Non-transcriptional IRF7 interacts with NF-κB to inhibit viral inflammation.

Interferon (IFN) regulatory factors (IRF) are key transcription factors in cellular antiviral responses. IRF7, a virus-inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphorylation in the cytoplasm, leading to its translocation to the nucleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory transcription factor NF-κB-p65 and inhibited the induction of inflammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB-dependent inflammatory target genes, induced by virus infection or toll-like receptor stimulation. A mutant IRF7, defective in transcriptional activity, interacted with NF-κB-p65 and suppressed NF-κB-induced gene expression. A single-action IRF7 mutant, active in anti-inflammatory function, but defective in transcriptional activity, efficiently suppressed Sendai virus and murine hepatitis virus replication. We, therefore, uncovered an anti-inflammatory function for IRF7, independent of transcriptional activity, contributing to the antiviral response of IRF7.

Mouse hepatitis virus JHMV I protein is required for maximal virulence.

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

DYRK1A is a multifunctional host factor that regulates coronavirus replication in a kinase-independent manner.

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.

2-Bromopalmitate depletes lipid droplets to inhibit viral replication.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

Ifit2 restricts murine coronavirus spread to the spinal cord white matter and its associated myelin pathology.

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-ß-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine ß-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).

The Role of Coronavirus Spike Protein in Inducing Optic Neuritis in Mice: Parallels to the SARS-CoV-2 Virus.

BACKGROUND: Optic neuritis (ON), one of the clinical manifestations of the human neurological disease multiple sclerosis (MS), was also reported in patients with COVID-19 infection, highlighting one potential neurological manifestation of SARS-CoV-2. However, the mechanism of ON in these patients is poorly understood. EVIDENCE ACQUISITION: Insight may be gained by studying the neurotropic mouse hepatitis virus (MHV-A59), a ß-coronavirus that belongs to the same family as SARS-CoV-2. RESULTS: Mouse hepatitis virus-A59, or its isogenic spike protein recombinant strains, inoculation in mice provides an important experimental model to understand underpinning mechanisms of neuroinflammatory demyelination in association with acute stage optic nerve inflammation and chronic stage optic nerve demyelination concurrent with axonal loss. Spike is a surface protein that mediates viral binding and entry into host cells, as well as cell-cell fusion and viral spread. Studies have implicated spike-mediated mechanisms of virus-induced neuroinflammatory demyelination by comparing naturally occurring demyelinating (DM) and nondemyelinating (NDM) MHV strains. CONCLUSIONS: Here, we summarize findings in MHV-induced experimental ON and myelitis, using natural DM and NDM strains as well as engineered recombinant strains of MHV to understand the role of spike protein in inducing ON and demyelinating disease pathology. Potential parallels in human coronavirus-mediated ON and demyelination, and insight into potential therapeutic strategies, are discussed.

The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

Microglia depletion exacerbates demyelination and impairs remyelination in a neurotropic coronavirus infection.

Microglia are considered both pathogenic and protective during recovery from demyelination, but their precise role remains ill defined. Here, using an inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, and mice infected with a neurotropic coronavirus (mouse hepatitis virus [MHV], strain JHMV), we show that depletion of microglia during the time of JHMV clearance resulted in impaired myelin repair and prolonged clinical disease without affecting the kinetics of virus clearance. Microglia were required only during the early stages of remyelination. Notably, large deposits of extracellular vesiculated myelin and cellular debris were detected in the spinal cords of PLX5622-treated and not control mice, which correlated with decreased numbers of oligodendrocytes in demyelinating lesions in drug-treated mice. Furthermore, gene expression analyses demonstrated differential expression of genes involved in myelin debris clearance, lipid and cholesterol recycling, and promotion of oligodendrocyte function. The results also demonstrate that microglial functions affected by depletion could not be compensated by infiltrating macrophages. Together, these results demonstrate that microglia play key roles in debris clearance and in the initiation of remyelination following infection with a neurotropic coronavirus but are not necessary during later stages of remyelination.

Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication.

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in nonstructural protein 3 (nsp3) that binds and hydrolyzes mono-ADP-ribose (MAR) covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutation of a highly conserved asparagine residue, which makes contact with the distal ribose of ADP-ribose. To determine if additional Mac1 activities contribute to CoV replication, we compared the replication of murine hepatitis virus (MHV) Mac1 mutants, D1329A and N1465A, to the previously mentioned asparagine mutant, N1347A. These residues contact the adenine and proximal ribose in ADP-ribose, respectively. N1465A had no effect on MHV replication or pathogenesis, while D1329A and N1347A both replicated poorly in bone marrow-derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. Interestingly, D1329A was also significantly more attenuated than N1347A in all cell lines tested. Conversely, D1329A retained some ability to block beta interferon (IFN-ß) transcript accumulation compared to N1347A, indicating that these mutations have different effects on Mac1 functions. Combining these two mutations resulted in a virus that was unrecoverable, suggesting that the combined activities of Mac1 are essential for MHV replication. We conclude that Mac1 has multiple functions that promote the replication of MHV, and that these results provide further evidence that Mac1 is a prominent target for anti-CoV therapeutics. IMPORTANCE In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within nonstructural protein 3. It has received significant attention as a potential drug target, as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the functions of Mac1 during infection remain largely unknown. Here, using targeted mutations in different regions of Mac1, we found that Mac1 has multiple functions that promote the replication of MHV, a model CoV, and, therefore, is more important for MHV replication than previously appreciated. These results will help guide the discovery of these novel functions of Mac1 and the development of inhibitory compounds targeting this domain.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry.

Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

Murine Coronavirus Infection Activates the Aryl Hydrocarbon Receptor in an Indoleamine 2,3-Dioxygenase-Independent Manner, Contributing to Cytokine Modulation and Proviral TCDD-Inducible-PARP Expression.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1ß (IL-1ß), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis.IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus.

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.

Single-Cell RNA Sequencing Reveals the Diversity of the Immunological Landscape following Central Nervous System Infection by a Murine Coronavirus.

Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.

Neuronal Ablation of Alpha/Beta Interferon (IFN-α/ß) Signaling Exacerbates Central Nervous System Viral Dissemination and Impairs IFN-γ Responsiveness in Microglia/Macrophages.

Alpha/beta interferon (IFN-α/ß) signaling through the IFN-α/ß receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/ß pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/ß induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/ß pathway genes as well as Il6, Tnf, and Il1ß between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/ß signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/ß and IFN-γ pathways in achieving optimal antiviral responses.IMPORTANCE IFN-α/ß induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/ß functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/ß receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/ß pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/ß and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.

CD4 Deficiency Causes Poliomyelitis and Axonal Blebbing in Murine Coronavirus-Induced Neuroinflammation.

Mouse hepatitis virus (MHV) is a murine betacoronavirus (m-CoV) that causes a wide range of diseases in mice and rats, including hepatitis, enteritis, respiratory diseases, and encephalomyelitis in the central nervous system (CNS). MHV infection in mice provides an efficient cause-effect experimental model to understand the mechanisms of direct virus-induced neural-cell damage leading to demyelination and axonal loss, which are pathological features of multiple sclerosis (MS), the most common disabling neurological disease in young adults. Infiltration of T lymphocytes, activation of microglia, and their interplay are the primary pathophysiological events leading to disruption of the myelin sheath in MS. However, there is emerging evidence supporting gray matter involvement and degeneration in MS. The investigation of T cell function in the pathogenesis of deep gray matter damage is necessary. Here, we employed RSA59 (an isogenic recombinant strain of MHV-A59)-induced experimental neuroinflammation model to compare the disease in CD4-/- mice with that in CD4+/+ mice at days 5, 10, 15, and 30 postinfection (p.i.). Viral titer estimation, nucleocapsid gene amplification, and viral antinucleocapsid staining confirmed enhanced replication of the virions in the absence of functional CD4+ T cells in the brain. Histopathological analyses showed elevated susceptibility of CD4-/- mice to axonal degeneration in the CNS, with augmented progression of acute poliomyelitis and dorsal root ganglionic inflammation rarely observed in CD4+/+ mice. Depletion of CD4+ T cells showed unique pathological bulbar vacuolation in the brain parenchyma of infected mice with persistent CD11b+ microglia/macrophages in the inflamed regions on day 30 p.i. In summary, the current study suggests that CD4+ T cells are critical for controlling acute-stage poliomyelitis (gray matter inflammation), chronic axonal degeneration, and inflammatory demyelination due to loss of protective antiviral host immunity.IMPORTANCE The current trend in CNS disease biology is to attempt to understand the neural-cell-immune interaction to investigate the underlying mechanism of neuroinflammation, rather than focusing on peripheral immune activation. Most studies in MS are targeted toward understanding the involvement of CNS white matter. However, the importance of gray matter damage has become critical in understanding the long-term progressive neurological disorder. Our study highlights the importance of CD4+ T cells in safeguarding neurons against axonal blebbing and poliomyelitis from murine betacoronavirus-induced neuroinflammation. Current knowledge of the mechanisms that lead to gray matter damage in MS is limited, because the most widely used animal model, experimental autoimmune encephalomyelitis (EAE), does not present this aspect of the disease. Our results, therefore, add to the existing limited knowledge in the field. We also show that the microglia, though important for the initiation of neuroinflammation, cannot establish a protective host immune response without the help of CD4+ T cells.

Sub-second heat inactivation of coronavirus using a betacoronavirus model.

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and four proteins, three of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub-second heat inactivation of CoV. The system is composed of a stainless-steel capillary immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log10 reduction).

Viral mouse models used to study multiple sclerosis: past and present.

Multiple sclerosis (MS) is a common inflammatory demyelinating disease of the central nervous system. Although the etiology of MS is unknown, genetics and environmental factors, such as infections, play a role. Viral infections of mice have been used as model systems to study this demyelinating disease of humans. Three viruses that have long been studied in this capacity are Theiler's murine encephalomyelitis virus, mouse hepatitis virus, and Semliki Forest virus. This review describes the viruses themselves, the infection process, the disease caused by infection and its accompanying pathology, and the model systems and their usefulness in studying MS.