SARS-CoV infection results in severe disease with high morbidity and mortality. This is in contrast to other human coronaviruses, which usually cause a mild upper respiratory tract infection. As is the case with other coronaviruses, the novel SARS-CoV accessory proteins that are unique to SARS-CoV likely contribute to the highly pathogenic nature of the virus. Thus, further characterization of these proteins is likely to elucidate mechanisms by which SARS-CoV disrupts cellular functions and causes disease.
In detailed studies of the 3a protein, we discovered that it causes intracellular vesicle formation, which is a prominent feature of cells from SARS patients (15
) and is both necessary and sufficient for Golgi fragmentation during virus infection. Vesicles containing ORF 3a protein are acidified compartments that also contain the trans
-Golgi protein TGN38 and the marker of late endosomes and lysosomes, Lamp1. These data are consistent with previous findings that ORF 3a interacts with caveolin and thus the endocytic pathway (40
). It will be important to discover whether the 3a-mediated processes of Golgi fragmentation and vesicle formation are directly related or whether they are coincidental disruptions that occur during infection. Finally, Golgi fragmentation was reduced by overexpression of the Golgi regulator protein, Arf1, suggesting that the functioning of this cellular protein may be disrupted by ORF 3a.
Positive-strand RNA viruses utilize intracellular membranes on which to replicate their RNA (28
). It is thought that the membrane acts as a nucleation site for replicase proteins and polymerases. Many RNA viruses, including the human enterovirus poliovirus and the group II coronavirus, MHV, cause a proliferation in cellular membranes or the formation of intracellular vesicles, which is believed to be advantageous for virus replication (21
). Interestingly, poliovirus-induced membrane rearrangement occurs through activation of Arf1 (5
). Electron microscopy analysis of MHV-infected cells shows the presence of double-membrane vesicles (DMVs), which are thought to be produced by autophagy. In one study, inhibition of cellular autophagy reduced MHV replication (44
). We found that the formation of intracellular vesicles during SARS-CoV infection does not affect replication, since depletion of ORF 3a from SARS-CoV abrogated vesicle formation but did not reduce the levels of viral RNA isolated from the cells and from the media. Although SARS-CoV may use vesicular membranes as sites of RNA replication (54
), intracellular vesicles may not be necessary, and perhaps not even beneficial, to the virus.
These observations raise the question, if not for replication, why would SARS-CoV cause such dramatic membrane rearrangement? Several possible explanations exist. Evidence suggests that poliovirus, which also induces intracellular vesicles and DMVs, may utilize the vesicles for nonlytic release of virus particles (24
). One study reported a deficiency in SARS-CoV release when 3a expression was reduced by RNA interference (RNAi) (34
). However, targeted disruption of group-specific ORFs by RNAi may also reduce genomic RNA and confound this interpretation. Our data suggest that release of genomic RNA is not affected by depletion of ORF 3a, since RNA levels observed in the media were equivalent for Δ3a and WT virus. Alternatively, rearrangement of intracellular membranes can coincide with disruption of the secretory pathway, which could facilitate immune evasion by inhibiting antiviral cytokine secretion or antigen presentation by major histocompatibility complex on the cell surface. Disruption of the secretory pathway by ORF 3a may require additional viral proteins, since expression of SARS-CoV ORF 3a protein by replication-deficient particles of Venezuelan equine encephalitis virus does not inhibit secretion of beta interferon (14
). It will also be important to determine the membrane location on which the virus replicates in the absence of 3a. Mitochondrial membranes represent a potential site of coronavirus replication. Several SARS-CoV proteins, including 3b and 9b, localize to mitochondria (our unpublished observations) and have been demonstrated to bind replicase proteins nsp8 and nsp14 (62
Except for the recent study on the 7a protein (52
), studies on death induction by SARS proteins have been limited to transfection/overexpression studies of the individual proteins, most likely due to the difficulty of working with this lethal pathogen. Consistent with previous reports, we have observed that overexpression of 3a is sufficient to cause cell death (31
). Importantly, our experiments with SARS-CoV Δ3a clearly demonstrate that this protein contributes to the cytotoxicity of the virus and illustrate its biological significance. Residual cytotoxicity of the SARS-CoV Δ3a virus may be attributable to the 7a protein, which is intact in this mutant strain and also contributes to virus-induced cell death (52
). Future studies that incorporate additional deletion mutants will be necessary to discern the mechanism and importance of SARS-CoV-induced cell death. An animal model that recapitulates the characteristics of human SARS does not currently exist, and it therefore remains difficult to determine the relative importance of ORF 3a to SARS-CoV pathogenesis. However, recent adaptation of SARS-CoV in mice has led to a lethal model that can be used to investigate the contribution of group-specific ORFs to SARS disease (47
) and thus might provide further insight into the functioning of these novel proteins.