Identification of a novel structural protein of arteriviruses

Abstract

Arteriviruses are positive-stranded RNA viruses with an efficiently organized, polycistronic genome. A short region between the replicase gene and open reading frame (ORF) 2 of the equine arteritis virus (EAV) genome was previously assumed to be untranslated. However, here we report that this segment of the EAV genome contains the 5' part of a novel gene (ORF 2a) which is conserved in all arteriviruses. The 3' part of EAV ORF 2a overlaps with the 5' part of the former ORF 2 (now renamed ORF 2b), which encodes the GS glycoprotein. Both ORF 2a and ORF 2b appear to be expressed from mRNA 2, which thereby constitutes the first proven example of a bicistronic mRNA in arteriviruses. The 67-amino-acid protein encoded by EAV ORF 2a, which we have provisionally named the envelope (E) protein, is very hydrophobic and has a basic C terminus. An E protein-specific antiserum was raised and used to demonstrate the expression of the novel gene in EAV-infected cells. The EAV E protein proved to be very stable, did not form disulfide-linked oligomers, and was not N-glycosylated. Immunofluorescence and immunoelectron microscopy studies showed that the E protein associates with intracellular membranes both in EAV-infected cells and upon independent expression. An analysis of purified EAV particles revealed that the E protein is a structural protein. By using reverse genetics, we demonstrated that both the EAV E and GS proteins are essential for the production of infectious progeny virus.

FIG. 1

Overview of the replication cycle of arteriviruses based on the family prototype, EAV. The viral genome and the nested set of subgenomic mRNAs are indicated, with the small black boxes representing the common 5′ leader sequence. The previously identified genes (14, 16) are depicted, with the white boxes representing the translationally active ORF(s) for each mRNA.

FIG. 2

Sequence analysis of the ORF 2a-containing genomic region of EAV and the corresponding parts of the genomes of the three other arteriviruses. cDNA sequences as well as amino acid sequences of significant ORFs are given; potential translation initiation codons (ATG) are underlined. The arrows indicate the start sites of the newly identified ORFs in the sequences. The positions of the TRSs of the relevant mRNAs are also indicated.

FIG. 3

Schematic overview of the revised genomic organization of the four arteriviruses. (A) Schema of the partial genomic organization of EAV, LDV, PRRSV, and SHFV in which the positions of the novel gene (in black) and previously identified ORFs are indicated. The TRSs used for the synthesis of mRNAs 2 and 3 (or mRNAs 4 and 5 in SHFV) are also depicted (arrows). (B) Comparison of the genomic organizations of the complete 3′ portions of the EAV and SHFV genomes. The hatched ORFs 2a, 2b, and 3 in SHFV have been proposed to be derived from a three-gene duplication (29). The SHFV ORFs 4a to 9 are assumed to be the homologs of EAV ORFs 2a to 7 (see the text).

FIG. 4

Analysis of arterivirus E proteins. (A) Alignment of the sequences of the E proteins of EAV, LDV, PRRSV, and SHFV. Absolutely conserved residues are indicated by asterisks; basic amino acid residues are underlined, to show their clustering in the C-terminal domain of the protein; dashes correspond to gaps introduced for optimal alignment. The conserved potential N-terminal myristoylation site (G-[not E, D, R, K, H, P, F, Y, or W]-X-X-[S, T, A, G, N, or C]-[not P]) and the internal phosphorylation site for casein kinase II ([S or T]-X-X-[D or E]) are indicated by arrows. (B) Hydrophobicity analysis of the arterivirus E proteins, using the method of Kyte and Doolittle and a 9-residue moving window (35). aa, amino acids.

