Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syndrome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expression. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple subgenomic RNA species, and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed by the enormous size of the coronavirus genome. This review summarizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly.

The molecular biology of coronaviruses.

Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, boasts tremendous sensitivity, can be highly sequence-specific, has little to no post-amplification processing, and is amenable to increasing sample throughput. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment. Starting with the theory behind real-time PCR, this review discusses the key components of a real-time PCR experiment, including one-step or two-step PCR, absolute versus relative quantitation, mathematical models available for relative quantitation and amplification efficiency calculations, types of normalization or data correction, and detection chemistries. In addition, the many causes of variation as well as methods to calculate intra- and inter-assay variation are addressed.

https://www.future-science.com/doi/pdf/10.2144/05391RV01

Real-time PCR for mRNA quantitation.

Coronaviruses (CoVs) primarily cause enzootic infections in birds and mammals but, in the last few decades, have shown to be capable of infecting humans as well. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and, more recently, Middle-East respiratory syndrome (MERS) has demonstrated the lethality of CoVs when they cross the species barrier and infect humans. A renewed interest in coronaviral research has led to the discovery of several novel human CoVs and since then much progress has been made in understanding the CoV life cycle. The CoV envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus’ life cycle, such as assembly, budding, envelope formation, and pathogenesis. Recent studies have expanded on its structural motifs and topology, its functions as an ion-channelling viroporin, and its interactions with both other CoV proteins and host cell proteins. This review aims to establish the current knowledge on CoV E by highlighting the recent progress that has been made and comparing it to previous knowledge. It also compares E to other viral proteins of a similar nature to speculate the relevance of these new findings. Good progress has been made but much still remains unknown and this review has identified some gaps in the current knowledge and made suggestions for consideration in future research. The most progress has been made on SARS-CoV E, highlighting specific structural requirements for its functions in the CoV life cycle as well as mechanisms behind its pathogenesis. Data shows that E is involved in critical aspects of the viral life cycle and that CoVs lacking E make promising vaccine candidates. The high mortality rate of certain CoVs, along with their ease of transmission, underpins the need for more research into CoV molecular biology which can aid in the production of effective anti-coronaviral agents for both human CoVs and enzootic CoVs.

/pdf/coronavirus-envelope-protein-current-knowledge-ulubnioebv.pdf

Coronavirus envelope protein: current knowledge

Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV.

Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus

Coronaviruses infect many species of animals including humans, causing acute and chronic diseases. This review focuses primarily on the pathogenesis of murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory coronavirus (SARS-CoV). MHV is a collection of strains, which provide models systems for the study of viral tropism and pathogenesis in several organs systems, including the central nervous system, the liver, and the lung, and has been cited as providing one of the few animal models for the study of chronic demyelinating diseases such as multiple sclerosis. SARS-CoV emerged in the human population in China in 2002, causing a worldwide epidemic with severe morbidity and high mortality rates, particularly in older individuals. We review the pathogenesis of both viruses and the several reverse genetics systems that made much of these studies possible. We also review the functions of coronavirus proteins, structural, enzymatic, and accessory, with an emphasis on roles in pathogenesis. Structural proteins in addition to their roles in virion structure and morphogenesis also contribute significantly to viral spread in vivo and in antagonizing host cell responses. Nonstructural proteins include the small accessory proteins that are not at all conserved between MHV and SARS-CoV and the 16 conserved proteins encoded in the replicase locus, many of which have enzymatic activities in RNA metabolism or protein processing in addition to functions in antagonizing host response.

Chapter 4 - Coronavirus Pathogenesis

We demonstrated that infection of 17Cl-1 cells with the murine coronavirus mouse hepatitis virus (MHV) induced caspase-dependent apoptosis MHV-infected DBT cells did not show apoptotic changes, indicating that apoptosis was not a universal mechanism of cell death in MHV-infected cells Expression of MHV structural proteins by recombinant vaccinia viruses showed that expression of MHV E protein induced apoptosis in DBT cells, whereas expression of other MHV structural proteins, including S protein, M protein, N protein, and hemagglutinin-esterase protein, failed to induce apoptosis MHV E protein-mediated apoptosis was suppressed by a high level of Bcl-2 oncogene expression Our data showed that MHV E protein is a multifunctional protein; in addition to its known function in coronavirus envelope formation, it also induces apoptosis

Induction of Apoptosis in Murine Coronavirus-Infected Cultured Cells and Demonstration of E Protein as an Apoptosis Inducer

Rotaviruses are recognized as the leading cause of severe dehydrating diarrhea in infants and young children worldwide. Preventive and therapeutic strategies are urgently needed to fight this pathogen. In tissue culture and in vivo, rotavirus induces structural and functional alterations in the host cell. In order to better understand the molecular mechanisms involved in the events after rotavirus infection, we identified host cellular genes whose mRNA levels changed after infection. For this analysis, we used microarrays containing more than 38,000 human cDNAs to study the transcriptional response of the human intestinal cell line Caco-2 to rotavirus infection. We found that 508 genes were differentially regulated >2-fold at 16 h after rotavirus infection, and only one gene was similarly regulated at 1 h postinfection. Of these transcriptional changes, 73% corresponded to the upregulation of genes, with the majority of them occurring late, at 12 or more hours postinfection. Some of the regulated genes were classified according to known biological function and included genes encoding integral membrane proteins, interferon-regulated genes, transcriptional and translational regulators, and calcium metabolism-related genes. A new picture of global transcriptional regulation in the infected cell is presented and families of genes which may be involved in viral pathogenesis are discussed.

https://jvi.asm.org/content/jvi/76/9/4467.full.pdf

Gene expression pattern in Caco-2 cells following rotavirus infection.

Gene expression pattern in Caco-2 cells following rotavirus infection

We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously.

/pdf/rnase-l-independent-specific-28s-rrna-cleavage-in-murine-gcoarommoa.pdf

Sungwhan An

Papers

Induction of Apoptosis in Murine Coronavirus-Infected Cultured Cells and Demonstration of E Protein as an Apoptosis Inducer

Gene expression pattern in Caco-2 cells following rotavirus infection.

Gene expression pattern in Caco-2 cells following rotavirus infection

RNase L-Independent Specific 28S rRNA Cleavage in Murine Coronavirus-Infected Cells

Characterizations of coronavirus cis-acting rna elements and the transcription step affecting its transcription efficiency