Michael J. Buchmeier

Coronavirus Spike Proteins in Viral Entry and Pathogenesis

u t p r r b e m Coronaviruses comprise a large and diverse family of enveloped, positive-stranded RNA viruses. The Coronaviridae exhibit broad host range, infecting many mammalian and avian species and causing upper respiratory, gastrointestinal, hepatic, and central nervous system diseases. In humans and fowl, coronaviruses primarily cause upper respiratory tract infections, while porcine and bovine coronaviruses establish enteric infections that result in severe economic loss. Coronaviruses of laboratory mice are, for historical reasons, designated as mouse hepatitis viruses (MHVs), but among these only a subset are strictly hepatotropic. Enteric strains are commonly found in rodent colonies and neurotropic strains are exploited to study central nervous system infection and demyelinating disease (Perlman et al., 2000). The extraordinary variations in host range and tissue tropism among coronaviruses are in large part attributable to variations in the spike glycoprotein. The S protein is a large, type I membrane glycoprotein that contains distinct functional domains near the amino (S1) and carboxy (S2) termini. These spikes function to define viral tropism by their receptor specificity and perhaps also by their membrane fusion activity during virus entry into cells. Recently their natural variation has attracted the attention of researchers interested in determinants of viral host range, virus entry, and virus–receptor interactions and their relationship to tropism. Evidence supporting a role for spike protein projections as agents of organ tropism and pathogenesis began with comparative studies of different naturally occurring MHV strains. In essence, nucleotide sequencing revealed that alterations in virus virulence were most closely associated with differences in the spike gene. These correlative findings were recently reinforced using the new technology of targeted RNA recombination, a

Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease

The contribution of the T cell chemoattractant chemokine IFN-inducible protein 10 (IP-10) in host defense following viral infection of the CNS was examined. IP-10 is expressed by astrocytes during acute encephalomyelitis in mouse hepatitis virus-infected mice, and the majority of T lymphocytes infiltrating into the CNS expressed the IP-10 receptor CXCR3. Treatment of mice with anti-IP-10 antisera led to increased mortality and delayed viral clearance from the CNS as compared with control mice. Further, administration of anti-IP-10 led to a >70% reduction (p ≤ 0.001) in CD4+ and CD8+ T lymphocyte infiltration into the CNS, which correlated with decreased (p ≤ 0.01) levels of IFN-γ. These data indicate that IP-10 functions as a sentinel molecule in host defense and is essential in the development of a protective Th1 response against viral infection of the CNS.

A Central Role for CD4+ T Cells and RANTES in Virus-Induced Central Nervous System Inflammation and Demyelination

ABSTRACT Infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in a demyelinating encephalomyelitis characterized by mononuclear cell infiltration and white matter destruction similar to the pathology of the human demyelinating disease multiple sclerosis. The contributions of CD4+ and CD8+ T cells in the pathogenesis of the disease were investigated. Significantly less severe inflammation and demyelination were observed in CD4−/− mice than in CD8−/− and C57BL/6 mice (P ≤ 0.002 andP ≤ 0.001, respectively). Immunophenotyping of central nervous system (CNS) infiltrates revealed that CD4−/− mice had a significant reduction in numbers of activated macrophages/microglial cells in the brain compared to the numbers in CD8−/− and C57BL/6 mice, indicating a role for these cells in myelin destruction. Furthermore, CD4−/−mice displayed lower levels of RANTES (a C-C chemokine) mRNA transcripts and protein, suggesting a role for this molecule in the pathogenesis of MHV-induced neurologic disease. Administration of RANTES antisera to MHV-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination (P ≤ 0.001) compared to those in control mice. These data indicate that CD4+ T cells have a pivotal role in accelerating CNS inflammation and demyelination within infected mice, possibly by regulating RANTES expression, which in turn coordinates the trafficking of macrophages into the CNS, leading to myelin destruction.

Naive Precursor Frequencies and MHC Binding Rather Than the Degree of Epitope Diversity Shape CD8+ T Cell Immunodominance

The primary CD8+ T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8+ T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2Kb, resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F1 hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8+ T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33–41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8+ T cell responses.