FIG. 5

Identification and stability of the EAV E protein in infected cells and upon independent expression in the MVA-T7 system. (A) The left panel shows an immunoprecipitation analysis of lysates of EAV (V)- or mock (M)-infected BHK-21 cells that were ³⁵S labeled for 60 min at 7 h p.i. Immunoprecipitations were performed with the E protein-specific antiserum (E) or the corresponding preimmunization serum (pE). The positions of the EAV nucleocapsid protein (N), the full-length E protein (E), and a truncated version of the E protein (E_i), which was only observed after a long exposure of the gel, are indicated. The right panel shows the results of a pulse-chase experiment. Cells were pulse labeled for 15 min and subsequently chased for the time periods (in minutes) indicated above the lanes. Protein synthesis during the chase was inhibited by the addition of cycloheximide. The numbers on the right represent the sizes (in kilodaltons) of marker proteins (M_r) run in the same SDS–20% polyacrylamide gel. (B) Corresponding analysis of the independently expressed EAV E protein, using lysates of MVA-T7-infected and pAVI02a (2a)- or pAVI16 (6)-transfected OST-7.1 cells that were labeled at 6 h p.i., as described above for the EAV-infected cells. Ab, antibody.

FIG. 6

Subcellular localization of the EAV E protein. (A) Immunofluorescence analysis of the localization of the EAV E protein in infected BHK-21 cells (8 h p.i.) (left panel) and upon independent expression in the same cells, using the MVA-T7 system and expression vector pAVI02a (7 h p.i.) (right panel). Cells were double labeled for the EAV E protein and either an ER marker (PDI) or a marker of the Golgi complex (the EAV G_L glycoprotein in infected cells, or the protein recognized by MAb F20/65-1-4 in transfected cells). In both systems, part of the E protein was seen in the Golgi complex, but most of it colocalized with PDI. Staining of mock-infected cells and MVA-T7-infected, pAVI16-transfected cells with the E protein-specific antiserum did not yield a detectable signal (data not shown). Bar, 25 μm. (B) Immunogold labeling of cryosections of EAV-infected RK-13 cells (8 h p.i.) with the E protein-specific antiserum and protein A coupled to 10-nm-diameter gold particles. A specific but not very abundant labeling of the membranes of the Golgi complex and ER (not shown) was observed. Bar, 100 nm.

FIG. 7

Identification of the E protein in EAV particles. (A) Analysis of pellets obtained after ultracentrifugation through a 20% (wt/wt) sucrose cushion of supernatants from mock- or EAV-infected BHK-21 cells that were labeled with ³⁵S[Met]-³⁵S[Cys]. Pellets were analyzed directly (−) or resuspended and subjected to immunoprecipitation analysis with E protein-specific antiserum (E) or the preimmunization serum (pE). The positions of the EAV E protein (8 kDa), N protein (apparent molecular mass, 14 kDa), M protein (16 kDa), and the small (G_S) and large (G_L) glycoproteins (25 kDa and 30 to 42 kDa, respectively) are shown at the left. The positions and sizes (in kilodaltons) of marker proteins (M_r) analyzed in the same gel are indicated at the right. Ab, antibody. (B) Sucrose density gradient centrifugation of [³⁵S]Met- or [³⁵S]Cys-labeled EAV preparations. The numbers of the gradient fractions and the position of each sample relative to the top and bottom of the centrifuge tube are indicated, as are the positions of the EAV structural proteins E, N, M, G_S, and G_L. Note that the N protein does not contain any Cys residues.

FIG. 8

Viral protein synthesis in BHK-21 cells transfected with EAV030-2aKO (2a⁻), EAV030-2bKO (2b⁻), and wild-type (wt) EAV030 RNA. ³⁵S labeling was carried out from 10 to 14 h posttransfection, and immunoprecipitations were performed with antisera recognizing the replicase cleavage product nsp2 (56), the EAV E protein (see the text), the EAV G_S protein (16), and a mixture of antibodies recognizing the EAV M and N proteins (16). The positions of the different structural proteins and nsp2 are displayed at the left. Numbers on the right indicate sizes in kilodaltons. Ab, antibody.