Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Human cytomegalovirus wikipedia , lookup
Ebola virus disease wikipedia , lookup
West Nile fever wikipedia , lookup
Hepatitis C wikipedia , lookup
Marburg virus disease wikipedia , lookup
Influenza A virus wikipedia , lookup
Orthohantavirus wikipedia , lookup
Hepatitis B wikipedia , lookup
Journal of General Virology (2015), 96, 1027–1032 Short Communication DOI 10.1099/vir.0.000048 Annexin A2 is involved in the production of classical swine fever virus infectious particles Chun Sheng, Xiaoxiang Liu, Qiuyue Jiang, Bin Xu, Chenhao Zhou, Yujing Wang, Jun Chen and Ming Xiao Correspondence College of Life and Environment Sciences, Shanghai Normal University, Shanghai, PR China Ming Xiao [email protected] Received 16 October 2014 Accepted 9 January 2014 Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses. To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus (CSFV), an RNA interference (RNAi) approach was utilized. Knockdown of ANXA2 did not impair CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of NS5A were revealed to be pivotal for the ANXA2–NS5A interaction. Substitutions of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production, which might partly be due to these mutations destroying the ANXA2–NS5A interaction. These results suggested that ANXA2 might participate in CSFV production process by binding NS5A. Classical swine fever virus (CSFV) is an important livestock pathogen and is a member of the genus Pestivirus, along with bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and border disease virus (BDV). They belong to the family Flaviviridae, which also contains the closely related human hepatitis C virus (HCV) (Heinz et al., 2000; Becher & Thiel, 2002). CSFV is an enveloped positive-strand RNA virus. The virus contains a single-strand, positive-sense RNA genome, which is composed of a 59UTR, an ORF and a 39UTR. The 39UTR and the 59UTR are thought to regulate pestivirus genome replication (Isken et al., 2003, 2004; Xiao et al., 2004; Pankraz et al., 2005). The 59UTR includes an internal ribosome entry site that is responsible for translation initiation of viral genomes (Fletcher & Jackson, 2002; Xiao et al., 2011). The ORF codes for a resulting polyprotein that is subsequently processed into mature proteins (Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) by cellular and viral proteases (Moennig & Plagemann, 1992). The CSFV NS5B protein has RNA-dependent RNA polymerase (RdRp) activity and is able to bind its cognate 39UTR and initiates genome replication (Steffens et al., 1999; Xiao et al., 2004). NS5A is a component of the replication complex. CSFV NS5A comprises 497 aa and is able to regulate viral RNA replication and translation through binding to NS5B and 39UTR (Chen et al., 2012; Xiao et al., 2009). The viral life cycle requires various viral non-structural proteins as well as host cellular proteins. Annexin A2 (ANXA2) is known as a lipid raft-associated scaffold protein. It is calcium dependent and cytosolic and mediates essential biological processes, including membrane trafficking, endosome formation, and aggregation of vesicles (Drust & Creutz, 1988). ANXA2 has been identified as an important 000048 G 2015 The Authors host factor in the regulation of several key processes in cytomegalovirus (Wright et al., 1995), influenza virus (LeBouder et al., 2008), calicivirus (González-Reyes et al., 2009), bluetongue virus (Beaton et al., 2002), human immunodeficiency virus (Ryzhova et al., 2006) and HCV (Backes et al., 2010). Recently, a proteomic analysis revealed that CSFV infection led to a significant increase of ANXA2 protein in host cells (Sun et al., 2008). Haem oxygenase I, another cellular protein and ANXA2 were found to be colocalized with the CSFV E2 protein (Shi et al., 2009). The expression of annexin A1, another member of the annexin family, was also elevated in PK-15 cells following CSFV infection (Sun et al., 2010). However, the role of many cellular proteins in the life cycle of CSFV remains poorly understood. In this paper, we show that ANXA2 is involved in CSFV production. To evaluate the potential involvement of ANXA2 in the life cycle of CSFV, an RNA interference (RNAi) approach and CSFV subgenomic replicons and genomic replicons (Sheng et al., 2010) (Fig. 1a) were utilized. A small interfering RNA (siRNA) molecule targeted to porcine ANXA2 sequence (GenBank accession no. NM_001005726) and a negative control siRNA molecule that has no matches either in the viral or the porcine genome were synthesized (Shanghai Gene Chem). An anti-CSFV