7a Protein of Severe Acute Respiratory Syndrome Coronavirus Inhibits Cellular Protein Synthesis and Activates p38 Mitogen-Activated Protein Kinase

Abstract

It was recently shown that the 7a protein of severe acute respiratory syndrome coronavirus induces biochemical changes associated with apoptosis. In this study, the mechanism by which the 7a protein induces apoptosis was examined. The 7a protein was tested for the ability to inhibit cellular gene expression because several proapoptotic viral proteins with this function have previously been identified. 7a protein inhibited expression of luciferase from an mRNA construct that specifically measures translation, whereas inhibitors of transcription and nucleocytoplasmic transport did not. The inhibition of translation and other cellular processes of gene expression have been associated with the induction of a stress response in cells. Western blot analysis using phosphospecific antibodies indicated that 7a protein activated p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal protein kinase/stress-activated protein kinase. Taken together, these data indicate that the induction of apoptosis by the 7a protein may be related to its ability to inhibit cellular translation and activate p38 MAPK.

Severe Acute Respiratory Syndrome Coronavirus Gene 7 Products Contribute to Virus-Induced Apoptosis

“…Several cellular organelles are capable of sensing proapoptotic signals, including the ER, lysosomes, and mitochondria (16). Although ER stress can induce apoptosis, the ER stress-induced MAPK JNK is not activated after ORF7a expression; however, p38 MAPK is activated by ORF7a expression, suggesting that the inhibition of protein synthesis and induction of apoptosis are not due to misfolded protein accumulation in the ER (31). The Golgi complex has recently been implicated in stress signaling and may play a much larger role in induction of apoptosis than previously thought (22,39).…”

“…Serial passage of SARSCoV in Vero cells resulted in a 29-nucleotide deletion within ORF7b (68), suggesting that the protein is dispensable in vitro and can be rapidly mutated in the absence of selective pressures. Both ORF7a and ORF7b are Golgi-localized integral membrane proteins (31,44,57). The ORF7a protein has a 15-residue N-terminal signal sequence and is predicted to have an 81-residue luminal domain, a 21-residue transmembrane segment, and a 5-amino-acid cytoplasmic tail.…”

“…Cells were harvested and fixed 24 h later and protein expression quantified using flow cytometry. ORF7a was detected in 32.9% Ϯ 0.6% of the transfected cell population and in 42.5% Ϯ 2.0% of the infected cell population ( The ORF7b protein localizes to the Golgi apparatus in cDNA-transfected and virus-infected cells (57), but the ORF7a protein has been localized to the Golgi complex (31,44,49), to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartment (18), and to mitochondria (67). When expressed from cDNA, neither ORF7a nor ORF7b colocalized with MitoTracker dye, particularly compared to high degree of colocalization seen with GM130, a marker of the cis-Golgi (Fig.…”

“…It has been reported that ORF7a and ORF7b induce apoptosis when expressed from cDNA (31,66,67). To confirm and extend those observations, Vero cells were transfected with plasmids expressing the cDNAs for ORF7a, ORF7b, or the influenza A virus M2 protein and then screened for the induction of cell death by multiple assays.…”

“…The mechanisms for induction of cell death by the viral proteins are not clear, and it is unknown if these proapoptotic events play a significant role in induction of cell death during virus infection. In particular, the proteins encoded by SARSCoV gene 7, the ORF7a and ORF7b proteins, not only induce apoptosis but inhibit cellular protein synthesis, activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway, activate NF-B, block cell cycle progression, and interact with the cellular transcription factor small glutamine-rich tetratricopeptide repeat-containing protein (SGT) as well as the antiapoptotic regulator Bcl-X L (17,28,31,67,81). The relevance of these functions in context of viral infection is presently unknown.…”