Jason Netland

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. It also highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers.

Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.

A Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Protein Enhances Virulence of an Attenuated Murine Coronavirus

ABSTRACT Most animal species that can be infected with the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) do not reproducibly develop clinical disease, hindering studies of pathogenesis. To develop an alternative system for the study of SARS-CoV, we introduced individual SARS-CoV genes (open reading frames [ORFs]) into the genome of an attenuated murine coronavirus. One protein, the product of SARS-CoV ORF6, converted a sublethal infection to a uniformly lethal encephalitis and enhanced virus growth in tissue culture cells, indicating that SARS-CoV proteins function in the context of a heterologous coronavirus infection. Furthermore, these results suggest that the attenuated murine coronavirus lacks a virulence gene residing in SARS-CoV. Recombinant murine coronaviruses cause a reproducible and well-characterized clinical disease, offer virtually no risk to laboratory personnel, and should be useful for elucidating the role of SARS-CoV nonstructural proteins in viral replication and pathogenesis.

Very Diverse CD8 T Cell Clonotypic Responses after Virus Infections

We measured CD8 T cell clonotypic diversity to three epitopes recognized in C57BL/6 mice infected with mouse hepatitis virus, strain JHM, or lymphocytic choriomeningitis virus. We isolated epitope-specific T cells with an IFN-γ capture assay or MHC class I/peptide tetramers and identified different clonotypes by Vβ chain sequence analysis. In agreement with our previous results, the number of different clonotypes responding to all three epitopes fit a log-series distribution. From these distributions, we estimated that >1000 different clonotypes responded to each immunodominant CD8 T cell epitope; the response to a subdominant CD8 T cell epitope was modestly less diverse. These results suggest that T cell response diversity is greater by 1–2 orders of magnitude than predicted previously.

ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia

ABSTRACT Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

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

Abstract 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-ΔE or SARS-CoV-Δ[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. hACE2 Tg mice, which express the human SARS-CoV receptor, are extremely susceptible to infection. We also show that rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] induced anti-virus T cell and antibody responses. Further, the E-deleted viruses were stable after 16 blind passages through tissue culture cells, with only a single mutation in the surface glycoprotein detected. The passaged virus remained avirulent in mice. These results suggest that rSARS-CoV-ΔE is an efficacious vaccine candidate that might be useful if SARS recurred.

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Coronavirus Infections

ABSTRACT 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. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

Severe Acute Respiratory Syndrome Coronavirus Infection Causes Neuronal Death in the Absence of Encephalitis in Mice Transgenic for Human ACE2

ABSTRACT 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. We previously showed that the brain was a major target organ for infection in mice that are transgenic for the SARS-CoV receptor (human angiotensin-converting enzyme 2). Herein, we use these mice to show that virus enters the brain primarily via the olfactory bulb, and infection results in rapid, transneuronal spread to connected areas of the brain. This extensive neuronal infection is the main cause of death because intracranial inoculation with low doses of virus results in a uniformly lethal disease even though little infection is detected in the lungs. Death of the animal likely results from dysfunction and/or death of infected neurons, especially those located in cardiorespiratory centers in the medulla. Remarkably, the virus induces minimal cellular infiltration in the brain. Our results show that neurons are a highly susceptible target for SARS-CoV and that only the absence of the host cell receptor prevents severe murine brain disease.

Severe Acute Respiratory Syndrome Coronavirus Protein 6 Is Required for Optimal Replication

ABSTRACT 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.

The N-Terminal Region of Severe Acute Respiratory Syndrome Coronavirus Protein 6 Induces Membrane Rearrangement and Enhances Virus Replication

ABSTRACT 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. Consistent with a role in virus replication, p6 partially colocalized with nonstructural protein 3 (nsp3), a marker for virus replication complexes. Further, while the C-terminal region is required for preventing STAT1 translocation to the nucleus, our results also indicated that the N-terminal 18 amino acids were necessary for maximal inhibition. Collectively, these results support the notion that p6 is a two-domain protein, although the function of each is not completely independent of the other.