siRNA directed to the CSFV NS5B gene served as a positive control (Xu et al., 2008). Results showed that the ANXA2-specific siRNA reduced the amount of ANXA2 without a significant influence on the cell viability (results not shown) while the control siRNA, anti-CSFV siRNA and the mock transfection without siRNA addition did not (Fig. 1b). Silencing ANXA2 did not reduce viral protein expression Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 Printed in Great Britain 1027 C. Sheng and others pro ms 5′UTR N C E SM ms pro 5′UTR N ΔC E CSM pro SM– 5′UTRN CSM– 5′UTR N pro E1 E1 Ems C ΔC Ems E1 E1 (b) NS3 NS4A NS4B NS5A E2 p70 NS2 E2 p70 NS2 NS3 NA NA iR ts ou ith W 1.2 1.0 0.8 0.6 0.4 0.2 0 ro nt Co A2 X AN ti- An NA NA iR ls siR An V SF C ti- NS5B G9973A C9976T (d) RNA CSM RNA–1 β-Tubulin 3′UTR 3′UTR G9973A C9976T NS3 NS4ANS4B NS5A NS5B 3′UTR (c) ANXA2 NS5B NS3 NS4A NS4B NS5A E2 p70 NS2 3′UTR NS5B NS3 NS4A NS4B NS5A E2 p70 NS2 24 48 72 h ** **** iR ts ou ith W iR ls ro nt Co NA NA NA siR ti- An A2 X AN siR V SF C ti- An NA Log10 TCID50 ml–1 (a) 8 6 4 2 0 24 48 72 h ** ** iR ts ou ith W NA NA siR ro nt Co A2 X AN ti- An NA NA iR ls ** ** ** ** siR V SF siR C ti- An Fig. 1. Effect of siRNA-mediated knockdown of ANXA2 on CSFV production. (a) Schematic representation of genomic replicons SM, SM”, subgenomic replicons CSM and CSM”, used in this study (Sheng et al., 2010). (b, c) The indicated siRNAs were transfected into PK-15 cells using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s protocol. Seventy-two hours after siRNA transfection, the transfected cells were retransfected with CSM” and a second dose of the same siRNA. Viral particles were not formed and newly synthesized viral RNAs were detected in CSM” transfected cells. (b) Seventy-two hours after retransfection, cells were harvested. The cell lysates were subjected to 10 % SDS-PAGE. The amounts of ANXA2 (upper) and CSFV NS3 (below) in the cells transfected with the indicated siRNAs were evaluated by Western blot analysis (Sheng et al., 2012) with anti-ANXA2 or anti-NS3 antibody. b-Tubulin was used as an internal control and also for monitoring cytotoxicity (middle). (c) Cells were harvested at 24, 48 or 72 h after CSM” subgenomic replicon and siRNA transfection. Quantification of viral RNA was determined by using RT-qPCR (Wang et al., 2011). Relative replication efficiencies are shown. The CSM subgenomic replicon was used to normalize the data. (d) The indicated siRNAs were transfected into PK-15 cells. The transfected cells were retransfected with SM” and a second dose of the same siRNA. The retransfected cells were harvested at 24, 48 and 72 h post-transfection, and titres were determined by immunofluorescence (Xiao et al., 2011). The arrowhead represents the detection limit of the assay. All quantified results are shown as means±SD of at least three independent experiments. ** P,0.01. and RNA replication (Fig. 1b, c). In contrast, substantial reduction in viral protein expression and RNA replication was observed for anti-CSFV siRNA treatment, indicating that the RNAi approach is efficient in controlling viral protein expression and RNA replication. Furthermore, the anti-ANXA2 siRNA resulted in a significant reduction of virus titre compared to that with the control siRNA and the treatment without siRNA (Fig. 1d). These results showed that knockdown of ANXA2 did not impair CSFV RNA replication, but significantly reduced infectious CSFV production, suggesting that ANXA2 is involved in the production of infectious CSFV particles. To examine the potential role of ANXA2 in the production of infectious CSFV particles, we analysed the effect of siRNA-mediated knockdown of ANXA2 on the extracellular and intracellular amounts of viral Core protein. Lower levels of extracellular Core protein were detected for the cells transfected with anti-ANXA2 siRNA, while the amount of intracellular Core protein is comparable to the 1028 level for control siRNA treatment (Fig. 2a, b). The antiANXA2 siRNA resulted in a significant reduction of extracellular Core proteins by about 70 % compared to that with the control siRNA (Fig. 2b). These data indicated that ANXA2 is important for Core protein release from CSFV RNA transfected cells. The amount of Core protein released into cell supernatants is used as a surrogate measure of the production of infectious virus (Miyanari et al., 2007), thus, ANXA2 is important for the production of infectious CSFV particles. This reduction in the amount of extracellular CSFV particles was due to a block in assembly or release. To address this question, we determined