key: cord-0005534-u2bbgn8k
authors: Yun, Sang-Im; Lee, Young-Min
title: Overview: Replication of porcine reproductive and respiratory syndrome virus
date: 2013-12-19
journal: J Microbiol
DOI: 10.1007/s12275-013-3431-z
sha: f00a6fcd4d95f6e44809fa7e113d6ce4acce10eb
doc_id: 5534
cord_uid: u2bbgn8k

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNA-processing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS Wensvoort et al., 1991; Benfield et al., 1992; Collins et al., 1992) , an economically devastating, pandemic disease of swine that is typically characterized by reproductive failure in breeding herds and respiratory problems and growth retardation in growing pigs (Done and Paton, 1995; Botner, 1997; Van Reeth, 1997; Zimmerman et al., 1997; Rossow, 1998; Suarez, 2000; Rowland, 2010) . Two PRRS outbreaks were first reported in the late 1980s in North America (Keffaber, 1989; Hill, 1990) and central Europe (Paton et al., 1991) . The disease is now found in most pig-producing countries and affects the swine industry and food safety worldwide (Albina, 1997; Blaha, 2000; Lunney et al., 2010; Shi et al., 2010a) , causing enormous economic losses each year (Brouwer et al., 1994; Garner et al., 2001; Zimmerman et al., 2006; Nieuwenhuis et al., 2012) . In the US, the annual loss due to PRRS is estimated to exceed $500 million (Neumann et al., 2005) . In particular, the emergence of highly pathogenic PRRSVs in China and Vietnam in 2006 (Li et al., 2007; Tian et al., 2007; Feng et al., 2008; Zhou et al., 2008; and their rapid spread to several neighboring Asian countries (An et al., 2011) have raised a growing concern that new pathogenic PRRSVs can spread throughout the world, posing a significant threat to the global agricultural community (Normile, 2007; Lunney and Chen, 2010; Murtaugh et al., 2010; Zhou and Yang, 2010) . Because of the current burden of PRRS and the emergence of highly pathogenic forms of PRRSV on a global level, control of this virus remains a research priority in all pig-producing countries.

PRRSV belongs to the family Arteriviridae in the order Nidovirales, which also includes two other families, the Coronaviridae and Roniviridae (Gorbalenya et al., 2006; de Groot et al., 2011) . Within the Arteriviridae family, PRRSV forms a single genus Arterivirus, together with equine arteritis virus (EAV), lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus (Plagemann and Moennig, 1992; Cavanagh, 1997; de Vries et al., 1997; Faaberg et al., 2011) . Like other arteriviruses, PRRSV is an enveloped virus (Dokland, 2010) containing a non-isometric nucleocapsid core (Spilman et al., 2009 ) that encapsidates a plus-strand genomic RNA of ~15 kb in length (Meulenberg et al., 1993) . This genomic RNA consists of a 5 -untranslated region (UTR), 10 open reading frames (ORFs), and a 3 -UTR (Snijder and Spaan, 2007; Britton and Cavanagh, 2008; Firth et al., 2011; Johnson et al., 2011) (Fig. 1) .

