Identification of porcine reproductive and respiratory syndrome virus ORF1a-encoded non-structural proteins in virus-infected cells

Abstract

The porcine reproductive and respiratory syndrome virus (PRRSV) replicase gene consists of two large ORFs, ORF1a and ORF1b, the latter of which is expressed by ribosomal frameshifting. The ORF1a-encoded part of the resulting replicase polyproteins (pp1a and pp1ab) is predicted to be processed proteolytically into ten non-structural proteins (nsps), known as nsp1-8, with both the nsp1 and nsp7 regions being cleaved internally (yielding nsp1a and nsp1b, and nsp7a and nsp7b, respectively). The experimental verification of these predictions depends strongly on the ability to identify individual cleavage products with specific antibodies. In this study, a panel of monoclonal and polyclonal antibodies was generated, which together were able to recognize eight ORF1a-encoded PRRSV nsps. Using these reagents, replicase cleavage products were detected in PRRSV-infected MARC-145 cells using a variety of immunoassays. By immunofluorescence microscopy, most nsps could be detected by 6 h post-infection. During the early stages of infection, nsp1b, nsp2, nsp4, nsp7a, nsp7b and nsp8 co-localized in distinct punctate foci in the perinuclear region of the cell, which were determined to be the site of viral RNA synthesis by in situ labelling. Western blot and immunoprecipitation analysis identified most individual nsps and several long-lived processing intermediates (nsp3-4, nsp5-7, nsp5-8 and nsp3-8). The identification and subcellular localization of PRRSV nsps in virus-infected cells documented here provides a basis for the further structure-function studies. Thus, this PRRSV antibody panel will be an important tool for future studies on the replication and pathogenesis of this major swine pathogen. INTRODUCTIONPorcine reproductive and respiratory syndrome (PRRS), a disease first described in the USA in 1987(Keffaber, 1989 and in Europe in 1991 (Wensvoort et al., 1991), has caused tremendous economic losses to the swine industry since its appearance, with recent annual costs estimated to be at least US$600 million in the USA alone (Neumann et al., 2005). Hallmark symptoms of PRRS are mild to severe respiratory disease in infected newborn and growing pigs, and reproductive failure in pregnant sows. The aetiological agent, PRRS virus (PRRSV), was first identified in the Netherlands in 1991, and is represented by the European prototypic strain, Lelystad virus (Wensvoort et al., 1991). In the USA, PRRSV was first isolated in 1992, and the North American prototypic strain was designated VR-2332(Benfield et al., 1992Collins et al., 1992). At the genome level, these two genotypes, European (type I) and North American (type II), share~60 % sequence identity (Allende et al., 1999;Nelsen et al., 1999). PRRSV is classified in the family Arteriviridae, which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus and simian hemorrhagic fever virus (Snijder & Meulenberg, 1998).PRRSV is a small, enveloped virus containing a singlestranded, positive-sense RNA genome. The genome is 15 kb and contains at least ten ORFs...

The non-structural protein Nsp2TF of porcine reproductive and respiratory syndrome virus down-regulates the expression of Swine Leukocyte Antigen class I

“…Approximately 75% of the viral genome at 5' proximal region can translate into polyproteins 1a and 1ab, which are further co-translationally processed into at least 14 nonstructural proteins (Nsps) by auto-proteolytic cleavages (Fang and Snijder, 2010;Li et al, 2012). These Nsps mainly function in viral replication and transcription, and some Nsps are also involved in host immune modulation.…”

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin

“…PRRSV nsp2 is known to be an important nonstructural protein involved in viral replication and transcription complex (RTC) assembly for viral RNA genome synthesis [43]. Furthermore, previous studies have demonstrated that at least eight PRRSV nonstructural proteins, including nsp1β, nsp2, nsp3, nsp4, nsp7, nsp8, nsp9, and nsp10, may be important replicases in the viral RTC [44][45][46]. Our data suggest that nsp1α, nsp1β, nsp3, nsp4, nsp7, nsp9, and nsp10 potentially interact with nsp2.…”

Proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase

“…Prior to SDS-PAGE, samples were heated at 95 • C in Laemmli sample buffer for 1 min. Gels containing immunoprecipitation samples from cells metabolically labeled with [ 35 S] methionine were subjected to fluorography as described previously (de Vries et al, 1992;Li et al, 2012).…”