Mark R. Denison

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen.

Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP1,2) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

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

ABSTRACT 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.

Severe Acute Respiratory Syndrome Coronavirus Group-Specific Open Reading Frames Encode Nonessential Functions for Replication in Cell Cultures and Mice

ABSTRACT SARS coronavirus (SARS-CoV) encodes several unique group-specific open reading frames (ORFs) relative to other known coronaviruses. To determine the significance of the SARS-CoV group-specific ORFs in virus replication in vitro and in mice, we systematically deleted five of the eight group-specific ORFs, ORF3a, OF3b, ORF6, ORF7a, and ORF7b, and characterized recombinant virus replication and gene expression in vitro. Deletion of the group-specific ORFs of SARS-CoV, either alone or in various combinations, did not dramatically influence replication efficiency in cell culture or in the levels of viral RNA synthesis. The greatest reduction in virus growth was noted following ORF3a deletion. SARS-CoV spike (S) glycoprotein does not encode a rough endoplasmic reticulum (rER)/Golgi retention signal, and it has been suggested that ORF3a interacts with and targets S glycoprotein retention in the rER/Golgi apparatus. Deletion of ORF3a did not alter subcellular localization of the S glycoprotein from distinct punctuate localization in the rER/Golgi apparatus. These data suggest that ORF3a plays little role in the targeting of S localization in the rER/Golgi apparatus. In addition, insertion of the 29-bp deletion fusing ORF8a/b into the single ORF8, noted in early-stage SARS-CoV human and civet cat isolates, had little if any impact on in vitro growth or RNA synthesis. All recombinant viruses replicated to wild-type levels in the murine model, suggesting that either the group-specific ORFs play little role in in vivo replication efficiency or that the mouse model is not of sufficient quality for discerning the role of the group-specific ORFs in disease origin and development.

Identification and Characterization of Severe Acute Respiratory Syndrome Coronavirus Replicase Proteins

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) encodes proteins required for RNA transcription and genome replication as large polyproteins that are proteolytically processed by virus-encoded proteinases to produce mature replicase proteins. In this report, we generated antibodies against SARS-CoV predicted replicase protein and used the antibodies to identify and characterize 12 of the 16 predicted mature replicase proteins (nsp1, nsp2, nsp3, nsp4, nsp5, nsp8, nsp9, nsp12, nsp13, nsp14, nsp15, and nsp16) in SARS-CoV-infected Vero cells. Immunoblot analysis of infected-cell lysates identified proteins of the predicted sizes. Immunofluorescence microscopy detected similar patterns of punctate perinuclear and distributed cytoplasmic foci with all replicase antibodies and as early as 6 h postinfection. Dual-labeling studies demonstrated colocalization of replicase protein nsp8 with nsp2 and nsp3 in cytoplasmic complexes and also with LC3, a protein marker for autophagic vacuoles. Antibodies directed against mouse hepatitis virus (MHV) virions and against the putative RNA-dependent RNA polymerase (Pol) detected SARS-CoV nucleocapsid and nsp12 (Pol), respectively, in SARS-CoV-infected Vero cells. These results confirm the predicted protein processing pattern for mature SARS-CoV replicase proteins, demonstrate localization of replicase proteins to cytoplasmic complexes containing markers for autophagosome membranes, and suggest conservation of protein epitopes in the replicase and nucleocapsid of SARS-CoV and the group II coronavirus, MHV. Further, the results demonstrate the ability of replicase antibodies to detect SARS-CoV-infected cells as early as 6 h postinfection and thus represent important tools for studies of SARS-CoV replication, inhibition, and diagnosis.

High Fidelity of Murine Hepatitis Virus Replication Is Decreased in nsp14 Exoribonuclease Mutants

ABSTRACT Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3′-to-5′ exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.

Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing.

Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice

Defining prospective pathways by which zoonoses evolve and emerge as human pathogens is critical for anticipating and controlling both natural and deliberate pandemics. However, predicting tenable pathways of animal-to-human movement has been hindered by challenges in identifying reservoir species, cultivating zoonotic organisms in culture, and isolating full-length genomes for cloning and genetic studies. The ability to design and recover pathogens reconstituted from synthesized cDNAs has the potential to overcome these obstacles by allowing studies of replication and pathogenesis without identification of reservoir species or cultivation of primary isolates. Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic. To test a possible route of emergence from the noncultivable Bat-SCoV to human SARS-CoV, we designed a consensus Bat-SCoV genome and replaced the Bat-SCoV Spike receptor-binding domain (RBD) with the SARS-CoV RBD (Bat-SRBD). Bat-SRBD was infectious in cell culture and in mice and was efficiently neutralized by antibodies specific for both bat and human CoV Spike proteins. Rational design, synthesis, and recovery of hypothetical recombinant viruses can be used to investigate mechanisms of transspecies movement of zoonoses and has great potential to aid in rapid public health responses to known or predicted emerging microbial threats.

Coronavirus Replication Does Not Require the Autophagy Gene ATG5

Macroautophagy (herein autophagy) is a cellular process, requiring ATG5, by which cells deliver double membrane-bound packets containing cytoplasm or cytoplasmic organelles to the lysosome. This process has been reported in some cases to be antiviral, while in other cases it has been reported to be required for efficient viral replication or release. A role for autophagy in RNA virus replication has been an attractive hypothesis because of the association of RNA virus replication with complex membrane rearrangements in the cytoplasm that can generate opposed double membranes. In this study we demonstrate that ATG5 is not required for murine hepatitis virus (MHV) replication in either bone marrow derived macrophages (BMMφ) lacking ATG5 by virtue of Cre-recombinase mediated gene deletion or primary low passage murine ATG5-/- embryonic fibroblasts (pMEFs). We conclude that neither ATG5 nor an intact autophagic pathway are required for MHV replication or release.

Four Proteins Processed from the Replicase Gene Polyprotein of Mouse Hepatitis Virus Colocalize in the Cell Periphery and Adjacent to Sites of Virion Assembly

ABSTRACT The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.