Based on its genetic diversity and geographic distribution, PRRSV is divided into two major genotypes (Murtaugh et al., The ~15-kb plus-strand genomic RNA of PRRSV is shown on top. Two long 5 -proximal ORFs (ORF1a and ORF1b) are translated into two large polyprotein precursors, pp1a and pp1ab; the latter is synthesized by a -1 ribosomal frameshift. The two polyproteins are cleaved into at least 14 NSPs: 10 encoded in ORF1a (NSP1α, NSP1β, NSP2 to NSP6, NSP7α, NSP7β, and NSP8) and 4 encoded in ORF1b (NSP9 to NSP12). This proteolysis is regulated by four viral proteases, namely NSP1α, NSP1β, NSP2, and NSP4. An additional protein designated NSP2TF is translated by a -2 ribosomal frameshift in the NSP2-coding region. (B) Synthesis of the viral structural proteins from the six subgenomic mRNAs. Eight short 3 -proximal ORFs are translated from a nested set of six major subgenomic mRNAs: ORF2a (GP2/2a), ORF2b (E, envelope), ORF3 (GP3), ORF4 (GP4), ORF5 (GP5), ORF6 (M, membrane), ORF7 (N, nucleocapsid), and a newly discovered protein encoded in ORF5a that overlaps with the 5end of ORF5. 2010; Shi et al., 2010a) : Type 1, represented by the European prototype Lelystad strain ; and Type 2, exemplified by the North American prototype VR-2332 strain (Benfield et al., 1992; Collins et al., 1992) . Interestingly, despite their concurrent emergence and similar clinical symptoms (Halbur et al., 1995) , the two genotypes show ~40% genetic divergence (Mardassi et al., 1994; Kapur et al., 1996; Allende et al., 1999; Nelsen et al., 1999; Meng, 2000; Oleksiewicz et al., 2000; Forsberg et al., 2002) , with a high degree of antigenic variation (Wensvoort et al., 1992; Nelson et al., 1993; Drew et al., 1995; Wootton et al., 1998) . Over the last decade, this genetic/antigenic diversity has expanded continuously and rapidly (Murtaugh et al., 2001; Stadejek et al., 2002; Mateu et al., 2003; Pesch et al., 2005; Han et al., 2006; Stadejek et al., 2006 Stadejek et al., , 2008 Balka et al., 2008; Li et al., 2009 Li et al., , 2011 Shi et al., 2010b) , highlighting the dynamic nature of PRRSV evolution and epidemiology. At present, a larger number of genetically heterogeneous PRRSVs are widely co-circulating throughout the world than ever before (Dewey et al., 2000; Goldberg et al., 2003; Ropp et al., 2004; Thanawongnuwech et al., 2004; Fang et al., 2007) , posing a significant challenge for the diagnosis, prevention, and control of PRRSV infection.

PRRSV is transmitted both horizontally (pig-to-pig infection) and vertically (transplacental infection) to fetuses during mid-to-late gestation (Christianson et al., 1992 (Christianson et al., , 1993 Yaeger et al., 1993) ; horizontal transmission occurs through both direct and indirect contact (Cho and Dee, 2006; Zimmerman et al., 2006) . Direct contact is the most efficient route of PRRSV transmission, via a variety of porcine secretions from infected animals in which the virus has been detected: e.g., saliva (Wills et al., 1997a; Prickett et al., 2008) , milk (Wagstrom et al., 2001) , nasal fluids (Rossow et al., 1994) , and se-men (Swenson et al., 1994; Christopher-Hennings et al., 1995) . Although its mechanism(s) remains elusive (Mateu and Diaz, 2008; Lunney and Chen, 2010; Yoo et al., 2010; Murtaugh and Genzow, 2011) , PRRSV persistence in pigs plays an important role in viral transmission because the virus is present at low levels in the infected animals (Wills et al., 1997b (Wills et al., , 2003 Allende et al., 2000; Bierk et al., 2001; Batista et al., 2002 Batista et al., , 2004 Horter et al., 2002) . In addition to these direct routes of PRRSV transmission, indirect routes of a particular concern include contaminated fomites (Dee et al., 2002 (Dee et al., , 2003 Otake et al., 2002b) , needles (Otake et al., 2002c ), transport vehicles (Dee et al., 2004 , aerosols (Torremorell et al., 1997; Brockmeier and Lager, 2002; Mortensen et al., 2002; Otake et al., 2002a Otake et al., , 2010 Kristensen et al., 2004; Trincado et al., 2004; Fano et al., 2005; Dee et al., 2009; Pitkin et al., 2009) , and insects as a mechanical vector (Otake et al., 2002d (Otake et al., , 2003a (Otake et al., , 2003b Schurrer et al., 2004 Schurrer et al., , 2005 .

