Interferons and tuft cell numbers are bottlenecks for persistent murine norovirus infection.

Noroviruses (NoVs) are a leading cause of viral gastroenteritis. Despite global clinical relevance, our understanding of how host factors, such as antiviral cytokines interferons (IFNs), modulate NoV population dynamics is limited. Murine NoV (MNoV) is a tractable in vivo model for the study of host regulation of NoV. A persistent strain of MNoV, CR6, establishes a reservoir in intestinal tuft cells for chronic viral shedding in stool. However, the influence of host innate immunity and permissive cell numbers on viral population dynamics is an open question. We generated a pool of 20 different barcoded viruses (CR6BC) by inserting 6-nucleotide barcodes at the 3' position of the NS4 gene and used this pool as our viral inoculum for in vivo infections of different mouse lines. We found that over the course of persistent CR6 infection, shed virus was predominantly colon-derived, and viral barcode richness decreased over time irrespective of host immune status, suggesting that persistent infection involves a series of reinfection events. In mice lacking the IFN-λ receptor, intestinal barcode richness was enhanced, correlating with increased viral intestinal replication. IL-4 treatment, which increases tuft cell numbers, also increased barcode richness, indicating the abundance of permissive tuft cells to be a bottleneck during CR6 infection. In mice lacking type I IFN signaling (Ifnar1-/-) or all IFN signaling (Stat1-/-), barcode diversity at extraintestinal sites was dramatically increased, implicating different IFNs as critical bottlenecks at specific tissue sites. Of interest, extraintestinal barcodes were overlapping but distinct from intestinal barcodes, indicating that disseminated virus represents a distinct viral population than that replicating in the intestine. Barcoded viruses are a valuable tool to explore the influence of host factors on viral diversity in the context of establishment and maintenance of infection as well as dissemination and have provided important insights into how NoV infection proceeds in immunocompetent and immunocompromised hosts.

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Norovirus evolution in immunodeficient mice reveals potentiated pathogenicity via a single nucleotide change in the viral capsid.

Interferons (IFNs) are key controllers of viral replication, with intact IFN responses suppressing virus growth and spread. Using the murine norovirus (MNoV) system, we show that IFNs exert selective pressure to limit the pathogenic evolutionary potential of this enteric virus. In animals lacking type I IFN signaling, the nonlethal MNoV strain CR6 rapidly acquired enhanced virulence via conversion of a single nucleotide. This nucleotide change resulted in amino acid substitution F514I in the viral capsid, which led to >10,000-fold higher replication in systemic organs including the brain. Pathogenicity was mediated by enhanced recruitment and infection of intestinal myeloid cells and increased extraintestinal dissemination of virus. Interestingly, the trade-off for this mutation was reduced fitness in an IFN-competent host, in which CR6 bearing F514I exhibited decreased intestinal replication and shedding. In an immunodeficient context, a spontaneous amino acid change can thus convert a relatively avirulent viral strain into a lethal pathogen.

The Effects of "Dry Swab" Incubation on SARS-CoV-2 Molecular Testing.

BACKGROUND: Widespread testing of SARS-CoV-2 has resulted in shortages of collection devices and transport media. We evaluated the stability of flocked swabs inoculated with SARS-CoV-2-containing specimen incubated dry (i.e., without transport medium) at room temperature. METHODS: A pool of SARS-CoV-2 positive specimen was used to inoculate flocked swabs. Five swabs were placed immediately into universal transport media (UTM) following inoculation, and tested immediately (day 0). Fifteen of the swabs were placed into sterile 15-mL conical tubes and incubated at room temperature for 1, 2, or 7 days. Following incubation, swabs were hydrated in separate vials of UTM and tested. This protocol was repeated for viral transport media (VTM) and saline. As a comparison, a series of swabs was prepared and tested in parallel, but stored in the corresponding liquid transport media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed at 1, 2, and 7 days postinoculation in duplicate. All molecular testing was performed using the Roche cobas SARS-CoV-2 assay. RESULTS: All dry swabs tested on days 1, 2, and 7 provided results that were within 2 cycle thresholds (CTs) of the average CT values for swabs hydrated in the same media and tested on day 0. There was no statistical difference in CT values between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to hydration in liquid media. CONCLUSIONS: The utilization of "dry swabs" may simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks while preserving the accuracy of testing.

