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1.
J Virol ; 96(3): e0184221, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1691423

ABSTRACT

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.


Subject(s)
Middle East Respiratory Syndrome Coronavirus/physiology , Murine hepatitis virus/physiology , Protein Interaction Domains and Motifs , Viral Regulatory and Accessory Proteins/genetics , Animals , Coronavirus Infections/immunology , Coronavirus Infections/virology , Host-Pathogen Interactions/immunology , Immunity, Innate , Mice , Mortality , Viral Regulatory and Accessory Proteins/chemistry , Viral Tropism , Virulence/genetics , Virulence Factors/genetics
2.
J Virol ; 96(3): e0184221, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1532965

ABSTRACT

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.


Subject(s)
Middle East Respiratory Syndrome Coronavirus/physiology , Murine hepatitis virus/physiology , Protein Interaction Domains and Motifs , Viral Regulatory and Accessory Proteins/genetics , Animals , Coronavirus Infections/immunology , Coronavirus Infections/virology , Host-Pathogen Interactions/immunology , Immunity, Innate , Mice , Mortality , Viral Regulatory and Accessory Proteins/chemistry , Viral Tropism , Virulence/genetics , Virulence Factors/genetics
4.
J Virol ; 95(22): e0127621, 2021 10 27.
Article in English | MEDLINE | ID: covidwho-1494956

ABSTRACT

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.


Subject(s)
Coronavirus Infections/pathology , Disease Models, Animal , Lung/pathology , Murine hepatitis virus/pathogenicity , Animals , Cell Line , Containment of Biohazards , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cytokines/metabolism , Humans , Inflammation , Liver/pathology , Liver/virology , Lung/virology , Mice , Murine hepatitis virus/drug effects , Murine hepatitis virus/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Virus Replication/drug effects
5.
Viruses ; 13(9)2021 08 27.
Article in English | MEDLINE | ID: covidwho-1374535

ABSTRACT

Infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, causes a respiratory illness that can severely impact other organ systems and is possibly precipitated by cytokine storm, septic shock, thrombosis, and oxidative stress. SARS-CoV-2 infected individuals may be asymptomatic or may experience mild, moderate, or severe symptoms with or without pneumonia. The mechanisms by which SARS-CoV-2 infects humans are largely unknown. Mouse hepatitis virus 1 (MHV-1)-induced infection was used as a highly relevant surrogate animal model for this study. We further characterized this animal model and compared it with SARS-CoV-2 infection in humans. MHV-1 inoculated mice displayed death as well as weight loss, as reported earlier. We showed that MHV-1-infected mice at days 7-8 exhibit severe lung inflammation, peribronchiolar interstitial infiltration, bronchiolar epithelial cell necrosis and intra-alveolar necrotic debris, alveolar exudation (surrounding alveolar walls have capillaries that are dilated and filled with red blood cells), mononuclear cell infiltration, hyaline membrane formation, the presence of hemosiderin-laden macrophages, and interstitial edema. When compared to uninfected mice, the infected mice showed severe liver vascular congestion, luminal thrombosis of portal and sinusoidal vessels, hepatocyte degeneration, cell necrosis, and hemorrhagic changes. Proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and the vacuolation of renal tubules were observed. The heart showed severe interstitial edema, vascular congestion, and dilation, as well as red blood cell extravasation into the interstitium. Upon examination of the MHV-1 infected mice brain, we observed congested blood vessels, perivascular cavitation, cortical pericellular halos, vacuolation of neuropils, darkly stained nuclei, pyknotic nuclei, and associated vacuolation of the neuropil in the cortex, as well as acute eosinophilic necrosis and necrotic neurons with fragmented nuclei and vacuolation in the hippocampus. Our findings suggest that the widespread thrombotic events observed in the surrogate animal model for SARS-CoV-2 mimic the reported findings in SARS-CoV-2 infected humans, representing a highly relevant and safe animal model for the study of the pathophysiologic mechanisms of SARS-CoV-2 for potential therapeutic interventions.


Subject(s)
Coronavirus Infections/pathology , Coronavirus Infections/virology , Murine hepatitis virus/physiology , Animals , Biomarkers , Biopsy , COVID-19/pathology , COVID-19/virology , Coronavirus Infections/mortality , Disease Models, Animal , Female , Genome, Viral , Humans , Immunohistochemistry , Liver Function Tests , Mice , Mortality , Organ Specificity , SARS-CoV-2/physiology , Viral Load
6.
J Virol ; 95(15): e0076621, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1305511

ABSTRACT

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.


Subject(s)
Murine hepatitis virus/physiology , Mutation, Missense , Viral Nonstructural Proteins , Virus Replication/genetics , Amino Acid Substitution , Animals , HeLa Cells , Humans , Macrophages/metabolism , Macrophages/virology , Mice , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
7.
J Virol ; 95(15): e0076621, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1236420

ABSTRACT

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.


Subject(s)
Murine hepatitis virus/physiology , Mutation, Missense , Viral Nonstructural Proteins , Virus Replication/genetics , Amino Acid Substitution , Animals , HeLa Cells , Humans , Macrophages/metabolism , Macrophages/virology , Mice , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
8.
Biotechnol Bioeng ; 118(5): 2067-2075, 2021 05.
Article in English | MEDLINE | ID: covidwho-1092502

ABSTRACT

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).