PRRSV infection is initiated by the attachment of the virions to the highly sulfated, negatively charged glycosaminoglycans on the surface of susceptible cells (Jusa et al., 1997; Vanderheijden et al., 2001; Delputte et al., 2002) , followed by binding to CD169 (Duan et al., 1998a , 1998b Vanderheijden et al., 2003; Delputte and Nauwynck, 2004; Delputte et al., 2005 Delputte et al., , 2007 Van Breedam et al., 2010b) , which triggers receptor-mediated clathrin-dependent endocytosis (Kreutz and Ackermann, 1996; Nauwynck et al., 1999; Vanderheijden et al., 2003) . At the early endosomes, the viral genome is released into the cytoplasm through a reaction mediated by CD163 (Calvert et al., 2007; Van Gorp et al., 2008 Das et al., 2010; Van Gorp et al., 2010) and presumably other cellular factors (Misinzo et al., 2008) . Once the genome enters the cytoplasm, ORF1a and ORF1b, located in the 5 -proximal three-quarters of the viral genome, are translated to produce two large polyproteins, pp1a and pp1ab (Snijder and Meulenberg, 1998; Snijder and Spaan, 2007) , with the expression of pp1ab controlled by a -1 ribosomal frameshift (Brierley et al., 1989; den Boon et al., 1991) (Fig. 1A) . Autocatalytic processing of these precursors generates at least 14 nonstructural proteins (NSPs) (Ziebuhr et al., 2000; Fang and Snijder, 2010) : 10 encoded in ORF1a (NSP1α, NSP1β, NSP2 to NSP6, NSP7α, NSP7β, and NSP8) and 4 encoded in ORF1b (NSP9 to NSP12) (Snijder et al., 1992 (Snijder et al., , 1994 den Boon et al., 1995; van Dinten et al., 1996; Wassenaar et al., 1997; Chen et al., 2010a; Li et al., 2012) . This proteolytic processing is mediated by four viral proteases residing in NSP1α, NSP1β, NSP2, and NSP4 (den Boon et al., 1995; Snijder et al., 1996; van Aken et al., 2006b) . Also, an additional viral protein is synthesized by a -2 ribosomal frameshift in the NSP2-coding region, yielding a transframe fusion NSP2TF with the Nterminal two-thirds of NSP2 (Fang et al., 2012) . Most, if not all, of the NSPs assemble into a replication and transcription complex (RTC) that accumulates at the virus-induced ER-derived double-membrane vesicles (van der Meer et al., 1998; Pedersen et al., 1999; Kroese et al., 2008) . The RTC directs both genome amplification ("replication") and subgenomic mRNA synthesis ("transcription") (Fang and Snijder, 2010) ; the latter, a hallmark of all nidoviruses (Pasternak et al., 2006; Sawicki et al., 2007; Snijder and Spaan, 2007) , produces a nested set of six major subgenomic mRNAs that are both 5 -and 3 -coterminal with the genomic RNA and thus consist of nucleotide sequences that are non-contiguous in the genomic RNA (de Vries et al., 1990) . Through the six subgenomic mRNAs, eight mostly overlapping ORFs situated in the 3 -proximal region of the viral genome are expressed, presumably by utilizing each subgenomic mRNA for the translation of one or two of its most 5 -proximal ORFs (Conzelmann et al., 1993; Meng et al., 1996) (Fig. 1B) . These ORFs encode eight structural proteins that constitute the infectious virion (Snijder and Meulenberg, 1998; Snijder and Spaan, 2007; Dokland, 2010) : i.e., four minor components encoded in ORF2a (GP2/2a), ORF2b (E, envelope), ORF3 (GP3), and ORF4 (GP4); three major components encoded in ORF5 (GP5), ORF6 (M, membrane), and ORF7 (N, nucleocapsid) (Meulenberg et al., 1995; Meulenberg and Petersen-den Besten, 1996; van Nieuwstadt et al., 1996; Snijder et al., 1999; Wu et al., 2001) ; and a recently identified protein encoded in ORF5a that overlaps with the 5 -end of ORF5 (Firth et al., 2011; Johnson et al., 2011) . At the late stage of viral replication, multiple copies of the N proteins bind to the newly synthesized genomic RNA to form a nucleocapsid complex (Tijms et al., 2002) , which buds into the lumen of the smooth ER and/or Golgi complex (Wood et al., 1970; Stueckemann et al., 1982; Dea et al., 1995; Weiland et al., 1995; Pol et al., 1997) and acquires the six viral envelope proteins, i.e., E, M, and GP2 to GP5 (Snijder et al., 2003b; Wieringa et al., 2004; Zevenhoven-Dobbe et al., 2004; Wissink et al., 2005) . In this event, the role of the protein product of ORF5a is unclear. Finally, the progeny virions accumulated in the intracellular membrane compartments are released into the extracellular space through exocytosis (Dea et al., 1995) .