Consumptive coagulopathy of severe yellow fever occurs independently of hepatocellular tropism and massive hepatic injury.

Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year despite the long-standing deployment of an effective vaccine. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains uncertain. Hepatocytes are a major target of yellow fever virus (YFV) infection, and the coagulopathy in severe YF has long been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, suggestive of a concurrent consumptive process. To define the relationship between coagulopathy and hepatocellular tropism, we compared infection and disease in Fah-/-, Rag2-/-, and Il2rɣ-/- mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic disease and associated coagulopathy and suggest directions for clinical management of severe YF cases.

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2.

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

Publisher Correction: SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function.

Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Here, we evaluate transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurs 4 days after peak viral titer and correlates with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures.

Replication-Competent Vesicular Stomatitis Virus Vaccine Vector Protects against SARS-CoV-2-Mediated Pathogenesis in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections, and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin-converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice that expressed human ACE2 and were immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naive mice from SARS-CoV-2 challenge. These data support development of VSV-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

Replication-competent vesicular stomatitis virus vaccine vector protects against SARS-CoV-2-mediated pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and hundreds of thousands of deaths. Accordingly, an effective vaccine is of critical importance in mitigating coronavirus induced disease 2019 (COVID-19) and curtailing the pandemic. We developed a replication-competent vesicular stomatitis virus (VSV)-based vaccine by introducing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2). Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high titers of antibodies that neutralize SARS-CoV-2 infection and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung indicating protection against pneumonia. Finally, passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

SARS-CoV-2 infection in the lungs of human ACE2 transgenic mice causes severe inflammation, immune cell infiltration, and compromised respiratory function.

Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) emerged in late 2019 and has spread worldwide resulting in the Coronavirus Disease 2019 (COVID-19) pandemic. Although animal models have been evaluated for SARS-CoV-2 infection, none have recapitulated the severe lung disease phenotypes seen in hospitalized human cases. Here, we evaluate heterozygous transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lung tissues with additional spread to other organs. Remarkably, a decline in pulmonary function, as measured by static and dynamic tests of respiratory capacity, occurs 4 days after peak viral titer and correlates with an inflammatory response marked by infiltration into the lung of monocytes, neutrophils, and activated T cells resulting in pneumonia. Cytokine profiling and RNA sequencing analysis of SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with prominent signatures of NF-kB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection recapitulates many features of severe COVID-19 infection in humans and can be used to define the mechanistic basis of lung disease and test immune and antiviral-based countermeasures.

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes.

Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19 patients. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

Rapid Dissemination and Monopolization of Viral Populations in Mice Revealed Using a Panel of Barcoded Viruses.

The gastrointestinal tract presents a formidable barrier for pathogens to initiate infection. Despite this barrier, enteroviruses, including coxsackievirus B3 (CVB3), successfully penetrate the intestine to initiate infection and spread systemically prior to shedding in stool. However, the effect of the gastrointestinal barrier on CVB3 population dynamics is relatively unexplored, and the selective pressures acting on CVB3 in the intestine are not well characterized. To examine viral population dynamics in orally infected mice, we produced over 100 CVB3 clones harboring nine unique nucleotide "barcodes." Using this collection of barcoded viruses, we found diverse viral populations throughout each mouse within the first day postinfection, but by 48 h the viral populations were dominated by fewer than three barcoded viruses in intestinal and extraintestinal tissues. Using light-sensitive viruses to track replication status, we found that diverse viruses had replicated prior to loss of diversity. Sequencing whole viral genomes from samples later in infection did not reveal detectable viral adaptations. Surprisingly, orally inoculated CVB3 was detectable in pancreas and liver as soon as 20 min postinoculation, indicating rapid systemic dissemination. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues. These results underscore a complex dynamic between dissemination and clearance for an enteric virus.IMPORTANCE Enteric viruses initiate infection in the gastrointestinal tract but can disseminate to systemic sites. However, the dynamics of viral dissemination are unclear. In this study, we created a library of 135 barcoded coxsackieviruses to examine viral population diversity across time and space following oral inoculation of mice. Overall, we found that the broad population of viruses disseminates early, followed by monopolization of mouse tissues with three or fewer pool members at later time points. Interestingly, we detected virus in systemic tissues such as pancreas and liver just 20 min after oral inoculation. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues.

