP1 foci, suggesting that these N proteins can inhibit PKR using two distinct mechanisms. Collectively, our work reveals an unexpected level of functional and mechanistic diversity among the innate immune evasion abilities of human coronavirus N proteins. These findings challenge existing presumptions that observations made in one coronavirus can be extrapolated to others, because even conserved essential proteins such as N can exhibit considerable functional heterogeneity.

microbiology↗

bioRxiv2025

A truncated SARS-CoV-2 nucleocapsid protein enhances virus fitness by evading antiviral responses

Viruses face a selective pressure to evade cellular antiviral responses to control the outcome of an infection. However, due to their limited genome size, viruses must adopt unique strategies to confront cellular sensors. Since emerging in humans, SARS-CoV-2 has accrued multiple mutations throughout its genome, some of which enhanced virus replication and led to the rise of viral variants. However, the biological consequences of many of these changes remain to be discovered. Here, we show that SARS-CoV-2 produces a truncated form of the nucleocapsid protein, called N*M210. Due to the acquisition of a viral transcription regulatory sequence (TRS) in the N gene, certain variants, such as Omicron, produce a new viral mRNA that markedly increases N*M210 expression. We show that N*M210 is a dsRNA binding protein, which inhibits multiple arms of the cellular antiviral response, including blocking interferon induction and inhibiting stress granule formation. We created a panel of recombinant SARS-CoV-2 viruses (rSARS-2) with mutations in the N gene that increased or decreased N*M210 production. We show that N*M210 production increases virus fitness, as viruses that produce more N*M210 outcompeted wild-type rSARS-2. We demonstrate that the fitness advantage provided by N*M210 is partly due to its ability to potently block stress granules. We propose a model where, to evade the cellular antiviral response, SARS-CoV-2 has evolved a mechanism to increase the production of a truncated form of the N protein, which broadly limits the activation of dsRNA-induced antiviral responses, tipping the balance in favour of the virus in the battle for control of the cell. O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/638421v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@98766dorg.highwire.dtl.DTLVardef@186f69eorg.highwire.dtl.DTLVardef@1a903f4org.highwire.dtl.DTLVardef@142c799_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISARS-CoV-2 variants evolved to upregulate truncated N (N*) synthesis to increase virus fitness C_LIO_LIN*M210 is a potent dsRNA-binding protein that blocks cellular dsRNA sensing C_LIO_LIN*M210 inhibits stress granule formation independent of G3BP1 binding C_LI

microbiology↗

bioRxiv2022

Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2, and to a lesser extent, the alphacoronavirus HCoV-229E. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting synthesis of full-length and subgenomic HCoV RNAs. Furthermore, consistent with our previous report on the effects of thiopurines on influenza A virus glycoproteins, we observed that 6-TG inhibited accumulation of Spike glycoproteins from diverse HCoVs. Specifically, 6-TG treatment decreased the accumulation of Spike proteins and increased their electrophoretic mobility, consistent with Spike migration following the enzymatic removal of N-linked oligosaccharides with Peptide:N-glycosidase F (PNGaseF). SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on lentiviruses pseudotyped with SARS-CoV-2 Spike; lentiviruses could be harvested from cell supernatants but were deficient in Spike and unable to infect human cells bearing ACE2 receptors. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. At low micromolar doses, the primary known mode of action of 6-TG is selective inhibition of the small GTPase Rac1. However, we observed that selective chemical inhibitors of the small GTPases Rac1, CDC42 and Rho had no effect on Spike processing and accumulation. The GTPase agonist ML099 countered the effects of 6-TG, suggesting that an unknown GTPase could be the relevant 6-TG-target protein involved in regulating Spike processing and accumulation. Overall, these findings provide important clues about the mechanism of action of a candidate antiviral that can broadly target HCoVs and suggest that small GTPases are promising targets for host-targeted antivirals. AUTHOR SUMMARYThe COVID-19 pandemic has ignited efforts to repurpose existing drugs as safe and effective antivirals. Rather than directly inhibiting viral enzymes, host-targeted antivirals inhibit host cell processes to indirectly impede viral replication and/or stimulate antiviral responses. Here, we describe a new antiviral mechanism of action for the FDA-approved thiopurine 6-thioguanine. We demonstrate that this thiopurine is a pro-drug that must be metabolized by host enzymes to gain antiviral activity. We show that it can inhibit the replication of several human coronaviruses, including SARS-CoV-2, at least in part by interfering with the processing and accumulation of Spike glycoproteins, thereby impeding assembly of infectious progeny viruses. We provide evidence implicating host cell GTPase enzymes in the antiviral mechanism of action.

microbiology↗

bioRxiv2022

Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

microbiology↗

bioRxiv2021

SARS-CoV-2 infection of circulating immune cells is not responsible for virus dissemination in severe COVID-19 patients

In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a Trojan Horse to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination.

immunology↗

bioRxiv2020

Human coronaviruses disassemble processing bodies

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this report, no information was known about how human coronaviruses (hu CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold hu CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that N protein-mediated PB loss correlated with elevated RNA for PB-localized transcripts encoding TNF and IL-6. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV-1) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to enhanced proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

microbiology↗