Although considerable research has been focused on PRRSV, little is known about the proteolytic processing pathway and the structure and function of most of the PRRSV NSPs. The initial functional assignments of the PRRSV NSPs have primarily been based on the experimental data of EAV, the arterivirus prototype (Fig. 1A ). NSP1α and NSP1β: PRRSV NSP1α and NSP1β each contain a cysteine protease domain responsible for autocatalytic processing at the NSP1α/1β (den Boon et al., 1995; Sun et al., 2009; Chen et al., 2010a) and NSP1β/2 (den Boon et al., 1995; Chen et al., 2010a) junctions, respectively. The atomic structure of PRRSV NSP1α reveals three domains (Sun et al., 2009) : (i) a N-terminal zinc-finger domain, (ii) a papainlike cysteine protease (PCPα) domain with a zinc ion bound at the active site that is required for its proteolytic activity, and (iii) a C-terminal extension bound to the substrate binding site of the PCPα domain. Similarly, the crystal structure of PRRSV NSP1β reveals four domains (Xue et al., 2010) : (i) an N-terminal metal-dependent nuclease domain, (ii) a linker domain, (iii) a papain-like cysteine protease (PCPβ) domain, and (iv) a C-terminal extension bound to the substrate binding site of the PCPβ domain, as observed for PRRSV NSP1α. In the case of both NSP1α and NSP1β, their C-terminal extensions occupy the protease active site after their release from the polyprotein, suggesting that they function in cis (Sun et al., 2009; Xue et al., 2010) . In PRRSV, inactivation of the PCPα activity in NSP1α blocks subgenomic mRNA synthesis without altering genome replication, whereas when PCPβ activity is eliminated in NSP1β, no sign of viral RNA synthesis is seen; therefore, both PCP protease activities are apparently required for productive viral RNA synthesis (Kroese et al., 2008) . Similarly, mutagenesis studies have shown that EAV NSP1 (which contains a tandem of the PCPα and PCPβ domains, with PCPα having lost its enzymatic activity) is involved in regulating the accumulation of minusstrand templates to control the relative abundance of viral mRNAs, thereby coordinating genome replication, subgenomic mRNA synthesis, and virus production (Tijms et al., 2001 (Tijms et al., , 2007 Nedialkova et al., 2010) . Both PRRSV NSP1α/1β (Chen et al., 2010a) and EAV NSP1 (Tijms et al., 2002) are translocated to the nucleus in infected cells, but no consensus nuclear localization signal has yet been found. The interaction of EAV NSP1 with the cellular transcription co-factor p100 suggests that it might be important for viral and/or cellular transcription (Tijms and Snijder, 2003) . NSP2 and NSP3: PRRSV NSP2 is predicted to have four domains: (i) an N-terminal cysteine protease domain, (ii) a large hypervariable domain, (iii) a transmembrane domain, and (iv) a C-terminal tail (Han et al., 2009) . The cysteine protease belongs to the mammalian ovarian tumor domain (OTU)-containing protein superfamily (Makarova et al., 2000; Han et al., 2009) ; it cleaves at the NSP2/3 junction that functions both in cis and in trans (Snijder et al., 1995; Han et al., 2009) and is crucial for the viral replication cycle (Han et al., 2009) . In EAV-infected cells, NSP2 is localized to the perinuclear membranes, which are presumably derived from the ER and are involved in the formation of the membrane-bound RTC, where viral RNA synthesis occurs (van der Meer et al., 1998; Pedersen et al., 1999) . In the ab-sence of EAV replication, the co-expression of EAV NSP2 and NSP3 is both necessary and sufficient to modify host cell membranes during the formation of the RTC . Also, EAV NSP2 interacts with NSP3 (Snijder et al., 1994) , and NSP3 has been implicated in the process of remodeling intracellular membranes (Posthuma et al., 2008) . Biochemical and morphologic studies of EAV replication have shown that the NSPs containing transmembrane domains (e.g., NSP2, NSP3, and NSP5) are part of the membrane-bound RTC, suggesting that they play an important role in recruiting other viral components of the RTC that lack the membrane-spanning domains (van der Meer et al., 1998) . In vitro, the EAV RTCs isolated from infected cells require a cytosolic host factor for viral RNA synthesis, which reproduces the synthesis of both viral genome and subgenomic mRNAs . Interestingly, PRRSV NSP2 contains a cluster of linear B-cell epitopes that are dispensable for virus replication (Oleksiewicz et al., 2001; Chen et al., 2010b) but capable of modulating the host immune response (Chen et al., 2010b; Li et al., 2010) . NSP4: PRRSV NSP4 contains the main protease (3C-like serine proteinase) responsible for all NSP processing, except for the NSP1α/1β, NSP1β/2, and NSP2/3 junctions (van Dinten et al., 1999; Ziebuhr et al., 2000) . Cleavages at the NSP3/4, NSP4/5, and NSP11/12 junctions have been confirmed experimentally by the use of recombinant PRRSV NSP4 (Tian et al., 2009) . The crystal structure of both PRRSV and EAV NSP4s reveals a chymotrypsin-like fold with a canonical catalytic triad (S-H-D), as well as a novel α/β C-terminal extension (Barrette-Ng et al., 2002; Tian et al., 2009 ) that may be involved in regulating viral polyprotein processing (van Aken et al., 2006a) . NSP9: Arterivirus NSP9 includes the viral RNA-dependent RNA polymerase (RdRp) (den Boon et al., 1991) . In PRRSV, the RdRp domain is located in the C-terminal region, which contains an upstream N-terminal region of unknown function (Gorbalenya et al., 2006; Fang and Snijder, 2010) . Enzymatically active EAV RdRp can be purified from E. coli and initiates RNA synthesis by a de novo mechanism on homopolymeric templates in a template-dependent fashion (Beerens et al., 2007) . NSP10: PRRSV NSP10 is predicted to have three domains (Gorbalenya et al., 2006; Fang and Snijder, 2010) : (i) an N-terminal zinc-binding domain, (ii) a linker domain, and (iii) a nucleotide triphosphate binding or helicase domain (den Boon et al., 1991) . Bacterially expressed PRRSV and EAV NSP10s possess ATPase activity and can unwind dsRNA and dsDNA in a 5 -to-3 direction (Bautista et al., 2002; Seybert et al., 2005) . The zinc-binding domain of EAV NSP10 is also critical for this activity (Seybert et al., 2005) . In EAV, the zinc-binding domain contains a set of 13 conserved Cys and His residues and is critical for viral RNA synthesis (van Dinten et al., 2000) . The linker domain (S2429P) has been implicated in subgenomic mRNA synthesis (van Dinten et al., 1997; van Marle et al., 1999b) . NSP11: Arterivirus NSP11 contains the uridylate-specific endoribonuclease (NendoU) domain, which is a major genetic marker unique to nidoviruses (Snijder et al., 2003a; Gorbalenya et al., 2006; Fang and Snijder, 2010) . Bacterially expressed NSP11 has been used to show that the endoribo-nuclease activity of both PRRSV and EAV NendoUs exhibits broad substrate specificity in vitro, but its function in infected cells is elusive (Nedialkova et al., 2009) . Viruses with mutations in the EAV NendoU active site are viable but have a defect in subgenomic mRNA synthesis (Posthuma et al., 2006) . Recently, IFN-mediated host innate immunity has been shown to be modulated by a panel of PRRSV NSPs (i.e., NSP1α, NSP1β, NSP2, NSP4, and NSP11) with different intensities (Beura et al., 2010; Chen et al., 2010a; Li et al., 2010) . In the case of PRRSV NSP2, the OTU domain-containing cysteine protease has been shown to possess deubiquitinating and interferon antagonism activity, thereby evading ubiquitin-and ISG15-dependent innate immunity (Frias-Staheli et al., 2007; Sun et al., 2010) . Other NSPs: To date, no specific functions have been demonstrated for the other PRRSV NSPs (NSP5, NSP6, NSP7α, NSP7β, NSP8, and NSP12). Also, it should be noted that during the proteolytic processing of EAV NSPs, many cleavage intermediates of unknown function have been observed (Snijder et al., 1994; van Dinten et al., 1996) , and alternative major and minor processing pathways have also been characterized .

