Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP 2a , GP 3 , GP 4 and GP 5 , the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP 2a and GP 5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP 2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP 5 protein. In contrast, the oligosaccharide linked to N46 of the GP 5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP 2a or of the oligosaccharide attached to N53 of GP 5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP 2a and GP 5 for PRRSV assembly and infectivity are discussed.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production

Wissink

Kroese

Maneschijn-Bonsing

et al. 2004

“…This heterodimerization is critical to virus assembly and infectivity in EAV (Snijder et al, 2003), and a similar role is presumed for PRRSV (Xia et al, 2009). Selective pressure analysis of ORF5 indicates a varying number of positively selected sites, localized mainly on the N-terminal part and ectodomain of GP5 (Delisle et al, 2012;Storgaard et al, 1999;Xu et al, 2010), including a known antigenic site (Hanada et al, 2005;Hu et al, 2009), which probably relates to differing environmental/experimental conditions and sequence datasets among studies.…”

Section: Prrsv: the Virusmentioning

confidence: 99%

Evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus

Brar

Shī

Murtaugh

et al. 2015

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the leading swine pathogens causing tremendous economic loss to the global swine industry due to its virulence, pathogenesis, infectivity and transmissibility. Although formally recognized only two and half decades ago, molecular dating estimation indicates a more ancient evolutionary history, which involved divergence into two genotypes (type 1 and type 2) prior to the 'initial' outbreaks of the late 1980s. Type 2 PRRSV circulates primarily in North America and Asia. The relatively greater availability of sequence data for this genotype from widespread geographical territories has enabled a better understanding of the evolving genotype. However, there are a number of challenges in terms of the vastness of data available and what this indicates in the context of viral diversity. Accordingly, here we revisit the mechanisms by which PRRSV generates variability, describe a means of organizing type 2 diversity captured in voluminous ORF5 sequences in a phylogenetic framework and provide a holistic view of known global type 2 diversity in the same setting. The consequences of the expanding diversity for control measures such as vaccination are discussed, as well as the contribution of modified live vaccines to the circulation of field isolates. We end by highlighting some limitations of current molecular epidemiology studies in relation to inferring PRRSV diversity, and what steps can be taken to overcome these and additionally enable PRRSV sequence data to be informative about viral phenotypic traits such as virulence.

“…Among these, GP5 and M proteins form a disulfidelinked heterodimer, which is an essential requirement for infectivity in LDV and EAV (Faaberg et al, 1995;Snijder et al, 2003). A recent study shows that GP2, GP3 and GP4 proteins form a disulfide-linked heterotrimer in EAV and that this heterotrimerization is essential for EAV infectivity (Wieringa et al, 2003a, b).…”

mentioning

confidence: 99%

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

Lee

Yoo

2005

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...