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Short Communication |
1 College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, PR China
2 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, The Institute of Biodiversity Science, Fudan University, Shanghai 200433, PR China
Correspondence
Ming Xiao
xiaoming88{at}263.net
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ABSTRACT |
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; 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 5'- and 3'-untranslated regions (UTRs), and a single large open reading frame (ORF) that encodes a polyprotein of approximately 3900 aa. The polyprotein (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) is processed into mature proteins by cellular and viral proteases (Moennig & Plagemann, 1992). The 3'-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 5'-UTR is able to regulate translation of the viral genomes (Fletcher & Jackson, 2002). An internal ribosome entry site (IRES) is present in the 5'-UTR, and is located between nt 40 and 350 from the 5'-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 C-terminal 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 NS3 proteins, encode a homologous protein called 2C with helicase and NTPase activity (Pfister & Wimmer, 1999). Recent evidence indicates that the NS3 protein is also important in viral replication (Gu et al., 2000; Kolykhalov et al., 2000; Piccininni et al., 2002). However, little is known about whether the CSFV NS3 protein has an effect on viral IRES-mediated and cellular translation. In this report, we investigated the effect of this protein on both IRES-mediated and cellular translations.
There have been a number of studies on IRES-mediated translation (He et al., 2003; Kalliampakou et al., 2005; Kato et al., 2002; Shimoike et al., 1999; Song et al., 2006; Zhang et al., 2002). From these studies it was found that a monocistronic reporter RNA containing viral 5'-UTR and the firefly luciferase (FLuc) is more effective than its corresponding reporter DNA in the study of IRES-directed translation (Song et al., 2006). Using standard methods, we created a monocistronic reporter RNA. The CSFV 5'-UTR was cloned into pGEM-T vector (Promega) as described previously (Xiao et al., 2004), ligated with a PCR-generated FLuc ORF, and used as a template for T7-mediated in vitro transcription reactions as described previously (Xiao et al., 2004). Full-length NS3 (NS3F) and truncated NS3 cDNAs (NS3H) were obtained (Sheng et al., 2007) and cloned into the pcDNA-3.1 vector (Clontech) (Xiao et al., 2003). The monocistronic reporter RNA (1.0 µg) was co-transfected into PK-15 cells with the pcDNA-NS3 vector that expresses the NS3F protein. Cultivation of PK-15 cells and transfection were done as described previously (Xiao et al., 2003). As a control, an unrelated protein, β-galactosidase (LacZ) was expressed in the same vector. Cell extracts were prepared 10 h after transfection. The FLuc activity was measured with the luciferase assay kit (Promega) according to the manufacturer's instructions. As shown in Fig. 1(a), expression of the NS3F protein increased FLuc activity compared with that observed in the LacZ expressing cell, suggesting that the NS3F protein enhanced the CSFV IRES-directed translation. It was found that the increase in FLuc activity correlated with increased levels of NS3F protein expression vector, indicating the stimulative effect of NS3F on the IRES-directed translation in a dose-dependent manner. Expression of LacZ in the same manner did not significantly affect the FLuc activity.
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). Although there is no immediate answer to explain this apparent discrepancy, it is possible that the different reporter constructs used could contribute to the discrepancies. The IRES in the reporter RNA used in our experiments is located at the 5'-terminal end of the reporter construct, in a similar location to the authentic viral IRES. In contrast, a dicistronic reporter was used in the previous report in which the IRES was inserted between two reporter genes. It was observed that the predicted secondary structure of the IRES located at the 5'-terminal end of the reporter construct was different from that inserted between the two reporter genes (data not shown). Moreover, experiments have revealed that IRES activity can be influenced by long-range RNA–RNA interaction (Kim et al., 2003), which may have reduced the observed activity (He et al., 2003).
For further characterization of the stimulative effect of the NS3 protein on IRES-directed translation, a pcDNA-3.1 vector that expresses NS3H, a truncated NS3 helicase with the postulated helicase domain (Sheng et al., 2007), was co-transfected into PK-15 cells with the monocistronic reporter RNA. A dose-dependent enhancement of IRES-directed translation was also observed, although the stimulative effect of NS3H was weaker than that of NS3F (Fig. 1b
). Expression of NS3F, NS3H and LacZ proteins was confirmed by immunoblot analysis using the corresponding antibody (Fig. 1c), and a dose-dependent expression of the above proteins was observed (data not shown).
