key: cord-0717565-jcb9w8y9
authors: Meulenberg, J.J.M.; Petersen den Besten, A.; de Kluyver, E.; van Nieuwstadt, A.; Wensvoort, G.; Moormann, R.J.M.
title: Molecular characterization of Lelystad virus
date: 1997-04-30
journal: Veterinary Microbiology
DOI: 10.1016/s0378-1135(96)01335-1
sha: 415ef3e7683dab7d7233c907858afecd8b52bd07
doc_id: 717565
cord_uid: jcb9w8y9

Abstract Lelystad virus (LV), the prototype of porcine reproductive respiratory syndrome virus, is a small enveloped virus, containing a positive strand RNA genome of 15 kb. LV is tentatively classified in the family Arteriviridae, which consists of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization and replication strategy as coronaviruses, but the size of the genome is much smaller (12–15 kb) and they have different morphological and physicochemical properties. The genome of LV contains eight open reading frames (ORFs) that encode the replicase genes (ORFs 1a and 1b), envelope proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7). Genomic comparison of European and North American isolates has shown that the structural proteins encoded by ORFs 2 to 7 vary widely. The amino acid sequences of ORFs 2 to 7 of North American strains share only 55 to 79% identical amino acids with those of European strains. Using polyvalent porcine anti-LV serum, gene-specific anti-peptide sera and monoclonal antibodies, we have identified six structural proteins of LV and their corresponding genes. These are: the 15 kDa unglycosylated nucleocapsid protein (N) encoded by ORF7, an 18 kDa unglycosylated integral membrane protein M encoded by ORF6, a 25 kDa N-glycosylated protein encoded by ORF5, a 31–35 kDa N-glycosylated protein encoded by ORF4, a 45–50 kDa N-glycosylated protein encoded by ORF3 and a 29–30 kDa N-glycosylated protein encoded by ORF2. A nomenclature for these structural proteins is proposed.

The porcine reproductive respiratory syndrome (PRRS) was first described a few years ago and is characterized by reproductive failure of sows and respiratory distress of piglets. In 1991 the Lelystad virus (LV; Wensvoort et al., 1991) also known as PRRS virus (Ohlinger et al., 1991 : Collins et al.. 1992 was identified as the causal agent. LV is a small, enveloped positive strand RNA virus that replicates preferentially in porcine alveolar lung macrophages, but only to a limited extent in other cells. LV is tentatively classified in the family Arterkliridue, which consists of LDV, EAV and SHFV (Meulenberg et al., 1993a; Plagemann and Moennig, 199 I) . These viruses share many characteristics such as genome organization, strategy of gene expression, the propensity to grow preferentially in macrophages and a tendency to induce persistent infections. In this review the molecular properties of LV are briefly summarized and a nomenclature for the structural proteins is proposed.

LV contains a polyadenylated positive strand RNA genome of 15.1 kb (Fig. I >. Eight open reading frames (ORFSI that encode virus-specific proteins were identified. ORFs 1 a and I b comprise about 80% of the viral genome and are predicted to encode the viral RNA dependent RNA polymerase. Their amino acid sequences contain elements conserved in RNA polymerases of the torovirus Berne virus, EAV, LDV and coronaviruses (Conzelmann et al., 1993; Meulenberg et al., 1993a) . The ORFs 3 to 7 encode structural proteins as will be discussed further below. Analogous to RNA synthesis during replication of EAV and LDV, multiple subgenomic RNAs are synthesized in LV-infected alveolar macrophages. These subgenomic RNAs have been identified in Northern blot hybridization analysis using LV-specific oligonucleotides located in the unique part of the various ORFs and at the extreme 5' and 3' end (Meulenberg et al., 1993a) . They form a 3' coterminal nested set and all contain a leader sequence derived from the 5' end of the viral genome ( Fig. 1 ). The junction site i.e. the site where the leader is fused to the body of the subgenomic RNA was identified for all six subgenomic RNAs (Meulenberg et al., 1993b) . These junction sites contain a conserved six nucleotide motif, UCAACC, or a very similar sequence.