Subject(s)
Betacoronavirus/physiology , Hot Temperature , Virus Inactivation , Murine hepatitis virus/physiology , Viral Plaque Assay
9.
Virology ; 556: 9-22, 2021 04.
Article in English | MEDLINE | ID: covidwho-985483

ABSTRACT

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Subject(s)
Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Murine hepatitis virus/physiology , Viral Replication Compartments/metabolism , Animals , Cell Line , Electron Microscope Tomography , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum/virology , Lysosomes/metabolism , Lysosomes/ultrastructure , Lysosomes/virology , Mice , Viral Proteins/metabolism , Viral Replication Compartments/ultrastructure , Virion/metabolism , Virus Assembly , Virus Replication
10.
J Virol ; 94(24)2020 11 23.
Article in English | MEDLINE | ID: covidwho-941660

ABSTRACT

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.


Subject(s)
Central Nervous System Infections/immunology , Central Nervous System Infections/virology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Host-Pathogen Interactions/immunology , Murine hepatitis virus/physiology , Animals , Central Nervous System Infections/genetics , Central Nervous System Infections/pathology , Computational Biology/methods , Coronavirus Infections/genetics , Coronavirus Infections/pathology , Encephalomyelitis/genetics , Encephalomyelitis/immunology , Encephalomyelitis/pathology , Encephalomyelitis/virology , Gene Expression Profiling , H-2 Antigens/genetics , H-2 Antigens/immunology , Host-Pathogen Interactions/genetics , Immunity, Innate , Mice , Sequence Analysis, RNA , Single-Cell Analysis
11.
J Virol ; 94(20)2020 09 29.
Article in English | MEDLINE | ID: covidwho-840609

ABSTRACT

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.


Subject(s)
Central Nervous System/virology , Interferon Type I/metabolism , Interferon-gamma/metabolism , Macrophages/metabolism , Microglia/metabolism , Neurons/metabolism , Signal Transduction , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Central Nervous System/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Models, Animal , Encephalomyelitis/immunology , Encephalomyelitis/virology , Macrophages/virology , Mice , Mice, Mutant Strains , Microglia/virology , Murine hepatitis virus/physiology , Neurons/virology , Neutrophil Infiltration , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Virus Replication
12.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-823496

ABSTRACT

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.


Subject(s)
Axons/immunology , Axons/metabolism , CD4 Antigens/deficiency , Coronavirus Infections/immunology , Coronavirus Infections/virology , Murine hepatitis virus/physiology , Poliomyelitis/etiology , Animals , Axons/pathology , Brain/immunology , Brain/metabolism , Brain/pathology , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Coronavirus Infections/pathology , Cytokines/metabolism , Disease Models, Animal , Disease Susceptibility/immunology , Ganglia, Spinal/immunology , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Immunohistochemistry , Inflammation Mediators/metabolism , Mice
13.
Viruses ; 12(8)2020 08 12.
Article in English | MEDLINE | ID: covidwho-717762

ABSTRACT

The fatal acute respiratory coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since COVID-19 was declared a pandemic by the World Health Organization in March 2020, infection and mortality rates have been rising steadily worldwide. The lack of a vaccine, as well as preventive and therapeutic strategies, emphasize the need to develop new strategies to mitigate SARS-CoV-2 transmission and pathogenesis. Since mouse hepatitis virus (MHV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2 share a common genus, lessons learnt from MHV and SARS-CoV could offer mechanistic insights into SARS-CoV-2. This review provides a comprehensive review of MHV in mice and SARS-CoV-2 in humans, thereby highlighting further translational avenues in the development of innovative strategies in controlling the detrimental course of SARS-CoV-2. Specifically, we have focused on various aspects, including host species, organotropism, transmission, clinical disease, pathogenesis, control and therapy, MHV as a model for SARS-CoV and SARS-CoV-2 as well as mouse models for infection with SARS-CoV and SARS-CoV-2. While MHV in mice and SARS-CoV-2 in humans share various similarities, there are also differences that need to be addressed when studying murine models. Translational approaches, such as humanized mouse models are pivotal in studying the clinical course and pathology observed in COVID-19 patients. Lessons from prior murine studies on coronavirus, coupled with novel murine models could offer new promising avenues for treatment of COVID-19.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Murine hepatitis virus/physiology , Pneumonia, Viral/virology , Animals , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Coronavirus Infections/transmission , Disease Models, Animal , Host Specificity , Humans , Mice , Murine hepatitis virus/genetics , Murine hepatitis virus/pathogenicity , Pandemics , SARS Virus/genetics , SARS Virus/pathogenicity , SARS Virus/physiology , SARS-CoV-2 , Virus Internalization , Virus Replication
14.
Science ; 369(6509): 1395-1398, 2020 09 11.
Article in English | MEDLINE | ID: covidwho-712737

ABSTRACT

Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo-electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target.


