Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 73 aa, termed E protein.The function of E protein is currently unknown. The E protein possesses two cysteines at positions 49 and 54 that are highly conserved among North American isolates. In the present study, it was shown that E protein did not homodimerize with itself nor did it heterodimerize with the nucleocapsid (N) protein. However, E protein was interactive non-covalently with itself or with the N protein as shown by pull-down assays. The significance of the E protein cysteine residues on virus replication was determined using an infectious clone. Each cysteine was substituted by serine and the mutations were introduced into a full-length clone of PRRSV. When transfected into Marc-145 cells, all cysteine mutant clones induced PRRSV-specific cytopathic effects and produced infectious progeny virus. The data indicate that cysteine residues in the E protein are not essential for replication of North American genotype PRRSV.Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae in the order Nidovirales, and it causes severe reproductive failure in pregnant sows and respiratory illness in pigs of all ages (Rossow et al., 1999). Other members of the family Arteriviridae include Lactate dehydrogenase-elevating virus (LDV) of mice, Equine arteritis virus (EAV) and Simian hemorrhagic fever virus (SHFV) (Cavanagh, 1997). The PRRSV genome is a singlestranded, positive-sense RNA of approximately 15 kb in size that encodes two large polyproteins, 1a and 1ab, in the 59-terminal 12 kb region and seven structural proteins in the 39-terminal 3 kb region: GP2 (glycoprotein 2), small envelope (E) protein, GP3, GP4, GP5, membrane (M) protein and nucleocapsid (N) protein (Meulenberg et al., 1993;Snijder & Meulenberg, 1998;Nelsen et al., 1999). Based on antigenic and genetic differences, PRRSV isolates are divided into two distinct genotypes, European type and North American type. The genetic similarity between the two groups is approximately 63 % (Meng et al., 1995; Nelson et al., 1993;Nelsen et al., 1999;Wootton et al., 2000).The viral genome is enclosed in the isomeric capsid structure composed of N proteins. The N protein, as the sole protein component of the viral capsid, interacts with itself through covalent and non-covalent interactions . North American PRRSV N proteins contain three highly conserved cysteine residues at amino acid positions 23, 75 and 90. By mutational analysis using the expressed protein, the cysteine at position 23 has been shown to be responsible for disulfide linkages for N-N interactions . Subsequently, using an infectious cDNA clone, the N-N interaction has been shown to be essential for virus infectivity (Lee et al., 2005). Lee et al. (2005) also showed that the cysteine at position 90 appeared to be essential for virus infectivity, while the cysteine at position 75 was not. Unlike North America...

Cysteine residues of the porcine reproductive and respiratory syndrome virus ORF5a protein are not essential for virus viability

“…Recently, it has been shown that disulphide bond formation between GP5 and M of PRRSV in insect cells co-expressing GP5 and M (Veit et al, 2014). Furthermore, E(2b) protein has been proved to be interactive noncovalently with itself or with the N protein (Lee and Yoo, 2005). In present study, we showed that ORF5a and 2b, or GP4 were interactive noncovalently, and there interactions may be a basis for virion assembly.…”

Pathogenicity and genetic characteristics associated with cell adaptation of a virulent porcine reproductive and respiratory syndrome virus nsp2 DEL strain CA-2

“…The PRRSV CA-2 strain was described previously and was propagated in MARC-145 or PAM-pCD163 cells (Choi et al, 2014). The CA-2 virus was plaque purified twice in MARC-145 cells, and the purified virus was serially passaged on MARC-145 cells as described previously with some modifications (Lee and Yoo, 2005). Briefly, sub-confluent MARC-145 cells grown in 100-mm diameter tissue culture dishes were inoculated with 1 ml of 100-fold diluted PRRSV CA-2.…”

Disulfide Linkages Mediating Nucleocapsid Protein Dimerization Are Not Required for Porcine Arterivirus Infectivity

“…For example, the type II PRRSV E protein contains two cysteines at positions 49 and 54, while type I isolates contain only a single cysteine at position 51 (56,60). However, data from a previous study disproved the hypothesis that the E and N proteins interact through their cysteine residues (32). The functional significance of Cys90 in type II N proteins is still undefined.…”