Structure and Intracellular Targeting of the SARS-Coronavirus Orf7a Accessory Protein

Abstract

The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 A resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded beta sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

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

“…For indirect immunofluorescence analysis (IFA), cells were stained and mounted as previously described (44,57). The primary antibodies used were rabbit anti-ORF7b polyclonal serum (1:1,000) (57), anti-ORF7a mouse monoclonal antibody (MAb) 2E11 (1:1,000 dilution) (44), anti-GM130 mouse MAb (1:100 dilution; BD Biosciences), rabbit anticalnexin immunoglobulin G (IgG) (1:100 dilution; Chemicon), and rabbit anti-ERGIC53 IgG (1:100 dilution; Sigma). Mitochondrial staining was performed using MitoTracker Red CMXRos (Invitrogen) at 250 nM per the manufacturer's protocol.…”

“…Fluorescence-activated cell sorting (FACS) was performed as described previously (44,57). Cells were incubated with the anti-ORF7a murine MAb 2E11 (1:1,000 dilution) (44), anti-ORF7b rabbit serum (1:1,000 dilution) (57), anti-SARS S MAb cocktail (1:100 dilution; kindly provided by CDC), anti-SARS mouse hyperimmune serum (1:1,000 dilution; kindly provided by CDC), or anti-active caspase 3 rabbit MAb (1:1,000 dilution; R&D Systems) followed by goat anti-mouse IgG (1:500 dilution, AlexaFluor 488 or AlexaFluor 647 labeled; Molecular Probes) or goat anti-rabbit IgG (1:500 dilution, AlexaFluor 488 or AlexaFluor 647 labeled; Molecular Probes) secondary antibody.…”

“…At approximately 100% confluence, cells were infected at an MOI of 5.0 and incubated for 24, 48, and 72 h. Infected cells were washed three times with PBS, fixed with 2% paraformaldehyde in PBS for 10 min, and then processed for IFA as previously described (44,57), followed by TUNEL staining performed as described above, with staining solution added directly atop the coverslips. Coverslips were mounted onto microscope slides using Prolong Antifade Gold (Molecular Probes) and visualized on a Zeiss LSM 510 META confocal microscope.…”

The search for a structural basis for therapeutic intervention against the SARS coronavirus

“…Orf7a, an integral membrane protein expressed on the membrane surface of host cells infected with the SARS virion, has also been suggested to be a structural protein . The structure of the soluble luminal domain of orf7a has been determined, although the function of the full-length protein remains unclear (Nelson et al, 2005). Don Wiley and colleagues used their comprehensive study of influenza hemagglutinin (HA) to propose the classical mechanism of class I fusion proteins for mediating enveloped virus and host-cell membrane fusion (Skehel & Wiley, 2000;Eckert & Kim, 2001).…”

“…As the accessory proteins vary among different coronaviruses, they almost certainly would not be targets for the design of broad-spectrum anti-virals. Two accessory protein structures have been determined to date: the orf7a luminal domain (Nelson et al, 2005) and orf9b, a lipid-binding protein (Meier et al, 2006).…”