Mouse Hepatitis Virus Replicase Protein Complexes Are Translocated to Sites of M Protein Accumulation in the ERGIC at Late Times of Infection

AbstractThe coronavirus mouse hepatitis virus (MHV) directs the synthesis of viral RNA on discrete membranous complexes that are distributed throughout the cell cytoplasm. These putative replication complexes are composed of intimately associated but biochemically distinct membrane populations, each of which contains proteins processed from the replicase (gene 1) polyprotein. Specifically, one membrane population contains the gene 1 proteins p65 and p1a-22, while the other contains the gene 1 proteins p28 and helicase, as well as the structural nucleocapsid (N) protein and newly synthesized viral RNA. In this study, immunofluorescence confocal microscopy was used to define the relationship of the membrane populations comprising the putative replication complexes at different times of infection in MHV-A59-infected delayed brain tumor cells. At 5.5 h postinfection (p.i.) the membranes containing N and helicase colocalized with the membranes containing p1a-22/p65 at foci distinct from sites of M accumulation. By 8 to 12 h p.i., however, the membranes containing helicase and N had a predominantly perinuclear distribution and colocalized with M. In contrast, the p1a-22/p65-containing membranes retained a peripheral, punctate distribution at all times of infection and did not colocalize with M. By late times of infection, helicase, N, and M each also colocalized with ERGIC p53, a specific marker for the endoplasmic reticulum–Golgi–intermediate compartment. These data demonstrated that the putative replication complexes separated into component membranes that relocalized during the course of infection. These results suggest that the membrane populations within the MHV replication complex serve distinct functions both in RNA synthesis and in delivery of replication products to sites of virus assembly

Coronavirus Replicase-Reporter Fusions Provide Quantitative Analysis of Replication and Replication Complex Formation

The replication of coronaviruses occurs in association with multiple virus-induced membrane structures that evolve during the course of infection; however, the dynamics of this process remain poorly understood. Previous studies of coronavirus replication complex organization and protein interactions have utilized protein overexpression studies and immunofluorescence of fixed cells. Additionally, live-imaging studies of coronavirus replicase proteins have used fluorescent reporter molecules fused to replicase proteins, but expressed from nonnative locations, mostly late-transcribed subgenomic mRNAs, in the presence or absence of the native protein. Thus, the timing and targeting of native replicase proteins expressed in real time from native locations in the genome remain unknown. In this study, we tested whether reporter molecules could be expressed from the replicase polyprotein of murine hepatitis virus as fusions with nonstructural protein 2 or 3 and whether such reporters could define the targeting and activity of replicase proteins during infection. We demonstrate that the fusion of green fluorescent protein and firefly luciferase with either nonstructural protein 2 or 3 is tolerated and that these reporter-replicase fusions can be used to quantitate replication complex formation and virus replication. The results show that the replicase gene has flexibility to accommodate a foreign gene addition and can be used directly to study replicase complex formation and evolution during infection as well as to provide highly sensitive and specific markers for protein translation and genome replication

SARS coronavirus replicase proteins in pathogenesis

Much progress has been made in understanding the role of structural and accessory proteins in the pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) infections. The SARS epidemic also brought new attention to the proteins translated from ORF1a and ORF1b of the input genome RNA, also known as the replicase/transcriptase gene. Evidence for change within the ORF1ab coding sequence during the SARS epidemic, as well as evidence from studies with other coronaviruses, indicates that it is likely that the ORF1ab proteins play roles in virus pathogenesis distinct from or in addition to functions directly involved in viral replication. Recent reverse genetic studies have confirmed that proteins of ORF1ab may be involved in cellular signaling and modification of cellular gene expression, as well as virulence by mechanisms yet to be determined. Thus, the evolution of the ORF1ab proteins may be determined as much by issues of host range and virulence as they are by specific requirements for intracellular replication

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

Commentary: Essential Programs and Services Model

To further discussion about the Essential Programs and Services (EPS) model for funding public education in Maine, Maine Policy Review asked eight superintendents—representing districts across the state— to provide their views. We also asked each to discuss the needs of his district and whether additional state policy options were necessary to tackle the most pressing issues. The districts represented by these superintendents are a cross section of urban and rural high-receivers and low-receivers. Still, several commonalities emerge: the need for a state commitment that does not wax and wane with the business cycle; the urgency of professional development for new and experienced teachers; and, the importance of linking student outcomes with student assessment measures and student funding. In short, EPS is not seen as a solution to the state’s ongoing debate over public-education funding, but is recognized as a necessary first step

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

Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society

Evaluation of Serologic and Antigenic Relationships Between Middle Eastern Respiratory Syndrome Coronavirus and Other Coronaviruses to Develop Vaccine Platforms for the Rapid Response to Emerging Coronaviruses

Background. Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs

Seeking Membranes: Positive-Strand RNA Virus Replication Complexes

How much do we really understand about how +RNA viruses usurp and transform the intracellular architecture of host cells when they replicate