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. 2006 Aug 15;103(33):12540-5.
doi: 10.1073/pnas.0605402103. Epub 2006 Aug 7.

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Wei Lu  1 , Bo-Jian Zheng, Ke Xu, Wolfgang Schwarz, Lanying Du, Charlotte K L Wong, Jiadong Chen, Shuming Duan, Vincent Deubel, Bing Sun
Affiliations

Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release

Wei Lu et al. Proc Natl Acad Sci U S A. .

Abstract

Fourteen ORFs have been identified in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome. ORF 3a of SARS-CoV codes for a recently identified transmembrane protein, but its function remains unknown. In this study we confirmed the 3a protein expression and investigated its localization at the surface of SARS-CoV-infected or 3a-cDNA-transfected cells. Our experiments showed that recombinant 3a protein can form a homotetramer complex through interprotein disulfide bridges in 3a-cDNA-transfected cells, providing a clue to ion channel function. The putative ion channel activity of this protein was assessed in 3a-complement RNA-injected Xenopus oocytes by two-electrode voltage clamp. The results suggest that 3a protein forms a potassium sensitive channel, which can be efficiently inhibited by barium. After FRhK-4 cells were transfected with an siRNA, which is known to suppress 3a expression, followed by infection with SARS-CoV, the released virus was significantly decreased, whereas the replication of the virus in the infected cells was not changed. Our observation suggests that SARS-CoV ORF 3a functions as an ion channel that may promote virus release. This finding will help to explain the highly pathogenic nature of SARS-CoV and to develop new strategies for treatment of SARS infection.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
3a protein presence in vivo and in vitro. (A) Serological assay for detecting specific IgG Abs against 3a protein in sera from SARS patients (n = 13) and healthy controls (n = 13) by ELISA (∗, P < 0.01). Virus-infected FRhK-4 cells were used for Western blot (B) and confocal microscopy assay (C). Anti-LH21 Ab was used in both Western blot and confocal microscopy assays.
Fig. 2.
Fig. 2.
Orientation of the 3a protein on the cell membrane. (A) Two specific antibodies were used for 3a protein orientation analysis. Anti-LH21 was directed against the N terminus of 3a protein, whereas anti-HA Ab was directed against the HA tag linked to the C terminus of 3a protein. (B) 3a- and vector-transfected FRhK-4 cells were permeabilized or nonpermeabilized, and 3a protein expressed on the cell surface was immunolabeled with anti-LH21 and anti-HA Abs, respectively. (C) An orientation model of the 3a protein at the cell surface. The extracellular N terminus, the intracellular C terminus, and the transmembrane domains are depicted in the model.
Fig. 3.
Fig. 3.
The 3a protein forms homodimers and tetramers. (A) A complex by immunoprecipitation using anti-HA Ab was detected by anti-LH21 Ab. The control group of HEK293 cells was transfected only with vector (Vec), and the 3a group was transfected with 3a plasmid fused with HA tag (3aHA). DTT was used to disrupt the disulfide bond formation. (B) Eight point mutants on different cysteines in the 3a protein are marked in the map (amino acid residues 81–160). (C) Eight mutants of 3a protein, M1–M8, were tested by immunoprecipitation. (D) FRET (pseudocolor) analysis of the homooligomeric 3a protein in HeLa cells. Emission spectra of 3aCFP and 3aYFP before and after photobleaching are presented. The averaged FRET efficiency of YFP–CFP fusion protein (as a positive control), 3aCFP and 3aYFP, M6CFP and M6YFP, as well as CFP and 3aYFP (as a negative control) of 10 cells each was calculated. Ef, FRET efficiency of a photobleaching region; Cf, same calculations for a nonbleaching region (n = 10).
Fig. 4.
Fig. 4.
The 3a protein forms an ion channel in Xenopus oocytes. Water-injected (A), 3a-cRNA-injected (B), and M6-cRNA-injected (C) oocytes were immunolabeled with anti-LH21 and monitored by confocal microscopy. (D) The oocytes were also lysed, and 3a protein was analyzed by Western blot. (E) Voltage dependencies of steady-state currents in control oocytes (open circles) and in oocytes with expressed 3a (open squares) or M6 protein (open triangles) in potassium (100 mM) buffer. (F) Inhibition of the 3a-mediated potassium-sensitive current by barium (open squares, 100 mM potassium buffer; open circles, in the presence of 10 mM BaCl2). (G) Dependence of the barium-sensitive current at −120 and −60 mV on barium concentration. The solid line represents a fit of Eq. 1. (H) Voltage dependencies of potassium current in bath solutions with different potassium concentrations (100 mM, open triangles; 50 mM, open circles; 10 mM, open squares). IK = ITotal − IBa; IK, the barium-inhibited potassium current; ITotal, the total current; IBa, current with the presence of 10 mM barium. For concentrations <100 mM, potassium was substituted with tetramethylammonium. All data represent averages of at least three oocytes + SEM.
Fig. 5.
Fig. 5.
SARS-CoV release is inhibited in 3a protein-suppressed FRhK-4 cells. (A) Three siRNAs targeting the 3a gene were cotransfected with the 3a expression plasmid 3aHA into FRhK-4 cells, and the suppressing effect of these siRNAs on 3a protein expression was detected by Western blot assay. Different concentrations (100, 50, 25, and 12.5 nM) of the most effective siRNA (si-003) and an unrelated siRNA (si-GFP, as negative control) were transfected into FRhK-4 cells. After incubation for 6 h, cells were infected with 100 TCID50 of SARS-CoV. Seventy-two hours after infection, intracellular SARS-CoV was measured by real-time quantitative RT-PCR in triplicate to determine copies of viral N gene (B) and P gene (C). The results were expressed as viral RNA copies per copy of β-actin, and the SDs are given. The relative virus yield in cell supernatant was titrated by TCID50 (D), and its genome was quantified by real-time RT-PCR in triplicate (E). The results from siRNA-pretreated cultures were compared with those from control transfectant (TC, defined as 100%). SDs are indicated. The virus release into the cell culture of the si-003-pretreated group was significantly reduced as compared with the si-GFP-pretreated group (P < 0.01).
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