The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function

Abstract

The severe acute respiratory syndrome-coronavirus (SARS-CoV) caused an outbreak of atypical pneumonia in 2003. The SARS-CoV viral genome encodes several proteins which have no homology to proteins in any other coronaviruses, and a number of these proteins have been implicated in viral cytopathies. One such protein is 3a, which is also known as X1, ORF3 and U274. 3a expression is detected in both SARS-CoV infected cultured cells and patients. Among the different functions identified, 3a is a capable of inducing apoptosis. We previously showed that caspase pathways are involved in 3a-induced apoptosis. In this study, we attempted to find out protein domains on 3a that are essential for its pro-apoptotic function. Protein sequence analysis reveals that 3a possesses three major protein signatures, the cysteine-rich, Yxx phi and diacidic domains. We showed that 3a proteins carrying respective mutations in these protein domains exhibit reduced pro-apoptotic activities, indicating the importance of these domains on 3a's pro-apoptotic function. It was previously reported that 3a possesses potassium ion channel activity. We further demonstrated that the blockade of 3a's potassium channel activity abolished caspase-dependent apoptosis. This report provides the first evidence that ion channel activity of 3a is required for its pro-apoptotic function. As ion channel activity has been reported to regulate apoptosis in different pathologic conditions, finding ways to modulate the ion channel activity may offer a new direction toward the inhibition of apoptosis triggered by SARS-CoV.

Viroporins: structure, function and potential as antiviral targets

“…3a mediates the production of infectious viral progeny, potentially linked to cellular trafficking of the spike glycoprotein (Tan, 2005), but has also been proposed to comprise a structural component of the infectious virion (Shen et al, 2005). 3a is pro-apoptotic in a number of cell lines, which appears directly dependent upon channel function, and may be linked to the induction of an ER stress response (Chan et al, 2009;Freundt et al, 2010;Law et al, 2005;Minakshi et al, 2009;Padhan et al, 2008). Two studies have reported small-molecule inhibitors targeting 3a.…”

The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

“…Antibodies to ORF3a also stain the plasma membrane [24,27,29], where it is either secreted out of the cell [30,31] or undergoes endocytosis [24]. Following ORF3a translation, the diacidic domain ensures that the protein is efficiently exported from the endoplasmic reticulum [32] to the Golgi apparatus, where ORF3a undergoes posttranslational modifications in the form of O-glycosylation [33] before being inserted into the plasma membrane.…”

“…The S protein is involved in viral assembly and packaging of coronaviruses (reviewed by [42]) and so the formation of inter-chain disulfide bonds between ORF3a and the S protein in the interior of the viral envelope may suggest that ORF3a is integrated into viral particles (reviewed by [20]). Indeed, ORF3a is incorporated into newly packaged mature SARS-CoV virions, suggesting that ORF3a is a structural protein [29,30,43]. Furthermore, the co-localization of ORF3a with M and E proteins, which are essential for viral assembly [44], suggests that ORF3a is important in SARS-CoV assembly or budding [27].…”

“…In addition to a possible role in viral replication, ORF3a is likely to modulate viral release, as a study has shown that there is a significant reduction in viral release in FRhK-4 cells transfected with ORF3a specific siRNAs [34]. Taking into account that ORF3a forms a potassium-sensitive ion channel in the plasma membrane [29,34], and ion channels for viral proteins control virus entry or release [46,47], the mechanism whereby ORF3a influences viral release warrants further investigation [34]. …”

“…Overexpression of ORF3a in several different cell culture models has demonstrated that it induces apoptosis [29,48,49,50], upregulates mRNA and protein levels of fibrinogen in lung epithelial cells [51], activates C-Jun N-terminal kinase (JNK) and the transcription factor nuclear factor kappa B (NF-kappaB), which is involved in the activation of pro-inflammatory genes [20,41,52] and downstream from that, up-regulates the production of pro-inflammatory cytokines and chemokines such as interleukin 8 (IL-8) and RANTES (CCL5) [52]. Taken together, these changes to host cellular homeostasis imply a role for ORF3a in the pathogenesis of SARS.…”