Neurovirulent Murine Coronavirus JHM.SD Uses Cellular Zinc Metalloproteases for Virus Entry and Cell-Cell Fusion.

The coronavirus (CoV) S protein requires cleavage by host cell proteases to mediate virus-cell and cell-cell fusion. Many strains of the murine coronavirus mouse hepatitis virus (MHV) have distinct, S-dependent organ and tissue tropisms despite using a common receptor, suggesting that they employ different cellular proteases for fusion. In support of this hypothesis, we found that inhibition of endosomal acidification only modestly decreased entry, and overexpression of the cell surface protease TMPRSS2 greatly enhanced entry, of the highly neurovirulent MHV strain JHM.SD relative to their effects on the reference strain, A59. However, TMPRSS2 overexpression decreased MHV structural protein expression, release of infectious particles, and syncytium formation, and endogenous serine protease activity did not contribute greatly to infection. We therefore investigated the importance of other classes of cellular proteases and found that inhibition of matrix metalloproteinase (MMP)- and a disintegrin and metalloprotease (ADAM)-family zinc metalloproteases markedly decreased both entry and cell-cell fusion. Suppression of virus by metalloprotease inhibition varied among tested cell lines and MHV S proteins, suggesting a role for metalloprotease use in strain-dependent tropism. We conclude that zinc metalloproteases must be considered potential contributors to coronavirus fusion.IMPORTANCE The family Coronaviridae includes viruses that cause two emerging diseases of humans, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as a number of important animal pathogens. Because coronaviruses depend on host protease-mediated cleavage of their S proteins for entry, a number of protease inhibitors have been proposed as antiviral agents. However, it is unclear which proteases mediate in vivo infection. For example, SARS-CoV infection of cultured cells depends on endosomal acid pH-dependent proteases rather than on the cell surface acid pH-independent serine protease TMPRSS2, but Zhou et al. (Antiviral Res 116:76-84, 2015, doi:10.1016/j.antiviral.2015.01.011) found that a serine protease inhibitor was more protective than a cathepsin inhibitor in SARS-CoV-infected mice. This paper explores the contributions of endosomal acidification and various proteases to coronavirus infection and identifies an unexpected class of proteases, the matrix metalloproteinase and ADAM families, as potential targets for anticoronavirus therapy.

The nsp1, nsp13, and M proteins contribute to the hepatotropism of murine coronavirus JHM.WU.

UNLABELLED: Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-ß expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor. IMPORTANCE: The Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.

The nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis.

UNLABELLED: All coronaviruses encode a macrodomain containing ADP-ribose-1"-phosphatase (ADRP) activity within the N terminus of nonstructural protein 3 (nsp3). Previous work showed that mouse hepatitis virus strain A59 (MHV-A59) with a mutated catalytic site (N1348A) replicated similarly to wild-type virus but was unable to cause acute hepatitis in mice. To determine whether this attenuated phenotype is applicable to multiple disease models, we mutated the catalytic residue in the JHM strain of MHV (JHMV), which causes acute and chronic encephalomyelitis, using a newly developed bacterial artificial chromosome (BAC)-based MHV reverse genetics system. Infection of mice with the macrodomain catalytic point mutant virus (N1347A) resulted in reductions in lethality, weight loss, viral titers, proinflammatory cytokine and chemokine expression, and immune cell infiltration in the brain compared to mice infected with wild-type virus. Specifically, macrophages were most affected, with approximately 2.5-fold fewer macrophages at day 5 postinfection in N1347A-infected brains. Tumor necrosis factor (TNF) and interferon (IFN) signaling were not required for effective host control of mutant virus as all N1347A virus-infected mice survived the infection. However, the adaptive immune system was required for protection since N1347A virus was able to cause lethal encephalitis in RAG1(-/-) (recombination activation gene 1 knockout) mice although disease onset was modestly delayed. Overall, these results indicate that the BAC-based MHV reverse genetics system will be useful for studies of JHMV and expand upon previous studies, showing that the macrodomain is critical for the ability of coronaviruses to evade the immune system and promote viral pathogenesis. IMPORTANCE: Coronaviruses are an important cause of human and veterinary diseases worldwide. Viral processes that are conserved across a family are likely to be good targets for the development of antiviral therapeutics and vaccines. The macrodomain is a ubiquitous structural domain and is also conserved among all coronaviruses. The coronavirus macrodomain has ADP-ribose-1"-phosphatase activity; however, its function during infection remains unclear as does the reason that coronaviruses have maintained this enzymatic activity throughout evolution. For MHV, this domain has now been shown to promote multiple types of disease, including hepatitis and encephalitis. These data indicate that this domain is vital for the virus to replicate and cause disease. Understanding the mechanism used by this enzyme to promote viral pathogenesis will open up novel avenues for therapies and may give further insight into the role of macrodomain proteins in the host cell since these proteins are found in all living organisms.

