A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry.

Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors.

Autocrine interferon priming in macrophages but not dendritic cells results in enhanced cytokine and chemokine production after coronavirus infection.

Coronaviruses efficiently inhibit interferon (IFN) induction in nonhematopoietic cells and conventional dendritic cells (cDC). However, IFN is produced in infected macrophages, microglia, and plasmacytoid dendritic cells (pDC). To begin to understand why IFN is produced in infected macrophages, we infected bone marrow-derived macrophages (BMM) and as a control, bone marrow-derived DC (BMDC) with the coronavirus mouse hepatitis virus (MHV).

T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice.

A dysregulated innate immune response and exuberant cytokine/chemokine expression are believed to be critical factors in the pathogenesis of severe acute respiratory syndrome (SARS), caused by a coronavirus (SARS-CoV). However, we recently showed that inefficient immune activation and a poor virus-specific T cell response underlie severe disease in SARS-CoV-infected mice. Here, we extend these results to show that virus-specific T cells, in the absence of activation of the innate immune response, were sufficient to significantly enhance survival and diminish clinical disease.

Regulatory T cells inhibit T cell proliferation and decrease demyelination in mice chronically infected with a coronavirus.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) develop acute and chronic demyelinating diseases with histopathological similarities to multiple sclerosis. The process of demyelination is largely immune-mediated, as immunodeficient mice (RAG1(-/-) mice) do not develop demyelination upon infection; however, demyelination develops if these mice are reconstituted with either JHMV-immune CD4 or CD8 T cells. Because myelin destruction is a consequence of the inflammatory response associated with virus clearance, we reasoned that decreasing the amount of inflammation would diminish clinical disease and demyelination.

Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease.

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused substantial morbidity and mortality in 2002-2003. Deletion of the envelope (E) protein modestly diminished virus growth in tissue culture but abrogated virulence in animals. Here, we show that immunization with rSARS-CoV-DeltaE or SARS-CoV-Delta[E,6-9b] (deleted in accessory proteins (6, 7a, 7b, 8a, 8b, 9b) in addition to E) nearly completely protected BALB/c mice from fatal respiratory disease caused by mouse-adapted SARS-CoV and partly protected hACE2 Tg mice from lethal disease.

The N-terminal region of severe acute respiratory syndrome coronavirus protein 6 induces membrane rearrangement and enhances virus replication.

The severe acute respiratory syndrome coronavirus (SARS-CoV) accessory protein 6 (p6) is a 63-amino-acid multifunctional Golgi-endoplasmic reticulum (ER) membrane-associated protein, with roles in enhancing virus replication and in evading the innate immune response to infection by inhibiting STAT1 (signal transducer and activator of transcription factor 1) translocation to the nucleus. Here, we demonstrate that p6 has an N-terminal region-cytoplasm-C-terminal region-cytoplasm configuration with residues 2 to 37 likely membrane embedded. Expression of p6, or of its N-terminal 41-amino-acid region, in the absence of other viral proteins, induced the formation of membranous structures, some of which were similar to double membrane vesicles involved in virus replication.

Evasion by stealth: inefficient immune activation underlies poor T cell response and severe disease in SARS-CoV-infected mice.

Severe Acute Respiratory Syndrome caused substantial morbidity and mortality during the 2002-2003 epidemic. Many of the features of the human disease are duplicated in BALB/c mice infected with a mouse-adapted version of the virus (MA15), which develop respiratory disease with high morbidity and mortality. Here, we show that severe disease is correlated with slow kinetics of virus clearance and delayed activation and transit of respiratory dendritic cells (rDC) to the draining lymph nodes (DLN) with a consequent deficient virus-specific T cell response.

De novo recruitment of antigen-experienced and naive T cells contributes to the long-term maintenance of antiviral T cell populations in the persistently infected central nervous system.

Mice infected with attenuated strains of mouse hepatitis virus, strain JHM, develop a chronic infection in the brain and spinal cord characterized by low levels of viral Ag persistence and retention of virus-specific CD4 and CD8 T cells at the site of infection. It is not known whether these cells are maintained by proliferation of T cells that entered the CNS during acute infection or are newly recruited from Ag-experienced or naive T cell pools. In this study, using adoptive transfer experiments and bone marrow chimeras, we show that at least some of these cells are recruited from the periphery, predominantly from the viral Ag-experienced T cell pool.

Rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease.

We evaluated the efficacy of rhesus theta-defensin 1 (RTD-1), a novel cyclic antimicrobial peptide, as a prophylactic antiviral in a mouse model of severe acute respiratory syndrome (SARS) coronavirus (CoV) lung disease. BALB/c mice exposed to a mouse-adapted strain of SARS-CoV demonstrated 100% survival and modest reductions in lung pathology without reductions in virus titer when treated with two intranasal doses of RTD-1, while mortality in untreated mice was approximately 75%. RTD-1-treated, SARS-CoV-infected mice displayed altered lung tissue cytokine responses 2 and 4 days postinfection compared to those of untreated animals, suggesting that one possible mechanism of action for RTD-1 is immunomodulatory.

Coronaviruses post-SARS: update on replication and pathogenesis.

Although coronaviruses were first identified nearly 60 years ago, they only received notoriety in 2003 when one of their members was identified as the aetiological agent of severe acute respiratory syndrome. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis.

Role of regulatory T cells in coronavirus-induced acute encephalitis.

