Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

Abstract

An internal ribosome entry site (IRES) present in the 59 untranslated region (UTR) promotes translation of classical swine fever virus (CSFV) genomes. Using an in vitro system with monocistronic reporter RNA containing the CSFV 59UTR, this study found that CSFV NS5A decreased CSFV IRES-mediated translation in a dose-dependent manner. Deletion analysis showed that the region responsible for repressing CSFV IRES activity might cover aa 390-414, located in the C-terminal half of CSFV NS5A. Triple and single alanine-scanning mutagenesis revealed that the inhibitory effect on CSFV IRES-directed translation mapped to the K399, T401, E406 and L413 residues of NS5A. These important amino acids were also found to be present in the NS5A proteins of bovine viral diarrhea virus (BVDV)-1, BVDV-2, border disease virus and hepatitis C virus, indicating that NS5A may play an important role in the switch from translation to replication in these viruses. Classical swine fever virus (CSFV) is a member of the genusPestivirus, which also contains bovine viral diarrhea virus (BVDV)-1, BVDV-2 and border disease virus (BDV) (Becher & Thiel, 2002;Heinz et al., 2000). The genus Pestivirus belongs to the family Flaviviridae. Hepatitis C virus (HCV), the major cause of transfusion-associated hepatitis, also belongs to this family (Cuthbert, 1994). CSFV is a small enveloped virus. Its genome is a single, positive-sense RNA, which contains a single large open reading frame (ORF) and 59 and 39 untranslated regions (UTRs). The ORF encodes a polyprotein of approximately 3900 aa. The 12 CSFV proteins are organized in the polyprotein in the order NH 2 -N pro -C-E rns -E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Moennig & Plagemann, 1992). The NS5B protein serves as the viral RNAdependent RNA polymerase (RdRp) (Steffens et al., 1999;Xiao et al., 2002) and contains a conserved GDD motif necessary for catalytic activity (Wang et al., 2007). NS3 is a multifunctional protein possessing serine protease, RNA helicase and nucleoside triphosphatase activities, and is important for virus replication and even for promoting viral and cellular translation (Gu et al., 2000; Piccininni et al., 2002;Sheng et al., 2007;Suzich et al., 1993;Warrener & Collett, 1995;Xiao et al., 2008; Xu et al., 1997). The 39UTR is probably involved in initiation of pestiviral genome replication (Isken et al., 2003(Isken et al., , 2004Pankraz et al., 2005;Xiao et al., 2004;Yu et al., 1999), whilst the 59UTR contains an internal ribosome entry site (IRES), located between nt 40 and 350 at the 59 terminus of the genome, which is able to facilitate translation of the viral genome (Fletcher & Jackson, 2002).NS5A of CSFV strain Shimen comprises 497 aa (aa 2684-3180 of the genome) (Fig. 1a). The function of NS5A in the life cycle of CSFV remains unclear. It has been reported that the NS5A protein of HCV is an essential component of the viral RNA replication machinery and may also function in modulation of the host cell environment (Tellinghuisen et al., 2004(Tellinghuisen et al., , 2006. ...

The 3′-Terminal Hexamer Sequence of Classical swine fever virus RNA Plays a Role in Negatively Regulating the IRES-Mediated Translation

“…However, we have observed that the untranslated regions could cause the cap-dependent translation shutdown (unpublished data). Therefore, the capped reporter control used for normalization [32], [33] showed varied expression when co-transfected with the RNAs containing different structure of the CSFV 3′ UTR. To avoid the varied expression caused by cap-dependent translation shutdown, most of the transfection experiments with the cap-independent CSFV RNAs were repeated at least three times.…”

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

“…This observation is in good agreement with the essential function of domain I and our finding that the C-terminal part of domain II and the entire domain III are critical for pestiviral RNA replication. Previously, a negative regulatory effect of NS5A on CSFV IRES-mediated translation of a reporter mRNA was reported (20). This effect also depended on residues in the CSFV NS5A region of aa 390 to 414.…”

“…However, the structure and function of the downstream part of NS5A are still ill defined. Recently, CSFV NS5A has been shown to regulate CSFV viral RNA replication by either binding to the 3= UTR of the viral RNA genome via its RNA binding activity or by modulating the RdRp activity of NS5B by direct protein-protein interactions (19,20).…”

The Molecular Biology of Pestiviruses

“…This could indicate that NS5A does not need to associate with other components of the replicase cotranslationally which allows also molecules provided in trans to act as substitutes. CSFV NS5A has recently been shown to regulate viral RNA replication by either binding to the 3 0 -UTR of the viral RNA genome via its RNAbinding activity or the modulation of NS5B RdRp activity by direct protein-protein interactions Grassmann et al, 2001;Xiao et al, 2009). Xiao et al described a negative regulatory effect of NS5A on CSFV IRES-mediated translation of a reporter mRNA.…”