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor

BackgroundRecent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein.ResultsStructural models of the transmembrane glycoproteins (GP-2) of the Arenaviruses, lymphochoriomeningitis virus (LCMV) and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue.ConclusionsThese findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Structural Basis of Severe Acute Respiratory Syndrome Coronavirus ADP-Ribose-1″-Phosphate Dephosphorylation by a Conserved Domain of nsP3

Summary The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 Å resolution. The structure of this “X” domain, seen in many single-stranded RNA viruses, reveals a three-layered α/β/α core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1″-phosphate (Appr-1″-p). The SARS nsP3 domain readily removes the 1″ phosphate group from Appr-1″-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1″-p.

In Vivo Expression of Major Histocompatibility Complex Molecules on Oligodendrocytes and Neurons during Viral Infection

Demyelination in multiple sclerosis and in animal models is associated with infiltrating CD8+ and CD4+ T cells. Although oligodendrocytes and axons are damaged in these diseases, the roles T cells play in the demyelination process are not completely understood. Antigen-specific CD8+ T cell lysis of target cells is dependent on interactions between the T cell receptor and major histocompatibility complex (MHC) class I-peptide complexes on the target cell. In the normal central nervous system, expression of MHC molecules is very low but often increases during inflammation. We set out to precisely define which central nervous system cells express MHC molecules in vivo during infection with a strain of murine hepatitis virus that causes a chronic, inflammatory demyelinating disease. Using double immunofluorescence labeling, we show that during acute infection with murine hepatitis virus, MHC class I is expressed in vivo by oligodendrocytes, neurons, microglia, and endothelia, and MHC class II is expressed only by microglia. These data indicate that oligodendrocytes and neurons have the potential to present antigen to T cells and thus be damaged by direct antigen-specific interactions with CD8+ T lymphocytes.

The V5A13.1 envelope glycoprotein deletion mutant of mouse hepatitis virus type-4 is neuroattenuated by its reduced rate of spread in the central nervous system

Abstract Following intracerebral inoculation of adult Balb/c Byj mice, the MHV-4 strain of mouse hepatitis virus (MHV) had an LD50 of <0.1 PFU, whereas its monoclonal antibody resistant variant V5A13.1 had an LD50 of 104.2 PFU. To determine the basis for this difference in neurovirulence we have studied the acute central nervous system (CNS) infection of these two viruses by in situ hybridization. Both viruses infected the same, specific neuroanatomical areas, predominantly neurons, and spread via the cerebrospinal fluid, along neuronal pathways and between adjacent cells. The neuronal nuclei infected and the spread of virus within the brain are described. The main difference between the parental and variant viruses was the rate at which the infection spread. MHV-4 spread rapidly, destroying large numbers of neurons and the animals died within 4 days of infection. The variant virus spread to the same areas of the brain but at a slower rate. This difference in the rate of virus spread was also apparent from the brain virus titers. The slower rate of spread of the variant virus appears to allow intervention by the immune response. Consistent with this, the variant virus spread slowly in athymic nu/nu mice, but in the absence of an intact immune response, infection and destruction of neurons eventually reached the same extent as that of the parental virus and the mice died within 6 days of infection. We conclude that the V5A13.1 variant of MHV-4 is neuroattenuated by its slower rate of spread in the CNS.

Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day.

Cutting Edge: Neosynthesis Is Required for the Presentation of a T Cell Epitope from a Long-Lived Viral Protein

CTLs recognize peptide epitopes which are proteolytically generated by the proteasome and presented on MHC class I molecules. According to the defective ribosomal product (DRiP) hypothesis, epitopes originate from newly synthesized polypeptides which are degraded shortly after their translation. The DRiP hypothesis would explain how epitopes can be generated from long-lived proteins. We examined whether neosynthesis is required for presentation of the immunodominant epitope NP118 of the lymphocytic choriomeningitis virus nucleoprotein, which has a half-life of >3 days. Two days after nucleoprotein biosynthesis was terminated in a tetracycline-regulated transfectant, the presentation of the NP118 epitope ceased. This indicates that NP118 epitopes are generated from newly synthesized nucleoproteins rather than from the long-lived pool of nucleoproteins in the cell. Therefore, the lymphocytic choriomeningitis virus nucleoprotein is the first substrate for which a major prediction of the DRiP hypothesis, namely the requirement for neosynthesis, is shown to hold true.