C57BL/6 mice infected with mouse hepatitis virus, strain JHM (JHMV) develop a rapidly fatal acute encephalitis. Previously, we showed that this disease is partially CD4 T cell-mediated since infection with a recombinant JHMV (rJ) mutated in only a single immunodominant CD4 T cell epitope (epitope M133, rJ.M(Y135Q)) results in a nonlethal disease.

Severe acute respiratory syndrome coronavirus protein 6 is required for optimal replication.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.

IFN-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells.

The neurotropic JHM strain of mouse hepatitis virus (JHMV) replicates primarily within glial cells following intracranial inoculation of susceptible mice, with relative sparing of neurons. This study demonstrates that glial cells derived from neural progenitor cells are susceptible to JHMV infection and that treatment of infected cells with IFN-gamma inhibits viral replication in a dose-dependent manner. Although type I IFN production is muted in JHMV-infected glial cultures, IFN-beta is produced following IFN-gamma-treatment of JHMV-infected cells.

Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant.

High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant.

Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2.

Infection of humans with the severe acute respiratory syndrome coronavirus (SARS-CoV) results in substantial morbidity and mortality, with death resulting primarily from respiratory failure. While the lungs are the major site of infection, the brain is also infected in some patients. Brain infection may result in long-term neurological sequelae, but little is known about the pathogenesis of SARS-CoV in this organ.

Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice.

Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Delta[6-9b]), the structural gene E (rSARS-CoV-DeltaE), and a combination of both sets of genes (rSARS-CoV-Delta[E,6-9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.

Severe acute respiratory syndrome coronavirus protein 6 accelerates murine hepatitis virus infections by more than one mechanism.

The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes numerous accessory proteins whose importance in the natural infection process is currently unclear. One of these accessory proteins is set apart by its function in the context of a related murine hepatitis virus (MHV) infection. SARS-CoV protein 6 increases MHV neurovirulence and accelerates MHV infection kinetics in tissue culture.

Structural and biological basis of CTL escape in coronavirus-infected mice.

Cytotoxic T lymphocyte escape occurs in many human infections, as well as mice infected with the JHM strain of mouse hepatitis virus, which exhibit CTL escape variants with mutations in a single epitope from the spike glycoprotein (S510). In all CTL epitopes prone to escape, only a subset of all potential variants is generally detected, even though many of the changes that are not selected would result in evasion of the T cell response. It is postulated that these unselected mutations significantly impair virus fitness.

Role of IFN-gamma responsiveness in CD8 T-cell-mediated viral clearance and demyelination in coronavirus-infected mice.

Immunocompetent, but not RAG1(-/-) mice infected with MHV-JHM develop demyelination. Transferred CD8 T cell-enriched splenocytes reconstitute demyelination, and this ability is dependent on donor IFN-gamma. We used IFN-gammaR1(-/-) mice to examine the target of IFN-gamma in CD8 T cell-mediated demyelination.

Congenital viral infections of the brain: lessons learned from lymphocytic choriomeningitis virus in the neonatal rat.

The fetal brain is highly vulnerable to teratogens, including many infectious agents. As a consequence of prenatal infection, many children suffer severe and permanent brain injury and dysfunction. Because most animal models of congenital brain infection do not strongly mirror human disease, the models are highly limited in their abilities to shed light on the pathogenesis of these diseases.

Maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus.

Macrophages and microglia are critical in the acute inflammatory response and act as final effector cells of demyelination during chronic infection with the neutrotropic MHV-JHM strain of mouse hepatitis virus (MHV-JHM). Herein, we show that "immature" F4/80(+)Ly-6C(hi) monocytes are the first cells, along with neutrophils, to enter the MHV-JHM-infected central nervous system (CNS). As the infection progresses, macrophages in the CNS down-regulate expression of Ly-6C and CD62L, consistent with maturation, and a higher frequency express CD11c, a marker for dendritic cells (DCs).

Pathogenesis of acute and chronic central nervous system infection with variants of mouse hepatitis virus, strain JHM.

Infection of mice with variants of mouse hepatitis virus, strain JHM (MHV-JHM), provide models of acute and chronic viral infection of the central nervous system (CNS). Through targeted recombination and reverse genetic manipulation, studies of infection with MHV-JHM variants have identified phenotypic differences and examined the effects of these differences on viral pathogenesis and anti-viral host immune responses. Studies employing recombinant viruses with a modified spike (S) glycoprotein of MHV-JHM have identified the S gene as a major determinant of neurovirulence.

Antiviral antibodies are necessary to prevent cytotoxic T-lymphocyte escape in mice infected with a coronavirus.

Mutation within virus-derived CD8 T-cell epitopes can effectively abrogate cytotoxic T-lymphocyte (CTL) recognition and impede virus clearance in infected hosts. These so-called "CTL escape variant viruses" are commonly selected during persistent infections and are associated with rapid disease progression and increased disease severity. Herein, we tested whether antiviral antibody-mediated suppression of virus replication and subsequent virus clearance were necessary for preventing CTL escape in coronavirus-infected mice.

Enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the N-terminal hydrophobic region but not C-terminal sorting motifs.

Severe acute respiratory syndrome coronavirus encodes several accessory proteins of unknown function. We previously showed that one such protein, encoded by ORF6, enhanced the growth of mouse hepatitis virus in tissue culture cells and in mice. Protein 6 consists of an N-terminal hydrophobic peptide and a C-terminal region containing intracellular protein sorting motifs.

Severe acute respiratory syndrome coronavirus protein 6 accelerates murine coronavirus infections.

One or more of the unique 3'-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H.