Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production

Abstract

The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 39-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 59 end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection. INTRODUCTIONArteriviruses are enveloped viruses (approx. 60 nm diameter) with a 13-16 kb positive-sense RNA genome. The family Arteriviridae (Snijder & Meulenberg, 1998; currently comprises four members: equine arteritis virus (EAV, the family prototype), lactate dehydrogenase-elevating virus (LDV) of mice, porcine reproductive and respiratory syndrome virus (PRRSV) and simian haemorrhagic fever virus (SHFV). The consequences of arterivirus infection can range from an asymptomatic, persistent or acute infection to abortion or lethal haemorrhagic fever (Maclachlan et al., 2007). Three of the four arteriviruses (EAV, LDV and SHFV) were first isolated and characterized approximately 50 years ago.In the late 1980s, two distantly related PRRSV genotypes, European (PRRSV-EU) and North American (PRRSV-NA), sharing a genome-wide mean nucleotide identity of only~63 % (Allende et al., 1999; Suárez et al., 1996), emerged simultaneously on both sides of the Atlantic (Collins et al., 1992;Wensvoort et al., 1991). Since then, the virus has been causing worldwide epidemics of a previously unknown reproductive and respiratory disease and has become one of the most economically important swine diseases. Recently, a large outbreak of highly virulent PRRSV affected the Asian swine industry, causing considerable economic losses (Zhou & Yang, 2010).The unification of the ...

Porcine reproductive and respiratory syndrome virus: Genetic diversity of recent British isolates

“…The cysteine residue at position 30 was maintained in every isolate. A putative transmembrane domain (Firth et al, 2011) spanning positions 12-30 is well conserved overall, with hypervariable amino acids at positions 18 and 26.…”

“…All sequences were 132 base pairs long, encoding the complete 43 amino acid long protein recently described (Firth et al, 2011;Johnson et al, 2011). The degree of nucleic acid similarity ranged from 100% to 82.5% (mean 94.2%).…”

Sequence analysis of minor protein genes of equine arteritis virus during persistent infection

“…The 2 most 5'-proximal ORFs (1a and 1b) occupy approximately three-quarters of the genome and encode 2 replicase polyproteins. The remaining 3' quarter contains eight ORFs which encode 8 structural proteins: E (envelope protein), GP2 (glycoprotein 2), GP3 (glycoprotein 3), GP4 (glycoprotein 4), 5a, GP5 (glycoprotein 5), M (matrix protein), and N (nucleocapsid protein) (8,10,22).…”

The N-N non-covalent domain of the nucleocapsid protein of type 2 porcine reproductive and respiratory syndrome virus enhances induction of IL-10 expression

“…Among them, ORF7 encodes the nucleocapsid (N) protein (Firth et al, 2011;Johnson et al, 2011;Snijder et al, 2013). PRRSV is divided into two genotypes: European (type 1 virus) and American (type 2 virus) (Meng et al, 1995).…”