Based on the "discontinuous RNA transcription" model (Sawicki and Sawicki, 1995) , the plus-strand genomic RNA of PRRSV is thought to serve as a template for either (i) continuous minus-strand RNA synthesis, which produces the genome-length minus-strand template for genome replication; or (ii) discontinuous minus-strand RNA synthesis, which generates a nested set of six major subgenome-length minus-strand templates, one for each subgenomic mRNA synthesis. All the subgenomic mRNAs are both 5 -and 3coterminal with the genomic RNA, with a common short "leader" sequence corresponding to the 5 -proximal region of the genome joined to different "body" segments that are co-linear with its 3 -proximal region (Pasternak et al., 2006; Sawicki et al., 2007) . This leader-body joining is guided by regulatory transcription-regulating sequences (TRSs); in the genomic RNA, these RNA motifs are located at the 3 -end of the leader sequence (leader TRS) and upstream of each structural protein-coding region (body TRS) ( van Marle et al., 1999a; Pasternak et al., 2001; Van Den Born et al., 2004) . In PRRSV, the 5 -proximal one or two ORFs of each subgenomic mRNA are translated to produce eight viral structural proteins that constitute an infectious virion (Meulenberg et al., 1995; Meulenberg and Petersen-den Besten, 1996; van Nieuwstadt et al., 1996; Snijder et al., 1999; Dea et al., 2000; Molenkamp et al., 2000; Wu et al., 2001; Johnson et al., 2011) : GP2 (GP2a), E, GP3, GP4, GP5, M, N, and a protein product of ORF5a (Fig. 1B) . The viral envelope contains the two major (GP5 and M) and four minor (E, GP2, GP3, and GP4) membrane proteins that are all required for the production and infectivity of infectious virions; however, the four minor proteins are dispensable for virus assembly (Wieringa et al., 2004; Wissink et al., 2005) . E protein has an ion channel protein-like property and is embedded in the viral membrane, presumably promoting uncoating of the virion and release of the viral genome into the cytoplasm (Lee and Yoo, 2006) . GP3 is heavily glycosylated (Dea et al., 2000; Das et al., 2011) , and its glycans on the viral surface prevent the recognition of epitopes by neutralizing antibodies (Vu et al., 2011) ; a subset of the GP3 proteins is secreted from the cells as a nonvirion-associated soluble form (Mardassi et al., 1998) . GP4 has a neutralizing epitope in the hypervariable region (Meulenberg et al., 1997) that might be associated with the E, GP2, and GP3 proteins through non-covalent interactions (Wieringa et al., 2004; Wissink et al., 2005; Das et al., 2010) . GP5 is a triple membrane-spanning protein with a short ectodomain (~40 aa) and a long cytoplasmic tail (~50-70 aa) (Meulenberg et al., 1995; Mardassi et al., 1996) , which contains major neutralizing epitopes (Wissink et al., 2003; Ansari et al., 2006) . M is the most conserved membrane protein and has a membrane topology similar to that of GP5 (Dea et al., 2000) . N is a serine phosphoprotein that forms a dimer and is distributed in the cytoplasm and the nucleus (Rowland and Yoo, 2003; You et al., 2008) . In the viral membrane, the GP5 and M proteins are embedded as disulfidelinked heterodimers, whereas the E, GP2, GP3, and GP4 proteins are associated with each other through non-covalent interactions (Meulenberg et al., 1993 (Meulenberg et al., , 1995 Mardassi et al., 1995 Mardassi et al., , 1996 Meulenberg and Petersen-den Besten, 1996; van Nieuwstadt et al., 1996; Wu et al., 2001; Wissink et al., 2005) . PRRSV has a very restricted cell tropism. In vivo, it targets specific subsets of porcine macrophages, primarily alveolar macrophages (Lawson et al., 1997; Duan et al., 1997a Duan et al., , 1997b Teifke et al., 2001) ; in vitro, it can also infect monocyte-or bone marrow-derived porcine dendritic cells when stimulated with GM-CSF/ IL-4 (Loving et al., 2007; Wang et al., 2007; Chang et al., 2008; Flores-Mendoza et al., 2008; Silva-Campa et al., 2009 ), but not lung dendritic cells (Loving et al., 2007) . PRRSV entry into porcine macrophages is the first step in a highly coordinated process of virus-host interactions. Based on recent findings (Welch and Calvert, 2010; Van Breedam et al., 2010a) , highly sulfated, negatively charged glycosaminoglycans such as heparan sulfates can be used as low-affinity attachment factors that concentrate virus particles on the cell surface (Jusa et al., 1997; Vanderheijden et al., 2001; Delputte et al., 2002 Delputte et al., , 2005 . Once this interaction has taken place, the viral GP5/M complex binds to the Nterminal portion of CD169 (also called sialoadhesin or siglec-1) (Duan et al., 1998a (Duan et al., , 1998b Vanderheijden et al., 2003; Delputte et al., 2005; Van Gorp et al., 2008; Van Breedam et al., 2010b) . This interaction is directed by the sialic acid-binding domain at the N-terminus of CD169 and sialic acids on the virion surface (Delputte and Nauwynck, 2004; Delputte et al., 2007; Van Breedam et al., 2010b) , which trigger receptor-mediated, clathrin-dependent endocytosis (Kreutz and Ackermann, 1996; Nauwynck et al., 1999; Vanderheijden et al., 2003) . Once internalized, the particles are transported to early endosomes, where the viral genome is released into the cytoplasm in a reaction that depends on both the acidic environment and scavenger receptor CD163 (Nauwynck et al., 1999; Calvert et al., 2007; Van Gorp et al., 2008 ). The role of CD163 is mediated through its cysteine-rich domain 5 (Van Gorp et al., 2010) and by interaction with GP2 and GP4 (Das et al., 2010) . The protease cathepsin E and an additional serine protease are also implicated in this process (Misinzo et al., 2008) . Other host factors, such as simian vimentin (Kim et al., 2006) and CD151 (Shanmukhappa et al., 2007) , have been identified in MARC-145 cells, a cell line susceptible to PRRSV infection (Kreutz, 1998) .

