Virus Host Interactions in SARS Coronavirus Infection

The global outbreak of Severe Acute Respiratory Syndrome which began in 2002 and ended in 2003 resulted in over 8000 cases in 29 countries, causing an atypical pneumonia which resulted in a case fatality rate of ~10%. The etiological agent of this disease was found to be a novel coronavirus dubbed SARS Coronavirus (SARS-CoV). One of the distinguishing features of this virus is the presence of several accessory genes in its positive-stranded RNA genome which have no known homologs in the coronavirus family. In this thesis we examine virus host interactions in the context of SARS coronavirus in order that some insight may be gained into the biology of this virus, which is still the subject of investigation. A lot of work has been done to study the function of SARS-CoV accessory proteins. One of these is the ORF6 accessory protein, which has been shown to accelerate infection, interfere with interferon production and signaling by impeding nuclear translocation, induce membrane rearrangements in the host cell and induce ER stress and subsequent caspase-mediated apoptosis. Examination of the ability of ORF6 to interact with other virus proteins by a yeast-two-hybrid assay showed an interaction with the nsp8 replicase protein. A direct interaction was shown using a cell-free system, and subsequently, transient transfection and immunoprecipitation experiments in Vero E6 cells showed that these proteins interacted in mammalian cells, which was confirmed in SARS-CoV infected cells. Examination of the co-localisation of these proteins in infected Vero E6 cells also showed a subcellular localization of the ORF6 protein to a intracellular vesicular population which was Lamp1- and CD63-positive. The impedance of nuclear translocation by the ORF6 protein is linked to its ability to function as an interferon antagonist. A plasmid-based system comprising a CMV-derived promoter was used to replicate this impedance, and it was found that the impedance is dose-dependent. Additionally, plasmidexpressed ORF6 was shown by immunofluorescence to colocalise with an intracellular vesicular population positive for Lamp1 and CD63. Alanine substitution of a putative diacidic motif in the ORF6 protein showed a reduction in the impedance of nuclear translocation and when examined by immunofluorescence also showed clustering of ORF6-stained vesicles, suggesting that the impedance of nuclear import by ORF6 and its localisation to this specific vesicular population are linked. In order to characterize the differential expression of genes in host cells which might lead to ORF6- mediated apoptosis, a defective recombinant vaccinia virus expressing ORF6 was generated. Analyses were performed using a QPCR array specific to human apoptotic signaling as well as microarray analysis for examination of genome-wide differential expression. Subsequent analysis of these results suggested that the extrinsic pathway of apoptosis was upregulated in cells expressing ORF6, and that intracellular calcium flux might have a role to play in ORF6-mediated apoptosis. The introduction of an exogenous nitric oxide donor into Vero E6 cells infected with SARS-CoV has been shown to exert a negative effect on viral replication. Here an attempt was made to explore the mechanism of this inhibition. Nitric oxide can exert an effect on viruses either directly or through several redox intermediates, one of which is peroxynitrite, which is formed through the reaction of nitric oxide and superoxide. It was shown that the effect of the exogenous nitric oxide donor SNAP on SARSCoV replication was not due to the effect of peroxynitrite, but probably through direct action of nitric oxide or another derivative. Examination of the palmitoylation of the S protein in SNAP-treated cells showed that SNAP treatment was able to reduce S palmitoylation and consequently its binding to the ACE-2 receptor in an in vitro cell-cell fusion assay. Also, QPCR analysis of viral RNA production showed a significant reduction in SNAP-treated cells. Examination of the cleavage of viral replicase polyproteins in these cells showed an alteration in the cleavage pattern of the replicase polyprotein

A putative diacidic motif in the SARS-CoV ORF6 protein influences its subcellular localization and suppression of expression of co-transfected expression constructs

<p>Abstract</p> <p>Background</p> <p>The ORF6 protein is one of the eight accessory proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV). Numerous properties of ORF6 have been documented and this study focuses on two of these, namely, its ability to suppress the expression of co-transfected expression constructs and its subcellular localization to vesicular structures.</p> <p>Results</p> <p>Using a transient transfection system, ORF6's ability to suppress the expression of co-transfected expression constructs was measured in a quantitative manner. While ORF6 does not have a global effect on protein synthesis, quantitative real-time PCR revealed that it down-regulated the mRNA level of the co-transfected myc-nsp8 gene. Furthermore, alanine substitution of a diacidic cluster motif (aa53-56) in the ORF6 gene caused a reduction in the suppression of expression of co-transfected myc-nsp8 gene. Our previous study revealed that ORF6 localized to vesicular structures in SARS-CoV infected Vero E6 cells. Here, ORF6 was observed to be localized to similar vesicular structures in Vero E6 cells which have been transiently transfected with a mammalian expression plasmid encoding for untagged ORF6. ORF6 showed partial colocalization with cellular proteins CD63 and Lamp1, suggesting that the vesicular structures may be a subpopulation of endosomal/lysosomal vesicles. The alanine substitution of the diacidic cluster motif also altered the subcellular localization of the ORF6 protein, indicating a potential relationship between the subcellular localization of the ORF6 protein and its ability to suppress the expression of co-transfected expression constructs.</p> <p>Conclusions</p> <p>By combining quantitative real-time PCR and transient transfection system, a simple and safe method is established to measure ORF6's ability to suppress the expression of co-transfected myc-nsp8. In addition, immunofluorescence analysis revealed that the subcellular localization of ORF6 when expressed on its own is similar to that observed in SARS-CoV infected cells. Through the use of these two assays, a putative diacidic motif in the ORF6 protein was found to influence its subcellular localization and ability to suppress the expression of co-transfected expression constructs.</p

Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT

Severe acute respiratory syndrome coronavirus (SARS-CoV) 7a is an accessory protein with no known homologues. In this study, we report the interaction of a SARS-CoV 7a and small glutamine-rich tetratricopeptide repeat-containing protein (SGT). SARS-CoV 7a and human SGT interaction was identified using a two-hybrid system screen and confirmed with interaction screens in cell culture and cellular co-localization studies. The SGT domain of interaction was mapped by deletion mutant analysis and results indicated that tetratricopeptide repeat 2 (aa 125-158) was essential for interaction. We also showed that 7a interacted with SARS-CoV structural proteins M (membrane) and E (envelope), which have been shown to be essential for virus-like particle formation. Taken together, our results coupled with data from studies of the interaction between SGT and HIV-1 vpu indicated that SGT could be involved in the life-cycle, possibly assembly of SARS-CoV.IS

A RT-qPCR system using a degenerate probe for specific identification and differentiation of SARS-CoV-2 Omicron (B.1.1.529) variants of concern

Fast surveillance strategies are needed to control the spread of new emerging SARS-CoV-2 variants and gain time for evaluation of their pathogenic potential. This was essential for the Omicron variant (B.1.1.529) that replaced the Delta variant (B.1.617.2) and is currently the dominant SARS-CoV-2 variant circulating worldwide. RT-qPCR strategies complement whole genome sequencing, especially in resource lean countries, but mutations in the targeting primer and probe sequences of new emerging variants can lead to a failure of the existing RT-qPCRs. Here, we introduced an RT-qPCR platform for detecting the Delta- and the Omicron variant simultaneously using a degenerate probe targeting the key ΔH69/V70 mutation in the spike protein. By inclusion of the L452R mutation into the RT-qPCR platform, we could detect not only the Delta and the Omicron variants, but also the Omicron sub-lineages BA.1, BA.2 and BA.4/BA.5. The RT-qPCR platform was validated in small- and large-scale. It can easily be incorporated for continued monitoring of Omicron sub-lineages, and offers a fast adaption strategy of existing RT-qPCRs to detect new emerging SARS-CoV-2 variants using degenerate probes.</p

A Candidate DNA Vaccine Encoding the Native SARS-CoV-2 Spike Protein Induces Anti-Subdomain 1 Antibodies

The ideal vaccine against viral infections should elicit antibody responses that protect against divergent strains. Designing broadly protective vaccines against SARS-CoV-2 and other divergent viruses requires insight into the specific targets of cross-protective antibodies on the viral surface protein(s). However, unlike therapeutic monoclonal antibodies, the B-cell epitopes of vaccine-induced polyclonal antibody responses remain poorly defined. Here we show that, through the combination of neutralizing antibody functional responses with B-cell epitope mapping, it is possible to identify unique antibody targets associated with neutralization breadth. The polyclonal antibody profiles of SARS-CoV-2 index-strain-vaccinated rabbits that demonstrated a low, intermediate, or high neutralization efficiency of different SARS-CoV-2 variants of concern (VOCs) were distinctly different. Animals with an intermediate and high cross-neutralization of VOCs targeted fewer antigenic sites on the spike protein and targeted one particular epitope, subdomain 1 (SD1), situated outside the receptor binding domain (RBD). Our results indicate that a targeted functional antibody response and an additional focus on non-RBD epitopes could be effective for broad protection against different SARS-CoV-2 variants. We anticipate that the approach taken in this study can be applied to other viral vaccines for identifying future epitopes that confer cross-neutralizing antibody responses, and that our findings will inform a rational vaccine design for SARS-CoV-2

The highly pathogenic H7N3 avian influenza strain from July 2012 in Mexico acquired an extended cleavage site through recombination with host 28S rRNA

Background: A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico. Methods: Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains. Results: The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly. Conclusions: This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can infect humans but currently lacks critical host receptor adaptations that would facilitate human to human transmission.Published versio

Reviewers: This article was reviewed by Prof Xiufan Liu (nominated by Dr Purificacion Lopez-Garcia) and Prof

A new piece in the puzzle of the novel avian-origin influenza A (H7N9) viru

Potential Human Adaptation Mutation of Influenza A(H5N1) Virus, Canada

10.3201/eid2009.140240Emerging Infectious Diseases2091580-158