Virus morphology and genome organization

Pestiviruses are enveloped viruses with a diameter of 40-60nm. The single stranded RNA with positive polarity and a length of approximately 12.3 kb is located within the viral particle and consists of one open reading frame (ORF), which encodes for a polyprotein of about 3900 amino acids (Knipe and Howley, 2013; Tautz et al., 2015). The ORF is flanked by 5’ and 3’

nontranslated regions (NTRs), which play an important role in translation and RNA replication (Becher et al., 2000; Frolov et al., 1998; Isken et al., 2004, 2003; Yu et al., 2000).

The translation of pestiviral RNA is initiated cap-independent by an internal ribosome entry site (IRES) in the 5’ NTR (Brock et al., 1992; Pestova et al., 1998; Poole et al., 1995). No poly(A)-tail can be found at the 3’NTR of pestiviruses. The coordination of translation and RNA replication in pestiviruses is facilitated by interaction of the 3’NTR with host proteins of the NFAR (nuclear factors associated with dsRNA) group (Isken et al., 2004, 2003).

The polyprotein is processed by viral and host proteases, resulting in the generation of four structural proteins, the core protein (C) and the envelope (E) glycoproteins E^rns, E1 and E2 as well as the eight nonstructural proteins (NS) N^pro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Figure 1) (Becher et al., 1998; Collett et al., 1988; Meyers et al., 1989; Rümenapf et al., 1993; Stark et al., 1990; Tautz et al., 2015; Thiel et al., 1991).

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Figure 1: Genome organization of pestiviruses: The genome of pestivirures is a single stranded RNA with positive polarity and a single open reading frame (ORF), which encodes for one polyprotein. Viral and cellular proteases process the polyprotein into the single structural and nonstructural proteins. Structural proteins are highlighted in grey

The N-terminal protease (N^pro) is the first protein encoded by the ORF and constitutes its own family (C53) of cysteine proteases (Rawlings et al., 2012). N^pro is an autoprotease and releases its own C-terminus, thereby creates the N-terminus of the C protein (core protein) (Rümenapf et al., 1998; Stark et al., 1993; Wiskerchen, 1991). Further processing of the pestiviral polyprotein between C/Erns, Erns/E1, E1/E2, E2/p7 and p7/NS2 is conducted by cellular signal peptidases (SPase) and signal peptide peptidase (SPPase) (Figure 1) (Elbers et al., 1996; Harada et al., 2000; Heimann et al., 2006; Rümenapf et al., 1993).

E^rns is an envelope glycoprotein unique to the genus Pestivirus (Peterhans and Schweizer, 2013). E^rns can be found on the surface of viral particles and in virus-free supernatant (Rümenapf et al., 1993; Thiel et al., 1999). Its unusual features include an amphipathic helix as a membrane anchor (Fetzer et al., 2005; Tews and Meyers, 2007) and an endoribonucleolytic activity (Schneider et al., 1993). E^rns is followed by the glycoproteins E1 and E2. Neutralizing antibodies against infections in animals are induced against E2 and to a lower degree E^rns (Boulanger et al., 1991; Weiland et al., 1992, 1990). The three glycoproteins

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build complexes connected by disulfide bounds. E^rns – homodimer, E1-E2-heterodimer and E2-homodimer were detected in infected cells (Thiel et al., 1991).

The nonstructural proteins p7, NS2-3, NS4A, NS4B, NS5A and NS5B follow the three glycoproteins. Protein p7 is a small protein of 7 kDa and appears as a mature protein or as part of an E2-p7 precursor (Elbers et al., 1996). It is necessary for virus morphogenesis but dispensable for viral replication (Harada et al., 2000). The cleavage of NS2-3 is mediated by an autoprotease within NS2, which requires the interaction with the cellular cofactor DNAJC14 (also called Jiv: J-domain protein interacting with viral protein) (Lackner et al., 2005, 2004). NS3 is a viral protein with a multitude of functions. A chymotrypsin-like serine protease can be found in the N-terminal domain of the protein, whereas a helicase and an RNA nucleoside triphosphatase can be found in the C-terminal part (Bazan and Fletterick, 1988; Gorbalenya et al., 1989; Tamura et al., 1993). The NS3 serine protease requires the central domain of NS4A as a cofactor to obtain its full activity. This NS3-4A protease is not only responsible for the cleavage of the C-terminus of NS2-3, but also for further processing of the polyprotein and therefore for the release of NS4B, NS5A and NS5B (Tautz et al., 2000;

Wiskerchen and Collett, 1991; Xu et al., 1997). Uncleaved NS2-3 plays an essential role in virion morphogenesis, but pestiviruses can adapt and form infectious particles also in the absence of uncleaved NS2-3 (Agapov et al., 2004; Lattwein et al., 2012; Moulin et al., 2007).

Furthermore, uncleaved NS2-3 cannot substitute for NS3 in viral replication (Lackner et al., 2004).

NS4A has a size of approximately 10 kDa. It serves as a cofactor for the NS3 serine protease and plays a role in efficient virion packaging (Agapov et al., 2004; Liang et al., 2009; Moulin et al., 2007; Tautz et al., 2000; Xu et al., 1997).

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NS4B has a size of about 35 kDa and exhibits an essential but yet uncharacterized role in viral replication (Grassmann et al., 2001). Comparable to HCV (hepatitis C virus) two conserved regions, termed Walker A and Walker B were identified in pestiviral NS4B. The sequence of these two regions showed similarities to nucleotide-binding motifs (Einav et al., 2004; Gladue et al., 2011; Walker et al., 1982).

NS5A has a size of 58 kDa and as the only part of the replicase complex, can be complemented in trans (Grassmann et al., 2001). NS5A is comprised of three domains connected by two low complexity sequences (LCS) (Isken et al., 2014). The domain I contains an amphipathic α-helix at the N-terminus, which serves as a membrane anchor and a zinc-binding site, important for viral RNA replication (Brass et al., 2007; Tellinghuisen et al., 2006). A mutagenesis study revealed that only LCS I, the N-terminal part of domain II and domain III tolerate deletions and thus are suited for receiving tags applicable for instances in live-cell imaging. In this study, fluorescent labeled BVDV NS5A was shown to localize to the surface of lipid droplets (Isken et al., 2014). Recent reports demonstrated a role for NS5A in viral RNA replication. NS5A was shown to regulate the replication of the viral genome by either binding to the 3’NTR of the virus or to NS5B (Chen et al., 2012; Sheng et al., 2012).

The identified regions in NS5A required for binding of NS5B as well as for viral RNA replication are conserved in BVDV, BDV, CSFV and HCV (Chen et al., 2012).

NS5B constitutes the viral RNA-dependent RNA polymerase (RdRp) and has a size of about 77 kDa (Kao et al., 1999; Zhong et al., 1998). Furthermore, NS5B was shown to play a role in virion morphogenesis (Ansari et al., 2004).

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