PRRS is controlled by several different strategies, including management (e.g., herd depopulation/repopulation, herd closure, and regional elimination), biosecurity, and vaccination (Corzo et al., 2010; Thanawongnuwech and Suradhat, 2010) . Of these strategies, vaccination is the most cost-effective for controlling PRRS, but it does not completely prevent PRRSV infection. Two types of PRRSV vaccines are commercially available: modified-live virus (MLV) and killed virus (KV) vaccines (Yoo et al., 2004; Charerntantanakul, 2009; Kimman et al., 2009; Cruz et al., 2010; Huang and Meng, 2010) . The MLV vaccine confers effective protection against genetically homologous PRRSVs but only partial protection against genetically heterologous PRRSVs (Meng, 2000; Murtaugh et al., 2002; Labarque et al., 2003; Okuda et al., 2008) ; it is of particular concern that the live vaccine viruses have the potential to spontaneously revert to virulence and spread the disease (Botner et al., 1997; Madsen et al., 1998; Mengeling et al., 1999; Storgaard et al., 1999; Wesley et al., 1999; Nielsen et al., 2001; Opriessnig et al., 2002; Amonsin et al., 2009; grosse Beilage et al., 2009; Li et al., 2009) . The KV vaccine, on the other hand, is safe but offers limited protection at best against either homologous or heterologous PRRSVs (Scortti et al., 2007; Zuckermann et al., 2007; Vanhee et al., 2009) . Thus, the current vaccines fail to provide sustainable disease control and prevention, particularly against the genetically heterologous PRRSVs (Cano et al., 2007a (Cano et al., , 2007b , making it difficult to achieve global eradication.