Related Enteric Viruses Have Different Requirements for Host Microbiota in Mice.

Accumulating evidence suggests that intestinal bacteria promote enteric virus infection in mice. For example, previous work demonstrated that antibiotic treatment of mice prior to oral infection with poliovirus reduced viral replication and pathogenesis. Here, we examined the effect of antibiotic treatment on infection with coxsackievirus B3 (CVB3), a picornavirus closely related to poliovirus. We treated mice with a mixture of five antibiotics to deplete host microbiota and examined CVB3 replication and pathogenesis following oral inoculation. We found that, as seen with poliovirus, CVB3 shedding and pathogenesis were reduced in antibiotic-treated mice. While treatment with just two antibiotics, vancomycin and ampicillin, was sufficient to reduce CVB3 replication and pathogenesis, this treatment had no effect on poliovirus. The quantity and composition of bacterial communities were altered by treatment with the five-antibiotic cocktail and by treatment with vancomycin and ampicillin. To determine whether more-subtle changes in bacterial populations impact viral replication, we examined viral infection in mice treated with milder antibiotic regimens. Mice treated with one-tenth the standard concentration of the normal antibiotic cocktail supported replication of poliovirus but not CVB3. Importantly, a single dose of one antibiotic, streptomycin, was sufficient to reduce CVB3 shedding and pathogenesis while having no effect on poliovirus shedding and pathogenesis. Overall, replication and pathogenesis of CVB3 are more sensitive to antibiotic treatment than poliovirus, indicating that closely related viruses may differ with respect to their reliance on microbiota.IMPORTANCE Recent data indicate that intestinal bacteria promote intestinal infection of several enteric viruses. Here, we show that coxsackievirus, an enteric virus in the picornavirus family, also relies on microbiota for intestinal replication and pathogenesis. Relatively minor depletion of the microbiota was sufficient to decrease coxsackievirus infection, while poliovirus infection was unaffected. Surprisingly, a single dose of one antibiotic was sufficient to reduce coxsackievirus infection. Therefore, these data indicate that closely related viruses may differ with respect to their reliance on microbiota.

Caspase-mediated cleavage of murine norovirus NS1/2 potentiates apoptosis and is required for persistent infection of intestinal epithelial cells.

Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.

A Secreted Viral Nonstructural Protein Determines Intestinal Norovirus Pathogenesis.

Murine norovirus (MNoV) infects a low percentage of enteric tuft cells and can persist in these cells for months following acute infection. Both tuft-cell tropism and resistance to interferon-λ (IFN-λ)-mediated clearance during persistent infection requires the viral nonstructural protein 1/2 (NS1/2). We show that processing of NS1/2 yields NS1, an unconventionally secreted viral protein that is central for IFN-λ resistance. MNoV infection globally suppresses intestinal IFN-λ responses, which is attributable to secreted NS1. MNoV NS1 secretion is triggered by caspase-3 cleavage of NS1/2, and a secreted form of human NoV NS1 is also observed. NS1 secretion is essential for intestinal infection and resistance to IFN-λ in vivo. NS1 vaccination alone protects against MNoV challenge, despite the lack of induction of neutralizing anti-capsid antibodies previously shown to confer protection. Thus, despite infecting a low number of tuft cells, NS1 secretion allows MNoV to globally suppress IFN responses and promote persistence.

Norovirus Cell Tropism Is Determined by Combinatorial Action of a Viral Non-structural Protein and Host Cytokine.

Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1-/- mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV.

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication.IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein.