Severe acute respiratory syndrome coronavirus accessory proteins 6 and 9b interact in vivo

Abstract

The 3'proximal one-third of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes the structural proteins and eight accessory proteins, including 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b, varying in length from 39 to 274aa which do not share significant homology with viral proteins of known coronaviruses. The SARS-CoV protein 6 is 63 amino acids in length and has been previously involved in virus pathogenicity and replication. To further analyze this functions, the interaction of SARS-CoV protein 6 with other viral and/or cellular factors has been analyzed during SARS-CoV infective cycle. Protein 6 immunoprecipitation from extracts of SARS-CoV infected cells and mass spectrometry analysis revealed an interaction of viral proteins 6 and 9b in biologically relevant conditions. This interaction has been reinforced by co-localization of both proteins in the cytoplasm of SARS-CoV infected cells.

Fig. 1

Amino acid sequence and transmembrane domains of SARS-coronavirus proteins 6 and 9b. (A) Amino acid sequence of protein 6 is shown in black. The transmembrane domain is highlighted in gray. (B) Amino acid sequence of protein 9b. The amino acids involved in the hydrophobic lipid-binding tunnel are highlighted in gray.

Fig. 2

SARS-CoV protein 6 expression and subcellular localization. Vero E6 cells were infected with SARS-CoV (S) or mock-infected (M). At the hours post-infection indicated in the figure (h.p.i), cells were either fixed for immunofluorescence or total cells extracts were obtained in Laemmli buffer. (A) Total cell extracts were separated by SDS-PAGE and expression of nucleoprotein (N) and protein 6 (6) was analyzed by Western blot. (B) SARS-CoV infected cell cultures were fixed at 17 h.p.i. Confocal immunofluorescence of protein 6 and ER was developed with anti-PDI rabbit antibody (ER marker) and anti-protein 6 rat antibody. Data were visualized with Alexa Fluor 488-conjugated anti-rabbit (green) and TxRed-conjugated anti-rat (red) antibodies. Co-localization of protein 6 in the ER is shown in yellow (merge). (C) Protein 6 expression was detected by immunofluorescence using an anti-protein 6 rabbit antibody and Tx-Red-conjugated anti-rabbit antibody (a–c). DAPI (blue) was used for cellular nuclei staining (d–f). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Immunoprecipitation of protein 6 in SARS-CoV infected cells. Immune matrices were prepared by incubation of protein A-Sepahrose with either anti-protein 6 (anti-protein 6) or preimmune rabbit sera (anti-control). These matrices were incubated with soluble extracts from SARS-CoV infected (S) or mock-infected cells (M). (A) After washing, aliquots of total extracts (Input) or the immunoprecipitates were analyzed by Western-blot using anti-protein 6 rat antibodies. (B) The complexity of the total extracts (Input) and immunoprecipitated samples was analyzed by silver-staining. The arrows show the IgG and the arrowhead shows the protein A.

Fig. 4

Identification of SARS-CoV interacting 9b protein by LC–MS. (A) MS/MS spectrum from the doubly-charged ion at m/z 581.8 Da spanning the sequence LGSQLSLSMAR, and corresponding to protein 9b from SARS-CoV. Figure displays the main fragmentation series (y-carboxy and b-amino). Water loss is marked with an (*). (B) Extracted Ion Chromatogram (EIC) of the SIM experiments monitoring the doubly-charged ions at m/z 581.8 (LGSQLSLSMAR, left) and 725.3 (AFQSTPIVVQMTK, right). Upper and lower panels shows respectively the results from control and anti-6-immunoprecipited SARS-infected cells preparations.

Fig. 5

Proteins 6 and 9b partially co-localize in the cytoplasm of SARS-CoV infected cells. Vero E6 cell cultures were SARS-CoV infected and were fixed and analyzed by confocal immunofluorescence. Three representative examples are shown (S.1–S.2 and S.3). SARS-CoV protein 6 was detected with rat anti-protein 6 antibody and visualized with Tx-Red-conjugated anti-rat antibody (a, f, k). SARS-CoV protein 9b was detected with mouse anti-protein 9b antibody and visualized with FITC-conjugated anti-mouse antibody (b, g, i). Merge images show the localization of proteins 6, 9b, and DAPI stained nucleus in blue (c, h, m). The masks show the partial co-localization of proteins 6 and 9b in the cytoplasm of SARS-CoV infected cells (d, i, n). The graphics show the quantification of co-localization signal (e, j, o). The asterisk shows a non-infected cell, as a control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)