Amino Acid Substitutions in the S2 Subunit of Mouse Hepatitis Virus Variant V51 Encode Determinants of Host Range Expansion

We previously described mouse hepatitis virus (MHV) variant V51 derived from a persistent infection of murine DBT cells with an expanded host range (R. S. Baric, E. Sullivan, L. Hensley, B. Yount, and W. Chen, J. Virol. 73:638-649, 1999). Sequencing of the V51 spike gene, the mediator of virus entry, revealed 13 amino acid substitutions relative to the originating MHV A59 strain. Seven substitutions were located in the amino-terminal S1 cleavage subunit, and six were located in the carboxy-terminal S2 cleavage subunit. Using targeted RNA recombination, we constructed a panel of recombinant viruses to map the mediators of host range to the six substitutions in S2, with a subgroup of four changes of particular interest. This subgroup maps to two previously identified domains within S2, a putative fusion peptide and a heptad repeat, both conserved features of class I fusion proteins. In addition to an altered host range, V51 displayed altered utilization of CEACAM1a, the high-affinity receptor for A59. Interestingly, a recombinant with S1 from A59 and S2 from V51 was severely debilitated in its ability to productively infect cells via CEACAM1a, while the inverse recombinant was not. This result suggests that the S2 substitutions exert powerful effects on the fusion trigger that normally passes from S1 to S2. These novel findings play against the existing data that suggest that MHV host range determinants are located in the S1 subunit, which harbors the receptor binding domain, or involve coordinating changes in both S1 and S2. Mounting evidence also suggests that the class I fusion mechanism may possess some innate plasticity that regulates viral host range

Klärung der Elternschaft der Apfelsorte ‘Meran’

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Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication

Heterologous Gene Expression from Transmissible Gastroenteritis Virus Replicon Particles

We have recently isolated a transmissible gastroenteritis virus (TGEV) infectious construct designated TGEV 1000 (B. Yount, K. M. Curtis, and R. S. Baric, J. Virol. 74:10600–10611, 2000). Using this construct, a recombinant TGEV was constructed that replaced open reading frame (ORF) 3A with a heterologous gene encoding green fluorescent protein (GFP). Following transfection of baby hamster kidney (BHK) cells, a recombinant TGEV (TGEV-GFP2) was isolated that replicated efficiently and expressed GFP. Replicon constructs were constructed that lacked either the ORF 3B and E genes or the ORF 3B, E, and M genes [TGEV-Rep(AvrII) and TGEV-Rep(EcoNI), respectively]. As the E and M proteins are essential for TGEV virion budding, these replicon RNAs should replicate but not result in the production of infectious virus. Following cotransfection of BHK cells with the replicon RNAs carrying gfp, GFP expression was evident by fluorescent microscopy and leader-containing transcripts carrying gfp were detected by reverse transcription-PCR (RT-PCR). Subsequent passage of cell culture supernatants onto permissive swine testicular (ST) cells did not result in the virus, GFP expression, or the presence of leader-containing subgenomic transcripts, demonstrating the single-hit nature of the TGEV replicon RNAs. To prepare a packaging system to assemble TGEV replicon particles (TGEV VRP), the TGEV E gene was cloned into a Venezuelan equine encephalitis (VEE) replicon expression vector and VEE replicon particles encoding the TGEV E protein were isolated [VEE-TGEV(E)]. BHK cells were either cotransfected with TGEV-Rep(AvrII) (E gene deletion) and VEE-TGEV(E) RNA transcripts or transfected with TGEV-Rep(AvrII) RNA transcripts and subsequently infected with VEE VRPs carrying the TGEV E gene. In both cases, GFP expression and leader-containing GFP transcripts were detected in transfected cells. Cell culture supernatants, collected ∼36 h posttransfection, were passed onto fresh ST cells where GFP expression was evident ∼18 h postinfection. Leader-containing GFP transcripts containing the ORF 3B and E gene deletions were detected by RT-PCR. Recombinant TGEV was not released from these cultures. Under identical conditions, TGEV-GFP2 spread throughout ST cell cultures, expressed GFP, and formed viral plaques. The development of infectious TGEV replicon particles should assist studies of TGEV replication and assembly as well as facilitate the production of novel swine candidate vaccines

A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease

Live-attenuated RNA virus vaccines are efficacious but subject to reversion to virulence. Among RNA viruses, replication fidelity is recognized as a key determinant of virulence and escape from antiviral therapy; increased fidelity is attenuating for some viruses. Coronavirus replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3′-5′ exonuclease activity (ExoN) that likely functions in RNA proofreading. In this study, we demonstrate that engineered inactivation of SARS-CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity in vivo and attenuation of pathogenesis in young, aged, and immunocompromised mouse models of human SARS. The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence, even after serial passage or long-term persistent infection in vivo. Our approach represents a strategy with potential for broad applications for the stable attenuation of coronaviruses and possibly other RNA viruses

Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ∼31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10- to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome

Zn2+ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture

Increasing the intracellular Zn2+ concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn2+ and PT at low concentrations (2 µM Zn2+ and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV—thus eliminating the need for PT to transport Zn2+ across the plasma membrane—we show that Zn2+ efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn2+ directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn2+ was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn2+ with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription

Norovirus GII.4 Strain Antigenic Variation

Noroviruses are the principal cause of epidemic gastroenteritis worldwide. Multiple reports have concluded that the major capsid proteins of GII.4 strains, which cause 80% of norovirus infections worldwide, are evolving rapidly, resulting in new epidemic strains. Surrogate neutralization assays using sera from outbreaks and from immunized mice suggest that, as with influenza virus, antigenic variation maintains GII.4 persistence in the face of human population herd immunity. To test this hypothesis, mice were hyperimmunized with virus-like particles (VLPs) representing an early (GII.4-1987) and a contemporary (GII.4-2006) GII.4 strain. Anti-GII.4-1987 IgG monoclonal antibodies (MAbs) strongly reacted with GII.4 VLPs derived between only 1987 and 2002. Ligand binding blockade was more efficient with GII.4-1987 and GII.4-1997 VLPs than with GII.4-2002. Anti-GII.4-2006 IgG MAbs recognized either a broad panel of GII.4 VLPs (1987 to 2006) or a subset of contemporary (2004 to 2006) VLPs. Most 2006 antibodies did not recognize or only poorly recognized GII.4 VLPs of 2007 or 2008, documenting rapid antigenic evolution of GII.4 capsids. Generally, 2006 MAbs blocked homotypic VLP-ligand binding but were unable to block VLPs representing strains primarily circulating during or earlier than 2002. These analyses demonstrate that both subtle and significant evolutionary change has occurred within antibody epitopes between epidemic strains, providing direct evidence that the GII.4 noroviruses are undergoing antigenic variation, likely in response to herd immunity. As with influenza virus, HIV, and hepatitis C virus, norovirus antigenic variation will significantly influence the design of efficacious vaccines and immunotherapeutics against these important human pathogens

Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans

Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS coronavirus

Recently, a novel coronavirus (2019-nCoV) has emerged from Wuhan, China, causing symptoms in humans similar to those caused by severe acute respiratory syndrome coronavirus (SARS-CoV). Since the SARS-CoV outbreak in 2002, extensive structural analyses have revealed key atomic-level interactions between the SARS-CoV spike protein receptor-binding domain (RBD) and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. Here, we analyzed the potential receptor usage by 2019-nCoV, based on the rich knowledge about SARS-CoV and the newly released sequence of 2019-nCoV. First, the sequence of 2019-nCoV RBD, including its receptor-binding motif (RBM) that directly contacts ACE2, is similar to that of SARS-CoV, strongly suggesting that 2019-nCoV uses ACE2 as its receptor. Second, several critical residues in 2019-nCoV RBM (particularly Gln493) provide favorable interactions with human ACE2, consistent with 2019-nCoV’s capacity for human cell infection. Third, several other critical residues in 2019-nCoV RBM (particularly Asn501) are compatible with, but not ideal for, binding human ACE2, suggesting that 2019-nCoV has acquired some capacity for human-to-human transmission. Last, while phylogenetic analysis indicates a bat origin of 2019-nCoV, 2019-nCoV also potentially recognizes ACE2 from a diversity of animal species (except mice and rats), implicating these animal species as possible intermediate hosts or animal models for 2019-nCoV infections. These analyses provide insights into the receptor usage, cell entry, host cell infectivity and animal origin of 2019-nCoV and may help epidemic surveillance and preventive measures against 2019-nCoV. IMPORTANCE The recent emergence of Wuhan coronavirus (2019-nCoV) puts the world on alert. 2019-nCoV is reminiscent of the SARS-CoV outbreak in 2002 to 2003. Our decade-long structural studies on the receptor recognition by SARS-CoV have identified key interactions between SARS-CoV spike protein and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. One of the goals of SARS-CoV research was to build an atomic-level iterative framework of virus-receptor interactions to facilitate epidemic surveillance, predict species-specific receptor usage, and identify potential animal hosts and animal models of viruses. Based on the sequence of 2019-nCoV spike protein, we apply this predictive framework to provide novel insights into the receptor usage and likely host range of 2019-nCoV. This study provides a robust test of this reiterative framework, providing the basic, translational, and public health research communities with predictive insights that may help study and battle this novel 2019-nCoV