Functional Properties of the Predicted Helicase of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the positive-strand RNA virus family Arteriviridae. Although considerable research has focused on this important pathogen, little is known about the function of most PRRSV proteins. To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. One region, nsp10, encoded a protein with a putative helicase domain and was further examined for functional helicase-like activities. PRRSV nsp10 was found to possess a thermolabile and pH-sensitive NTPase activity that was modulated by polynucleotides and to unwind dsRNA in a 5' to 3' polarity. These results provide the first evidence of the functional properties of PRRSV helicase and further support the finding that nidovirus helicases possess properties that distinguish them from other viral helicases.

Genomic characterization and pathogenicity of a strain of type 1 porcine reproductive and respiratory syndrome virus

“…den Boon et al, 1995;van Dinten et al, 1996;Wassenaar et al, 1997). These nsps are not only involved in viral replication and genomic transcription (Bautista et al, 2002;Fang and Snijder, 2010), but also play important roles in the modulation of host innate immune responses (Beura et al, 2010;He et al, 2015;Li et al, 2010a;Shi et al, 2011;Yoo et al, 2010), and in the pathogenicity and virulence of PRRSV (Kwon et al, 2008;Li et al, 2014a;Wang et al, 2008). ORFs 2a, 2b, ORF3 to 7, and ORF5a encode viral structural proteins including GP2a, E, GP3, GP4, GP5, M, N and ORF5a (Johnson et al, 2011;Mardassi et al, 1995;Meng et al, 1994;Meulenberg et al, 1995;Music and Gagnon, 2010;van Nieuwstadt et al, 1991;Wu et al, 2001).…”

Mutational analysis of the functional sites in porcine reproductive and respiratory syndrome virus non-structural protein 10

“…Some ZBD mutant proteins associated with different EAV phenotypes were found to be ATPase and helicase deficient (Seybert et al, 2005). PRRSV nsp10 has been shown to have ATPase activity and can unwind dsDNA or dsRNA in a 59A39 direction (Bautista et al, 2002), but is otherwise uncharacterized. In this study, a series of nsp10 mutants potentially affecting DNA binding, ATP hydrolysis and DNA unwinding were constructed, and their biochemical activities were tested to identify critical sites related to different enzymic functions.…”

Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

“…The viral genome contains, in the 5Ј to 3Ј direction, a 5Ј leader, at least nine open reading frames (ORF 1a, ORF 1b, and ORFs 2 to 7), and a 3Ј nontranslated region (14,23,32,39). ORF 1a and ORF 1b are located directly downstream of the 5Ј leader and encode a large replicase polyprotein, which is thought to be autoproteolytically cleaved into 13 smaller nonstructural proteins assumed to be involved in virus replication and transcription (4,35,38). ORFs 2 to 7 encode the structural proteins associated with the virion.…”