Comparison of the amino acid sequences encoded by the ORFs identified in the genome of LV with those of other viruses indicates that LV is distantly related to coronaviruses and toroviruses and closely related to LDV and EAV. As is shown in Fig.  2 , the amino acid sequences of ORFla (apart from a gap at the N-terminal half) and ORFlb of LV are highly identical with the corresponding ORFs of LDV (Chen et al., Table 1 Amino acid sequence identity of ORFs 2 to 7 of LV with those of other isolates of PRRSV and of other arteriviruses Murtaugh et al. (1993) . ' Data derived from Meng et al. (1994) . d Data derived from Mardassi et al. (1994) . ' Data derived from Chen et al. (1993) . ' Data derived from Godeny et al. (1993) . 1993; Godeny et al., 1993) and EAV (den Boon et al., 1991) . These data and the identity observed between the amino acid sequences of ORFs 2 to 7 of these viruses (Table 1) indicate that LV is more related to LDV than to EAV. Unfortunately the sequences of ORFs la and 1 b of LV could not be compared with those of other PRRSV isolates since these have not yet been determined. However, the nucleotide sequence has been determined for ORFs 2 to 7 of another European isolate, PRRSV-10 (Conzelmann et al., 1993) two U.S. isolates, VR2332 (Murtaugh et al., 1993) and VR2385 (Meng et al., 1994) and a Canadian isolate IAFexp-91 (Mardassi et al., 1994) . The amino acid sequences of ORFs 2 to 7 of LV are highly identical with those derived from the nucleotide sequences of the European isolate PRRSV-10, but vary widely with those derived from the nucleotide sequences of North American isolates VR2332, VR2385 and IAFexp-91 (Table 1 ). This variation is in line with the antigenic differences observed in the reactivity of European and U.S. isolates with polyclonal pig sera as well as mouse monoclonal antibodies (Wensvoort et al., 1992; Nelson et al., 1993; Drew et al., 1995) .

In cells infected with LV, or the North American isolate VR2332, at least three viral proteins of 15, 18 and 25 kDa have been detected by Western blotting with polyclonal pig sera directed against the respective strains (Table 2 ; Meulenberg et al., 1995; Nelson et al., 1993) . The corresponding ORFs from which these proteins are expressed were identified with gene-specific anti-peptide sera raised in rabbits. It was shown that the 15 kDa nucleocapsid protein N is encoded by ORF7, the 18 kDa M protein is encoded by ORF6 and the 25 kDa protein is encoded by ORF5 (Table 2 ; Meulenberg et al., 1995) . Using the cell line CL2621, which is susceptible for LV. we have been able to improve the virus growth and purification methods. Hence, using more viral antigen and LV-specific monoclonal antibodies, recently developed by van Nieuwstadt et al. (1996) we have identified three additional structural proteins in Western blot analysis. These were a 29-30 kDa protein encoded by 0RF2, a 45-50 kDa protein encoded by ORF3 and a 31-35 kDa protein encoded by 0RF4. Earlier findings of Drew et al. (1995) also indicated that the 45-50 kDa ORF3 protein is a structural protein. Endoglycosidase treatment of the LV proteins has shown that the 25, 29-30, 31-35 and 45-50 kDa proteins are N-glycosylated proteins whereas the N and M protein are not van Nieuwstadt et al., 1996) . Therefore, we may conclude that LV contains six structural proteins, four glycoproteins encoded by ORFs 2 to 5, an unglycosylated membrane protein M encoded by 0RF6 and the nucleocapsid protein N encoded by ORF7.

Since we have now identified six structural proteins of LV, we would like to propose a nomenclature for these proteins. In line with the nomenclature for coronaviruses, which are distantly related to arteriviruses, general concensus exists to also use N for the nucleocapsid protein encoded by 0RF7 and to use M for the nonglycosylated membrane protein M encoded by 0RF6 of arteriviruses. The other four structural proteins. the 29-30 kDa protein encoded by 0RF2, the 45-50 kDa protein encoded by ORF3, the 3 1-35 kDa protein encoded by ORF4 and the 25 kDa protein encoded by ORF5 are glycoproteins and therefore we propose to name these GP,, GP,, GP,, and GPs, respectively.

In this way the proteins are linked to the ORFs, from which they are expressed and no confusion exists about their molecular weights. Two structural N-glycosylated proteins were identified in virions of EAV. These were a 30-42 kDa glycoprotein designated GIClarge) encoded by ORF5 and a 25 kDa glycoprotein, designated GsCFmall) encoded by 0RF2 (De Vries et al., 1992) . Since the glycoprotein encoded by 0RF2 of LV is larger than the glycoprotein encoded by 0RF5 of LV. the names G, and G, used for EAV are not appropriate for LV. The major envelope glycoprotein of 24-44 kDa of LDV is expressed by 0RF5 and named VP3 (Faaberg et al., 1995) . In our opinion the nomenclature proposed here for the structural glycoproteins of LV may also be applied for proteins expressed by the corresponding ORFs of other arteriviruses, although their molecular masses may be different.

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