extracellular and intracellular infectivity levels by a TCID50 assay. Anti-ANXA2 siRNAs significantly decreased extracellular and intracellular infectivity levels and a comparable reduction of extracellular and intracellular infectivity levels was observed (Fig. 2c). We did not find an accumulation of intracellular infectious particles in the anti-ANXA2 siRNA transfected cells. Lower levels of specific infectivity (SI) of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 Journal of General Virology 96 Annexin A2 is involved in CSFV production (a) (b) 1.5 Extracellular Intracellular Extracellular core 1.0 0.5 Intracellular core NA A RN i ts ou l tro 0 A RN siR 2 XA n Co ith W A RN si AN FV NA iR ts si ou CS ith int int W A A (c) **** 3 ** ** 2 RNA SM RNA–1 Log10 TCID50 ml–1 4 X Co AN NA FV siR CS int int A 1.2 RNA SI 1.0 ** 0.8 0.6 0.6 0.4 i l tro A RN siR n Co 2 si A NX A ti- An ** 0 NA A RN 0.4 ** 0.2 0 W A2 0.8 1 it NA siR 1.0 5 ts r nt 1.2 Extracellular Intracellular 6 NA iR s ol A (d) 7 u ho ** ** ** si C ti- An V SF A RN NA iR it W 0.2 0 ts u ho SI SM SI–1 Core SM Core–1 β-Tubulin A RN A RN i ls ro t on C 2 XA AN ti- An si NA V F CS siR ti- An Fig. 2. Effect of siRNA-mediated knockdown of ANXA2 on the extracellular and intracellular amount of viral Core protein and infectivity. Seventy-two hours after the indicated siRNA transfection, the siRNA transfected PK-15 cells were retransfected with a second dose of the same siRNA together with a CSFV genomic replicon, SM- (Sheng et al., 2010). (a) At 72 h posttransfection, the cells were harvested and extracellular and intracellular Core proteins were detected by Western blot analysis with anti-Core antibody. b-Tubulin serves as an internal control. (b) Signal intensities quantified by Kodak ID 3.5 software were used to evaluate the levels of extracellular and intracellular Core. The relative values were shown by comparing densitometric values with the densitometric value for the SM replicon, which was set at 1. (c) Extracellular and intracellular infectivity levels were determined by TCID50 assay as described in Fig. 1(d). (d) Release of virus RNA from transfected cells 72 h posttransfection was determined by using reverse transcript-qPCR (RT-qPCR) as described in Fig. 1(c). Specific infectivity (SI) was calculated as TCID50 ml”1/RNA copies. The SM was used to normalize the data. All quantified results are shown as means±SD of at least three independent experiments. ** P,0.01. virions made in the presence of anti-ANXA2 siRNA were detected while release of virus RNA was comparable to the level for control siRNA treatment (Fig. 2d). Taken together, these results indicated that a decrease in the production of infectious CSFV particles might be attributable to defective virion assembly rather than to virus release or RNA replication. The anti-ANXA2 siRNA treatment might not impair virus RNA, but result in virions with lower infectivity. http://vir.sgmjournals.org Previous reports showed that HCV NS5A is necessary for HCV assembly and production and interacts with ANXA2 (Appel et al., 2008; Masaki et al., 2008; Saxena et al., 2012). To investigate whether ANXA2 interacts with CSFV NS5A, several NS5A mutants (Fig. 3a) formed in our previous work (Chen et al., 2012) were used for immunoprecipitation analysis. As shown in Fig. 3(b), ANXA2 specifically coprecipitated CSFV NS5A. Mutant D335–389 lost ANXA2 binding activity but other NS5A mutants retained Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 1029 137 173 224 268283335390414444 497 + + + + + – + – – + + + + + – + – + + + – – + + + WB:FLAG + + IP: ANXA2 WB:ANXA2 – + + D390–414 D415–413 CD53 + –33 IP: ANXA2 WB:FLAG –62 –48 + –33 348 348 348 349 389 389 390 389 389 * * (f) –62 –48 IP: ANXA2 WB:FLAG * (d) WT R338A N359A G378A G378L G378L 301 301 301 302 335 335 336 335 335 N359A G378A (e) HCV 52 HCV 1b HCV 1a HCV 64 BDV X818 BVDV1 NADL BVDV2 C413 CSFV Eystrup CSFV Shimen WT R338A (c) –62 –48 –62 –48 WB:FLAG (g) IP: ANXA2 WB:ANXA2 RNA SM RNA–1 1 WT ND136 D137–172 D173–223 D224–268 D269–282 D283–334 D335–389 (b) D390–414 D415–443 CD53 ANXA2 NS5B 3′UTR binding binding binding 1.2 1.0 0.8 0.6 0.4 0.2 0 24 48 72 h CSM-WT CSM-N359A CSM-G378L CSM-R338A CSM-G378A -Tubulin NS3 Log10 TCID50 ml–1 (a) WT ND136 D137–172 D173–223 D224–268 D269–282 D283–334 D335–389 C. Sheng and others CSM-WT CSM-N359A CSM-G378L CSM-R338A CSM-G378A 8 6 4 2 0 24 48 72 hrs SM-WT SM-N359A SM-G378L CSM-R338A CSM-G378A Fig. 3. ANXA2 interacts with CSFV NS5A. (a) Schematic of the CSFV NS5A protein and its mutant forms from previous reports (Chen et al., 2012). D335–389 indicates an