Although significant progress has been made in understanding the routes of PRRSV transmission and in developing and implementing control measures for PRRSV infection, there is clearly an urgent need for novel strategies that may be applicable to the development of a safer, more effective vaccine against PRRSV. Despite the clinical importance of PRRSV in animal health, only limited information is available to date regarding the biological functions of the viral nonstructural and structural proteins in replication and pathogenesis. In particular, the molecular characterization of the 14 replicase proteins and their roles in PRRSV RNA synthesis have represented a major challenge in PRRSV biology. We and others have established a reverse genetics system for PRRSV by constructing a full-length infectious cDNA that allows genetic manipulation of the viral genome and from which molecularly cloned viruses can be rescued. This system offers a unique opportunity to address some of the key questions in PRRSV biology. New information will give us a better understanding of the molecular and genetic basis of PRRSV replication and pathogenesis, a prerequisite for the development of new and promising strategies to Nauwynck, H.J., Duan, X., Favoreel, H.W., Van Oostveldt, P., and

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Comparative morphogenesis of three PRRS virus strains

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle

Formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes

Oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections

Characterization of emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States

Porcine reproductive and respiratory syndrome

Experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and 10-week-old pigs

The interaction between PRRSV and the late gestation pig fetus

Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences

Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands

A contemporary view of coronavirus transcription

Retention of ingested porcine reproductive and respiratory syndrome virus in houseflies

Spatial dispersal of porcine reproductive and respiratory syndrome virus-contaminated flies after contact with experimentally infected pigs

Failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with PRRSV

A complex zinc finger controls the enzymatic activities of nidovirus helicases

Role of CD151, A tetraspanin, in porcine reproductive and respiratory syndrome virus infection

Molecular epidemiology of PRRSV: a phylogenetic perspective

Phylogeny-based evolutionary, demographical, and geographical dissection of North American type 2 porcine reproductive and respiratory syndrome viruses