CSFV NS5B protein is the viral replicase with an RNA-dependent RNA polymerase activity (Steffens et al., 1999; Xiao et al., 2006). Recent evidence indicates an interaction between the NS3 and NS5B proteins (Ishido et al., 1998; Zhang et al., 2005). To examine whether the interaction between the NS3 and NS5B proteins has an effect on the viral IRES-directed translation, we first transfected the CSFV NS5B protein expression vector into PK-15 cells (Xiao et al., 2003) together with the monocistronic reporter RNA. CSFV IRES-directed translation was not significantly affected by increasing the concentration of NS5B protein (Fig. 2a). Similar results were obtained for the CSFV NS3 protease (NS3P), which is lacking the helicase domain (Fig. 2a). The combined effect of both CSFV NS3 and NS5B on CSFV IRES-mediated translation was also examined in vivo. Transfection experiments were performed in which reporter RNA (1.0 µg) was co-transfected into PK-15 cells with 0.4 µg plasmids encoding NS3F, NS3H or NS3P, together with increasing concentrations of the NS5B protein expression vector. Results showed that the NS5B protein, in a dose-dependent manner, increased the stimulative effect of the NS3F protein on CSFV IRES-directed translation, but had no effect on translation in the presence of NS3H or NS3P (Fig. 2b). Expression of both NS5B and NS3P was confirmed by immunoblot analysis using the corresponding antibody (Fig. 1c). A dose-dependent expression of both proteins was observed (data not shown).
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). The NS3F protein was stronger than the NS3H protein in the enhancement of cellular translation (Fig. 3a). Expression of each of these proteins had previously been confirmed by immunoblot (Fig. 1c). These data strongly suggest that the RNA helicase, and not the RNA replicase, could enhance cellular translation. Results also showed that the NS5B protein, in a dose-dependent manner, promoted the stimulative effect of NS3F on cellular translation, but did not enhance translation in the presence of NS3H or NS3P (Fig. 3c).
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; Li et al., 1999). Previous reports show that a mutant RNA helicase selectively inhibits the cap-directed translation of brome mosaic virus (Noueiry et al., 2000). Modulation of RNA structure is an essential step in many fundamental processes, including RNA synthesis, splicing, replication and translation (Kadare & Haenni, 1997). The positive effect of RNA helicase on CSFV IRES-directed translation might be beneficial to viral cytopathogenicity because of the amount that NS3 increases in cells infected with the cytopathogenic isolates (Meyers & Thiel, 1995).
NS3 RNA helicase also promotes cellular translation, consistent with previous reports in which the best characterized RNA helicase, the eukaryotic translation initiation factor eIF-4A, is believed to disrupt secondary structures in mRNA upstream of the initiation codon, thereby facilitating attachment of the 40S ribosome (Pause & Sonenberg, 1992; Rozen et al., 1990). Helicase activity is also required for efficient translation termination (Gross et al., 2007). Viral RNA helicase enhancing cellular translation has also been found in previous data (Kato et al., 2002). In fact, RNA helicases have been described as essential factors in cell development and differentiation, and some of them play a role in transcription, translation initiation and replication of viral RNA genomes (Luking et al., 1998).
Interestingly, our data show that the full-length NS3 protein is stronger than a truncated NS3 protein in promoting both virus IRES-mediated and cellular translation. Previous experiments have demonstrated that RNA helicase activity of full-length NS3 protein is higher compared with that of the truncated NS3 protein that retains the helicase domain (Sheng et al., 2007; Zhang et al., 2005). This may explain the enhanced activity of the full-length protein on translation. It is possible that higher RNA helicase activity is more efficient for translation.
This report shows that the NS5B protein is able to enhance the stimulative effect of the full-length NS3 protein on CSFV IRES-mediated translation and its host cellular translation, but that it does not affect translation in the presence of truncated NS3 proteins. This may be related to the interaction between NS3 and NS5B. It has been shown that HCV NS5B enhances RNA helicase activity of the full-length NS3 protein, but does not affect that of a truncated NS3 protein from which the protease domain has been deleted. The protease domain is required for specific NS3 and NS5B interaction, suggesting that the NS5B protein promotes RNA helicase activity of the full-length NS3 protein by binding to the NS3 protease domain (Zhang et al., 2005). Previous data have shown that NS5B forms a complex with NS3 through an amino-terminal portion of NS3 (Ishido et al., 1998). Our results suggest that the NS5B and NS3 proteins interact via the protease domain during the enhancement of translation. However, how the CSFV NS5B protein enhances the stimulative effect of the full-length NS3 protein on translation by binding to the NS3 protease domain remains unclear.
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ACKNOWLEDGEMENTS |
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Received 29 July 2007;
accepted 19 December 2007.
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