Biology subjects

Orchard, R. C.

Publications and source records attributed to Orchard, R. C..

8 recordsLinked to original sources

bioRxiv2026

Plscr1 inhibits murine norovirus entry

Noroviruses are a leading cause of gastroenteritis worldwide, yet host factors that restrict norovirus replication are not well understood. By mining both CRISPR activation and CRISPR knockout genome-wide screens, we identified the interferon-stimulated gene Plscr1 as a restriction factor for murine norovirus (MNV). Plscr1 inhibits the fusion of enveloped viruses with endocytic membranes, making its antiviral activity against the nonenveloped MNV surprising. Here, we demonstrate that Plscr1 is both necessary and sufficient to restrict MNV infection in vitro and contributes to the control of colonic infection in mice. Mechanistically, we determined that Plscr1 inhibits MNV entry at a post-attachment step. A single amino acid substitution in the minor capsid protein VP2 confers resistance to Plscr1. Because VP2 delivers viral RNA by puncturing endocytic membranes during calicivirus entry, these findings implicate a late entry step targeted by Plscr1. Our results expand the antiviral activity of PLSCR1 to a nonenveloped virus and identify an unexpected point of convergence between enveloped- and non-enveloped-virus entry pathways.

microbiology↗

bioRxiv2025

A single amino acid mutation in norovirus NS4 promotes viral spread

Viruses can rapidly adapt and evolve to new, unfavorable environments due to their decreased replication fidelity, large reproductive index, and short life cycle. Often these adaptations that enable increased fitness in a new, specialized environment comes with a trade-off of decreased fitness in a standard, general environment. Understanding the tradeoffs of generalist and specialist viruses has provided important insight into vaccine development, mechanism of action of antivirals, and function of viral proteins. Here, we sought to identify how a specialist murine norovirus (MNV) could be converted to a generalist without a simple reversion of a genetic mutation. Previously, we found that a mutation in MNV (NS6F182C) overcame restriction by host protein Trim7 but decreased the efficiency of viral polyprotein NS6-7 cleavage and resulted in attenuation of this specialist virus. Here, we find that a single valine-to-isoleucine mutation in MNV non-structural protein NS4 (NS4V11I) is sufficient to rescue the attenuated replication of specialist NS6F182C over multiple cycles of replication. However, NS4V11I did not affect the defective polyprotein cleavage but instead the NS4V11I mutation facilitates faster viral spread in vitro independent of interferon signaling. The emergence of this mutation in NS4V11I suggests an unappreciated connection between NS4 and NS6 during norovirus replication and provides a system to define the unknown role of norovirus NS4 during infection. ImportanceViruses and hosts are involved in a continuous arms race for survival. Often when viruses evolve to specialize in specific host environments, they lose their versatility and become specialists, only able to grow in one setting. This feature has been leveraged to create live-attenuated vaccines, identify the mechanism of action of antivirals, and to uncover fundamental aspects of viral replication. Here we aim to understand how a specialized virus can adapt again to become a generalist in the context of murine norovirus infection. We identify an unexpected connection between two murine norovirus non-structural proteins and uncover a role for the viral protein NS4 in viral spread. Taken together, these data provide new insight into viral evolution and the functions of norovirus proteins.

microbiology↗

bioRxiv2025

Trim47 inhibits murine norovirus replication in a strain-dependent manner

Human norovirus is the leading cause of gastroenteritis worldwide. Norovirus exhibits remarkable genetic diversity. Understanding the impact of genetic diversity on infection and immunity has been challenging due to the difficulties of in vitro cultivation and the current lack of a small animal model. Murine norovirus (MNV) has emerged as a premier model system to investigate norovirus biology. Here, we identify Trim47 as a host restriction factor that potently inhibits MNV infection in a strain dependent manner. We determine that Trim47 expression inhibits an early stage of the viral life cycle for the MNV strain CR6, while the replication of the closely related strain CW3 is not restricted by Trim47. Using a forward genetic screen we determine that genetic variation within the nonstructural gene NS1 accounts for this differential sensitivity to Trim47. While most TRIM containing proteins promote the ubiquitination and degradation of its targets, Trim47 does neither. Instead, Trim47 promotes the deubiquitination of the NS1/2 precursor protein. Our data provide new insight into a potential antiviral gene and mechanistic insight into norovirus evolution that may impact viral tropism. ImportanceViruses exist as genetically heterogeneous populations. Understanding the contribution of viral genetic variation on infection outcomes is critical in predicting emerging viruses and their variants. Noroviruses are genetically diverse but human norovirus has been technically challenging to study. In this study we use the model system murine norovirus to identify a viral strain specific restriction mechanism where a host gene can specifically restrict one strain of the virus but has no impact on a closely related strain. Dissecting the mechanism of this specificity provides insight into viral diversity and possible host restriction pathways.

microbiology↗

bioRxiv2024

Trim7 does not have a role in the restriction of murine norovirus infection in vivo

