Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus

Abstract

A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoV-infected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.

FIG. 1.

Genome organization of SARS-CoV. ORFs encoding the nonstructural proteins (black boxes), as well as ORFs encoding the structural polypeptides (gray boxes) are indicated Also, selected ORFs encoding for putative accessory genes (unshaded boxes) are shown. ORF7a (also called ORFX4 and ORF8) encoding peptide U122 is represented by the striated box. The ORFs shown are labeled according to Snijder et al. (23). S, spike; N, nucleocapsid.

FIG. 2.

U122 is expressed in SARS CoV-infected Vero E6 cells. (A) Analysis of the U122 putative sequence predicts a signal peptide sequence (underlined) at the N terminus, the cleavage site of which is indicated with an arrow. A putative membrane-spanning domain (boxed) and an ER retrieval motif (bold italics) are found at the C terminus. (B) Blots of Vero E6 cells probed with mouse anti-U122 antiserum. Lane 1, 20 μg of protein from uninfected cells; lanes 2 and 3, 20-μg samples of proteins from SARS-CoV-infected cells harvested at about 25% CPE and 50 to 75% CPE, respectively; lane 4, 15 μg of total protein from cells transfected with pXJU122 plasmid; lane 5, 20 μg of total lysate from an IBV-infected cell culture. (C) Uninfected and virus-infected cells at 25% CPE were fixed and stained with mouse anti-U122 antibody. WB, Western blot; mαU122, mouse anti-U122 antibody.

FIG. 3.

Processing of U122 expressed in Vero E6 cells. (A) Untagged U122 was translated in vitro and visualized by autoradiography. A single band of ∼17.5 kDa was detected. (B) Untagged U122-L (lane 1), matU122 (lane 2), and U122 (lane 3) proteins were expressed in Vero E6 cells. Total proteins were extracted by using RIPA buffer, and 20 μg of total protein was used for SDS-15% PAGE. Western blots of these proteins were probed with mouse anti-U122 antibodies. (C) Vero E6 cells were transfected with plasmid pXJU122 for pulse-chase analysis. At 6 h posttranfection, cells were starved for 30 min in cysteine- and methionine-deficient medium and subsequently labeled for 30 min with ³⁵S-labeled amino acids. Cells were either lysed directly (time zero) or chased for 30, 60, 90, or 120 min. Cell lysates were immunoprecipitated with mouse anti-U122 antibodies and separated on an SDS-15% PAGE gel, followed by autoradiography. (D) Quantification of the pulse-chase experiment. The amount of immature (▴) and mature (○) ³⁵S-labeled U122 protein was determined with a densitometer and expressed as a percentage of the total labeled protein at each time point (results are means ± standard error of the means for two experiments). IP, immunoprecipitation; WB, Western blot; mαU122, mouse anti-U122 antibody.

FIG. 4.

The U122K>E protein is rapidly processed in transfected cells. (A) Untagged U122 and U122K>E proteins were expressed in transfected Vero E6 cells. At 16 h posttransfection total proteins were extracted by using RIPA buffer and 20 μg of protein separated on an SDS-15% PAGE gel. Western blots of these proteins were probed with mouse anti-U122 antibodies. (B) Vero E6 cells were transfected with pXJU122K>E for pulse-chase analysis. At 6 h posttranfection, cells were starved for 30 min in cysteine- and methionine-deficient medium and subsequently labeled for 10 min with ³⁵S-labeled amino acids. Cells were either lysed directly (time zero) or chased for 10, 20, 30, or 60 min. Cell lysates were immunoprecipitated with mouse anti-U122 antibodies, followed by separation on an SDS-15% PAGE gel and autoradiography. IP, immunoprecipitation; WB, Western blot; mαU122, mouse anti-U122 antibody.

FIG. 5.

Intracellular localization of expressed U122 and retrieval signal mutant. Vero E6 cells were transfected with plasmid pXJU122 or pXJU122K>E. At 16 h posttransfection, cells were fixed with methanol and labeled with mouse anti-U122 antibody (left panels) and either antibodies to the ER marker GRP94 (rat anti-GRP94), the intermediate compartment marker Sec31 (rabbit anti-Sec31), or Golgi marker GS28 (rabbit anti-GS28) (middle panels). When anti-U122 was used with anti-GS28 or anti-Sec31, FITC-conjugated goat anti-mouse and Rh-conjugated anti-rabbit antibodies (Santa Cruz Biochemicals) were used as secondary antibodies. For double labeling with anti-U122 and anti-GRP94, FITC-conjugated anti-rat and Rh-conjugated anti-mouse (Santa Cruz Biochemicals) antibodies were used as secondary antibodies. Merged images showed colocalization of U122 and K>E proteins with the marker proteins (right panels).