Induction of T helper 3 regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus

Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage

Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity

The molecular biology of arteriviruses

Arteriviruses

Identification of a novel structural protein of arteriviruses

Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex

The 5 end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus

The arterivirus Nsp2 protease. An unusual cysteine protease with primary structure similarities to both papain-like and chymotrypsin-like proteases

The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases

Cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid

Porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern Europe support the definition of new genetic subtypes

Definition of subtypes in the European genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in Europe

Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses

Examination of the selective pressures on a live PRRS vaccine virus

Replication of lactate dehydrogenase-elevating virus in macrophages. 2. Mechanism of persistent infection in mice and cell culture

Ultrastructural pathogenesis of the PRRS virus

Crystal structure of porcine reproductive and respiratory syndrome virus leader protease Nsp1alpha

The cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions

Excretion of porcine reproductive and respiratory syndrome virus in semen after experimentally induced infection in boars

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridizationimmunohistochemistry double labelling

Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch's postulates fulfilled

Genetics and geographical variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand

Taming PRRSV: revisiting the control strategies and vaccine design

Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark

Structure and cleavage specificity of the chymotrypsin-like serine protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

Arterivirus subgenomic mRNA synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease

Equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mRNA synthesis, interacts with the cellular transcription co-factor p100

Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus

A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positivestranded RNA virus

Airborne transmission of Actinobacillus pleuropneumoniae and porcine reproductive and respiratory syndrome virus in nursery pigs

Attempts to transmit porcine reproductive and respiratory syndrome virus by aerosols under controlled field conditions

Mutagenesis analysis of the nsp4 main proteinase reveals determinants of arterivirus replicase polyprotein autoprocessing

Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7

Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage

The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner

Secondary structure and function of the 5 -proximal region of the equine arteritis virus RNA genome

ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex

An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription

Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication

The predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mRNA synthesis, genome replication, and virion biogenesis

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains

The porcine reproductive and respiratory syndrome virus requires trafficking through CD163-positive early endosomes, but not late endosomes, for productive infection

Sialoadhesin and CD163 join forces during entry of the porcine reproductive and respiratory syndrome virus

Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus

The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor

The arterivirus replicase

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion

Pathogenesis and clinical aspects of a respiratory porcine reproductive and respiratory syndrome virus infection

Effects of heparin on the entry of porcine reproductive and respiratory syndrome virus into alveolar macrophages

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Development of an experimental inactivated PRRSV vaccine that induces virus-neutralizing antibodies

Immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3

Shedding of porcine reproductive and respiratory syndrome virus in mammary gland secretions of sows

Porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability

Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease

Electron microscopic studies on the morphogenesis of PRRSV in infected cells-comparative studies

A brief review of CD163 and its role in PRRSV infection

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus

Mystery swine disease in The Netherlands: the isolation of Lelystad virus

Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus

Structural protein requirements in equine arteritis virus assembly

Duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus: routes of excretion

Porcine reproductive and respiratory syndrome virus: a persistent infection

Envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus

The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain

Electron microscopic study of tissue cultures infected with simian haemorrhagic fever virus

Antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b

The crystal structure of porcine reproductive and respiratory syndrome virus nonstructural protein Nsp1beta reveals a novel metal-dependent nuclease

Evidence for the transmission of porcine reproductive and respiratory syndrome (PRRS) virus in boar semen

Modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus

Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

Rescue of disabled infectious single-cycle (DISC) equine arteritis virus by using complementing cell lines that express minor structural glycoproteins

Porcine reproductive and respiratory syndrome in China

Highly virulent porcine reproductive and respiratory syndrome virus emerged in China

Virus-encoded proteinases and proteolytic processing in the Nidovirales

Porcine reproductive and respiratory syndrome virus (porcine arterivirus)

General overview of PRRSV: a perspective from the United States

Assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (PRRSV) vaccines based on measurement of serologic response, frequency of gamma-IFN-producing cells and virological parameters of protection upon challenge

This work was supported by a Utah Agricultural Experiment Station grant (UTA01102), Utah Science Technology and Research funds, and a Korean National Research Foundation grant . This research was approved as journal paper number UAES #8613. S.I.Y was supported by the Basic Science Research Program (2009-0069679) from the National Research Foundation funded by the Korean Ministry of Education, Science, and Technology. We thank Dr. Deborah McClellan for reading the manuscript.