Subject(s)
Cytoplasmic Vesicles/chemistry , Intracellular Membranes/chemistry , Murine hepatitis virus/physiology , RNA, Viral/biosynthesis , Virus Replication , Animals , Cryoelectron Microscopy , Cytoplasmic Vesicles/ultrastructure , Cytoplasmic Vesicles/virology , Electron Microscope Tomography , Intracellular Membranes/ultrastructure , Intracellular Membranes/virology , Mice , Viral Nonstructural Proteins/chemistry
15.
J Virol ; 94(3)2020 01 17.
Article in English | MEDLINE | ID: covidwho-124739

ABSTRACT

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.


Subject(s)
Cytokines/metabolism , Gene Expression Regulation, Enzymologic , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Murine hepatitis virus/physiology , Poly(ADP-ribose) Polymerases/biosynthesis , Proviruses/physiology , Receptors, Aryl Hydrocarbon/metabolism , Virus Replication/physiology , Animals , Cytokines/genetics , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Mice , Mice, Knockout , Poly(ADP-ribose) Polymerases/genetics , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction
16.
J Virol ; 94(11)2020 05 18.
Article in English | MEDLINE | ID: covidwho-10361

ABSTRACT

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.


Subject(s)
Coronavirus Infections/virology , Murine hepatitis virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Host-Pathogen Interactions , Interferon Type I/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Mice , Models, Molecular , Murine hepatitis virus/pathogenicity , Mutagenesis , Protein Conformation , Structure-Activity Relationship , Ubiquitination , Viral Proteins/chemistry , Virulence , Virus Replication
17.
PLoS Pathog ; 16(3): e1008392, 2020 03.
Article in English | MEDLINE | ID: covidwho-5756

ABSTRACT

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.


Subject(s)
Carcinoembryonic Antigen/chemistry , Murine hepatitis virus/chemistry , Murine hepatitis virus/physiology , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Animals , Carcinoembryonic Antigen/metabolism , Carcinoembryonic Antigen/ultrastructure , Cell Line, Tumor , Cryoelectron Microscopy , HEK293 Cells , Humans , Membrane Fusion , Mice , Models, Molecular , Protein Binding , Protein Conformation , Protein Conformation, alpha-Helical , Protein Domains , Protein Multimerization , Proteolysis , Receptors, Virus/metabolism , Receptors, Virus/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS Virus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure , Virus Attachment
18.
mBio ; 11(1)2020 02 11.
Article in English | MEDLINE | ID: covidwho-2427

ABSTRACT

Coronaviruses (CoVs) are common human and animal pathogens that can transmit zoonotically and cause severe respiratory disease syndromes. CoV infection requires spike proteins, which bind viruses to host cell receptors and catalyze virus-cell membrane fusion. Several CoV strains have spike proteins with two receptor-binding domains, an S1A that engages host sialic acids and an S1B that recognizes host transmembrane proteins. As this bivalent binding may enable broad zoonotic CoV infection, we aimed to identify roles for each receptor in distinct infection stages. Focusing on two betacoronaviruses, murine JHM-CoV and human Middle East respiratory syndrome coronavirus (MERS-CoV), we found that virus particle binding to cells was mediated by sialic acids; however, the transmembrane protein receptors were required for a subsequent virus infection. These results favored a two-step process in which viruses first adhere to sialic acids and then require subsequent engagement with protein receptors during infectious cell entry. However, sialic acids sufficiently facilitated the later stages of virus spread through cell-cell membrane fusion, without requiring protein receptors. This virus spread in the absence of the prototype protein receptors was increased by adaptive S1A mutations. Overall, these findings reveal roles for sialic acids in virus-cell binding, viral spike protein-directed cell-cell fusion, and resultant spread of CoV infections.IMPORTANCE CoVs can transmit from animals to humans to cause serious disease. This zoonotic transmission uses spike proteins, which bind CoVs to cells with two receptor-binding domains. Here, we identified the roles for the two binding processes in the CoV infection process. Binding to sialic acids promoted infection and also supported the intercellular expansion of CoV infections through syncytial development. Adaptive mutations in the sialic acid-binding spike domains increased the intercellular expansion process. These findings raise the possibility that the lectin-like properties of many CoVs contribute to facile zoonotic transmission and intercellular spread within infected organisms.


Subject(s)
Coronavirus Infections/virology , Receptors, Virus/metabolism , Sialic Acids/metabolism , Animals , Carcinoembryonic Antigen/metabolism , Coronavirus Infections/metabolism , Dipeptidyl Peptidase 4/metabolism , Humans , Membrane Fusion , Mice , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Middle East Respiratory Syndrome Coronavirus/physiology , Murine hepatitis virus/genetics , Murine hepatitis virus/metabolism , Murine hepatitis virus/physiology , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
19.
J Virol ; 94(4)2020 01 31.
Article in English | MEDLINE | ID: covidwho-2317

ABSTRACT

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.


Subject(s)
Murine hepatitis virus/physiology , Nucleocapsid Proteins/metabolism , RNA, Viral/biosynthesis , Transcription, Genetic/physiology , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology , Animals , Cell Line , Humans , Mice , Nucleocapsid Proteins/genetics , RNA, Viral/genetics , Viral Nonstructural Proteins/genetics
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