Higher-order structure of the Rous sarcoma virus SP assembly domain.

UNLABELLED: Purified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly. IMPORTANCE: The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

Cell-type-specific activation of the oligoadenylate synthetase-RNase L pathway by a murine coronavirus.

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2',5'-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types-neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.

A novel full-length isoform of murine pregnancy-specific glycoprotein 16 (psg16) is expressed in the brain but does not mediate murine coronavirus (MHV) entry.

The mouse pregnancy-specific glycoprotein 16 (PSG16) has been reported to be an alternative receptor for mouse hepatitis virus (MHV), some strains of which cause encephalitis in mice lacking the canonical receptor CEACAM1a. The known isoforms of PSG16 are N-terminally truncated relative to other PSG family proteins and are expressed in neurons as well as in the placenta. We have cloned a novel full-length isoform of psg16 that is also expressed in the brain, placenta, and retina but, like the truncated form, lacks MHV receptor activity when expressed on 293T cells, suggesting that PSG16 does not mediate CEACAM1a-independent spread of MHV.

Pathogenesis of neurotropic murine coronavirus is multifactorial.

Although coronavirus tropism is most often ascribed to receptor availability, increasing evidence suggests that for the neurotropic strains of the murine coronavirus mouse hepatitis virus (MHV), spike-receptor interactions cannot fully explain neurovirulence. The canonical MHV receptor CEACAM1a and its spike-binding site have been extensively characterized. However, CEACAM1a is poorly expressed in neurons, and the extremely neurotropic MHV strain JHM.SD infects ceacam1a(-/-) mice and spreads among ceacam1a(-/-) neurons. Two proposed alternative MHV receptors, CEACAM2 and PSG16, also fail to account for neuronal spread of JHM.SD in the absence of CEACAM1a. It has been reported that JHM.SD has an unusually labile spike protein, enabling it to perform receptor-independent spread (RIS), but it is not clear if the ability to perform RIS is fully responsible for the extremely neurovirulent phenotype. We propose that the extreme neurovirulence of JHM.SD is multifactorial and might include as yet unidentified neuron-specific spread mechanisms.

Conserved and variable features of Gag structure and arrangement in immature retrovirus particles.

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Murine coronavirus receptors are differentially expressed in the central nervous system and play virus strain-dependent roles in neuronal spread.

Coronavirus infection of the murine central nervous system (CNS) provides a model for studies of viral encephalitis and demyelinating disease. Mouse hepatitis virus (MHV) neurotropism varies by strain: MHV-A59 causes mild encephalomyelitis and demyelination, while the highly neurovirulent strain JHM.SD (MHV-4) causes fatal encephalitis with extensive neuronal spread of virus. In addition, while neurons are the predominant CNS cell type infected in vivo, the canonical receptor for MHV, the carcinoembryonic antigen family member CEACAM1a, has been demonstrated only on endothelial cells and microglia. In order to investigate whether CEACAM1a is also expressed in other cell types, ceacam1a mRNA expression was quantified in murine tissues and primary cells. As expected, among CNS cell types, microglia expressed the highest levels of ceacam1a, but lower levels were also detected in oligodendrocytes, astrocytes, and neurons. Given the low levels of neuronal expression of ceacam1a, primary neurons from wild-type and ceacam1a knockout mice were inoculated with MHV to determine the extent to which CEACAM1a-independent infection might contribute to CNS infection. While both A59 and JHM.SD infected small numbers of ceacam1a knockout neurons, only JHM.SD spread efficiently to adjacent cells in the absence of CEACAM1a. Quantification of mRNA for the ceacam1a-related genes ceacam2 and psg16 (bCEA), which encode proposed alternative MHV receptors, revealed low ceacam2 expression in microglia and oligodendrocytes and psg16 expression exclusively in neurons; however, only CEACAM2 mediated infection in human 293T cells. Therefore, neither CEACAM2 nor PSG16 is likely to be an MHV receptor on neurons, and the mechanism for CEACAM1a-independent neuronal spread of JHM.SD remains unknown.

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice.

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.