Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

Abstract

A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 59-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation. Classical swine fever virus (CSFV) is a member of the genusPestivirus of the family Flaviviridae. Bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV) and hepatitis C virus (HCV) also belong to this family (Becher & Thiel, 2002;Cuthbert, 1994;Heinz et al., 2000). CSFV is the leading cause of classical swine fever, a highly contagious and sometimes fatal viral disease of pigs, and it can cause a considerable amount of economic loss. CSFV possesses a single plus-strand RNA genome consisting of 59-and 39-untranslated regions (UTRs), and a single large open reading frame (ORF) that encodes a polyprotein of approximately 3900 aa. The polyprotein (NH2-N pro -C-E rns -E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) is processed into mature proteins by cellular and viral proteases (Moennig & Plagemann, 1992). The 39-UTR is most likely to be involved in the initiation of pestiviral genome replication (Isken et al., 2003;Pankraz et al., 2005;Xiao et al., 2004;Yu et al., 1999), while the 59-UTR is able to regulate translation of the viral genomes (Fletcher & Jackson, 2002). An internal ribosome entry site (IRES) is present in the 59-UTR, and is located between nt 40 and 350 from the 59-terminal end of the genome. The HCV and the pestiviral IRESs direct the binding of ribosomes to the start codon even in the absence of canonical translation initiation factors (Hellen & Sarnow, 2001).NS3 is a multifunctional protein possessing serine protease, RNA helicase and nucleoside triphosphatase (NTPase) activities, which are located in two functionally distinct domains. The N-terminal one-third of NS3 primarily serves as a protease to process the viral polyprotein. The helicase and NTPase activities are localized to the Cterminal end of the NS3 protein (Warrener & Collett, 1995; Xu et al., 1997). The HCV, BVDV and CSFV NS3 proteins have been demonstrated to have RNA helicase activity (Kim et al., 1995;Sheng et al., 2007;Tai et al., 1996;Warrener & Collett, 1995). Some plus-strand RNA viruses, such as poliovirus and rhinovirus, which lack ...

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

“…Our previous studies revealed that the NS3 protein could promote CSFV IRES-mediated translation. The CSFV NS5B protein did not significantly affect IRES-directed translation alone, but significantly enhanced the stimulative effect of NS3 protein on IRES-mediated translation (Xiao et al, 2008). It has been reported that HCV NS5A protein downregulates HCV IRES-dependent translation (Kalliampakou et al, 2005).…”

“…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).…”

Genetic features of a translation initiation system composed of IRES element, nucleotide context surrounding the initiation codon, and translation initiation region of classical swine fever virus RNA

“…Although genetic diversity in CSFV IRES elements was observed in this study, high conversation among these IRES sequences results in a stable secondary structure to facilitate an interaction between IRES elements and viral proteins. NS3 of CSFV can interact with the IRES element to increase translation initiation efficiency (Xiao et al, 2008;Zhu et al, 2010).…”

“…These positive sense viral RNAs function similarly to mRNAs, but they contain the 5'-UTR, which exhibits extensive secondary structure features and multiple AUG codons preceding the initiation codon (Firth and Brierley, 2012). The extensive secondary structure plays an important role in IRES activity during the CSFV life cycle, as some CSFV and host cell proteins combine with the proper secondary structure for the IRES (Xiao et al, 2008;Zhu et al, 2010). The function of CSFV IRES is strongly influenced by the coding sequences immediately downstream of the initiation codon (Fletcher and Jackson, 2002).…”

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses

“…However, these proteins also play an important role in internal ribosome entry site (IRES)-mediated translation of CSFV. CSFV NS3 is a multifunctional protein possessing serine protease, RNA helicase, and nucleoside triphosphatase (NTPase) activities [81]. The RNA helicase activity of NS3 could promote viral and cellular translation, whereas the protease domain of NS3 interacted with NS5B to enhance viral translation [81].…”