Trim7 is an E3 ubiquitin ligase that was recently identified as a central regulator of host- viral interactions with both pro-viral and anti-viral activity in cell culture. As an inhibitor, Trim7 overexpression ubiquitinates viral proteins by recognizing C-terminal glutamines that are hallmarks of 3C-like protease cleavage events. Here we sought to determine the physiological impact of Trim7 in resolving murine norovirus (MNV) infection of mice as MNV is potently inhibited by Trim7 in vitro. Utilizing two independently derived Trim7 deficient mouse lines we found no changes in the viral burden or tissue distribution of MNV in both an acute and persistent model of infection. Additionally, no changes in cytokine responses were observed after acute MNV infection of Trim7-deficient mice. Furthermore, removal of potentially confounding innate immune responses such as STING and STAT1 did not reveal any role for Trim7 in regulating MNV replication. Taken together, our data fails to find a physiological role for Trim7 in regulating MNV infection outcomes in mice and serves as a caution for defining Trim7 as a broad acting antiviral. ImportanceIntrinsic antiviral molecules that restrict viral replication are important drivers of viral evolution and viral tropism. Recently, Trim7 was shown to provide cell intrinsic protection against RNA viruses, including murine norovirus. Biochemically, Trim7 recognizes the cleavage product of viral proteases, suggesting a novel and broad mechanism to restrict viral replication. Here, we tested whether Trim7 had a physiological role in restricting murine norovirus replication in mice. Unexpectedly, we found no impact of viral replication or innate immune responses during murine norovirus infection. Our findings urge caution in defining Trim7 as a broad antiviral factor in the absence of in vivo evidence.

microbiology↗

bioRxiv2024

Metabolic reprogramming tips vaccinia virus infection outcomes by stabilizing interferon-g induced IRF1

Interferon (IFN) induced activities are critical, early determinants of immune responses and infection outcomes. A key facet of IFN responses is the upregulation of hundreds of mRNAs termed interferon-stimulated genes (ISGs) that activate intrinsic and cell-mediated defenses. While primary interferon signaling is well-delineated, other layers of regulation are less explored but implied by aberrant ISG expression signatures in many diseases in the absence of infection. Consistently, our examination of tonic ISG levels across uninfected human tissues and individuals revealed three ISG subclasses. As tissue identity and many comorbidities with increased virus susceptibility are characterized by differences in metabolism, we characterized ISG responses in cells grown in media known to favor either aerobic glycolysis (glucose) or oxidative phosphorylation (galactose supplementation). While these conditions over time had a varying impact on the expression of ISG RNAs, the differences were typically greater between treatments than between glucose/galactose. Interestingly, extended interferon-priming led to divergent expression of two ISG proteins: upregulation of IRF1 in IFN-{gamma}/glucose and increased IFITM3 in galactose by IFN- and IFN-{gamma}. In agreement with a hardwired response, glucose/galactose regulation of interferon-{gamma} induced IRF1 is conserved in unrelated mouse and cat cell types. In galactose conditions, proteasome inhibition restored interferon-{gamma} induced IRF1 levels to that of glucose/interferon-{gamma}. Glucose/interferon-{gamma} decreased replication of the model poxvirus vaccinia at low MOI and high MOIs. Vaccinia replication was restored by IRF1 KO. In contrast, but consistent with differential regulation of IRF1 protein by glucose/galactose, WT and IRF1 KO cells in galactose media supported similar levels of vaccinia replication regardless of IFN-{gamma} priming. Also associated with glucose/galactose is a seemingly second block at a very late stage in viral replication which results in reductions in herpes- and poxvirus titers but not viral protein expression. Collectively, these data illustrate a novel layer of regulation for the key ISG protein, IRF1, mediated by glucose/galactose and imply unappreciated subprograms embedded in the interferon response. In principle, such cellular circuitry could rapidly adapt immune responses by sensing changing metabolite levels consumed during viral replication and cell proliferation.

microbiology↗

bioRxiv2020

CD300lf conditional knockout mouse reveals strain-specific cellular tropism for murine norovirus

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lfF/F) mouse to elucidate the cell tropism of persistent and non-persistent strains of murine norovirus. Using this mouse model, we demonstrate that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoVCR6) in vivo. In contrast, the nonpersistent MNoV strain CW3 (MNoVCW3) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect CW3 viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoVCW3 to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoVCR6, that myelomonocytic cells are a major, but not exclusive, target cell of MNoVCW3, and that STAT1 signaling restricts the cellular tropism of MNoVCW3. This provides the first genetic system to study the cell type-specific role of CD300lf in norovirus pathogenesis. IMPORTANCEHuman noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoVCR6 requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for non-persistent MNoVCW3 infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoVCW3 infection. Mortality associated with MNoVCW3 strain in Stat1-/- mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

microbiology↗

bioRxiv2020

Cytidine monophosphate N-acetylneuraminic acid synthetase and solute carrier family 35 member A1 are required for reovirus binding and infection

Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, {sigma}1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCEAttachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

molecular biology↗

bioRxiv2019

CD300lf is the primary physiologic receptor of murine norovirus but not human norovirus

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. However, after high dose intraperitoneal challenge with MNoV in Cd300lf-/-Stat1-/- mice a single amino acid mutation in the MNoV capsid protein emerged. This substitution did not alter receptor utilization in vitro. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV. Author SummaryHuman norovirus is the leading cause of non-bacterial gastroenteritis causing up to 200,000 deaths each year. How human norovirus enters cells is unknown. Because human norovirus is difficult to grow in the laboratory and in small animals, we use mouse or murine norovirus as a model system. We recently discovered that murine norovirus can use the either CD300ld or CD300lf as a receptor in vitro. We also showed that CD300lf deficient mice were resistant to oral challenge with a single virus strain. Here we determined that CD300lf is essential for infection of diverse murine norovirus strains in cell lines and in mice with normal immune systems demonstrating its the primary physiologic receptor for diverse murine norovirus strains independent of infection route. However, in immunodeficient mice injected with high dose virus directly into the abdominal cavity, we observed a norovirus mutant that enabled CD300lf-independent infection. Finally, we demonstrated that human CD300lf is not the elusive receptor for human norovirus.

microbiology↗