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.

Equine arteritis virus

“…The structural proteins of the EAV virion include seven envelope proteins (E, GP2, GP3, GP4, ORF5a protein, GP5, and M) and the N protein, which respectively are encoded by ORFs 2a, 2b, 3-4, 5a, 5b, and 6-7 that are located at the 3 0 proximal quarter of the genome (Fig. 2;de Vries et al, 1992;Firth et al, 2011;Snijder and Spaan, 2007;Snijder et al, 1999;Wieringa et al, 2004). These structural proteins are expressed from six subgenomic viral messenger RNAs (sg mRNAs) that form a 3 0 -co-terminal nested set and contain a common leader sequence encoded by the 5 0 -end of the genome (Fig.…”

“…With the exception of the bicistronic sg mRNAs 2 and 5, the sg mRNAs are functionally monocistronic. Translation of proteins from sg mRNAs 2 (E and GP2 proteins) and 5 (ORF5a protein and GP5) occurs by leaky scanning of the 5 0 -proximal end of these sg mRNAs (Firth et al, 2011;Snijder et al, 1999). The ORFs 1a and 1b located at the 5 0 end of the genome and are translated into two polyproteins (pp1a and pp1ab) that are further processed into 12-13 nonstructural proteins by three viral proteases (nsps 1, 2, and 4).…”

“…The EAV sg mRNAs contain a common 5 0 sequence of 211 nt called ''leader'', which is identical to the 5 0 end of the viral genomic RNA (Snijder and Meulenberg, 1998). The leader TRS is located in a hairpin loop structure at the 3 0 end of the leader (van den Born et al, 2005a) and the body TRSs are present upstream of every structural protein gene except ORFs 2b and 5a (de Vries et al, 1990;den Boon et al, 1996;Firth et al, 2011;Snijder et al, 1999). The EAV genome contains multiple UCAACU sequences that could be transcriptionally silent.…”

“…With the exception of the last one, each sg mRNA, is structurally polycistronic, although they are functionally monocistronic and only the 5 0proximal ORF is translated from each sg mRNA. However, there are two exceptions, where sg mRNAs 2 and sg mRNA 5 are functionally bicistronic and each encode two structural proteins per sg mRNA (Firth et al, 2011;Snijder et al, 1999).…”

“…GP2 is abundant in EAV-infected cells, but is expressed in low amounts in viral particles. The GP2 protein is not essential for viral RNA replication and transcription (Molenkamp et al, 2000;Snijder et al, 1999). Sequence comparison analysis indicates that the GP2 protein is highly conserved between EAV isolates (Hedges et al, 1996).…”