internal deletion mutant lacking corresponding amino acids. The ANXA2, NS5B and 39UTR binding activity of these proteins are shown on the right. (b) Immunoprecipitation analysis of the interaction between ANXA2 and CSFV NS5A. PK-15 cells were transfected with the pcDNA3.1/N-FLAG vector containing NS5A (WT or mutant). At 72 h post-transfection, cell lysates were immunoprecitated using anti-ANXA2 antibody. Immunoprecipitation of NS5A-ANXA2 interaction was determined by Western blot analysis with anti-FLAG antibody (upper) or anti-ANXN A2 antibody (below). Total lysates were fractionated by 10 % SDS-PAGE and subjected to Western blot analysis with anti-FLAG antibody (middle). (c) The NS5A protein sequence of CSFV was aligned with that of BDV, BVDV and HCV using CLUSTAL_X (partially shown). The conserved amino acids are indicated by asterisks. (d) Schematic representation of NS5A and its substitution mutants used in this study. (e) Immunoprecipitation of the NS5A–ANXA2 interaction was determined by Western blot analysis with anti-FLAG antibody or antiANXN A2 antibody as described in Fig. 3(b). (f) PK-15 cells were transfected with the CSM” subgenomic replicon carrying WT or mutant NS5A proteins (R338A, N359A, G378A or G378L). Quantification of viral RNA was determined as described in Fig. 1(c). The amounts of NS3 (below) were evaluated as described in Fig. 1(b). (g) PK-15 cells were transfected with the SM” subgenomic replicon carrying WT or mutant NS5A proteins (R338A, N359A, G378A or G378L). Titres were determined as described in Fig. 1(d). All quantified results are shown as means±SD of at least three independent experiments. **P,0.01. the activity (Fig. 3b), indicating that amino acids 335–389 might be important for ANXA2 binding. As a comparison, we aligned the NS5A sequences of CSFV, BDV, BVDV and HCV. Three conserved amino acids R338, N359 and G378 were indicated (Fig. 3c), suggesting that these amino acids might be important for NS5A interactions with ANXA2. To corroborate this assumption, alanine or leucine substitutions were introduced into R338, N359 and G378 in CSFV NS5A. Four CSFV NS5A mutants, R338A, N359A, G378A and G378L were produced (Fig. 3d). Immunoprecipitation 1030 assays revealed that ANXA2 binding activity was lost for R338A, N359A and G378L and was substantially reduced for G378A (Fig. 3e). The results indicated that amino acids R338, N359 and G378 of CSFV NS5A are pivotal for the ANXA2–NS5A interaction. Furthermore, the same substitution mutations were incorporated into the CSM2 subgenomic replicon and the SM2 genomic replicon, respectively. These mutations had no substantial effect on viral RNA replication and viral protein expression (Fig. 3f) but substantially reduced CSFV production (Fig. 3g). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 Journal of General Virology 96 Annexin A2 is involved in CSFV production Host proteins participate in many aspects of viral infection. Vesicle-associated membrane protein-associated proteins mediate the formation of HCV RNA placation complex on lipid rafts (Gao et al., 2004). RNA helicase A, another host protein, has been found to be involved in the expression and replication of CSFV and bind CSFV 59UTR and 39UTR (Sheng et al., 2013). Cellular NFAR proteins associate specifically with both the termini of the BVDV RNA genome involving regulatory elements (Isken et al., 2003). ANXA2 is an important host protein that plays a role in different steps of viral life cycle (Backes et al., 2010; Beaton et al., 2002; González-Reyes et al., 2009; LeBouder et al., 2008; Ryzhova et al., 2006; Wright et al., 1995). In the report, we identified ANXA2 as an important factor in the production of the CSFV infectious particles. ANXA2 also is a key host factor that influences HCV yield (Backes et al., 2010). For the role of ANXA2 in viral RNA replication, scientists reported contradictory findings. Some people showed that ANXA2 knockdown reduced viral RNA replication (Saxena et al., 2012) while another observed that ANXA2 knockdown did not have this effect (Backes et al., 2010). Our present results suggested that ANXA2 plays a role in CSFV assembly rather than in genome replication and virion release. The discrepancy may partly be due to the differences in virus species and in siRNA sequences. The data for immunoprecipitation experiments showed that ANXA2 interacts with CSFV NS5A, in agreement with the previous reports in which ANXA2 binds HCV NS5A (Saxena et al., 2012). The NS5A protein of CSFV, BVDV and HCV has been shown to be an essential replicase component (Masaki et al., 2008; Tellinghuisen et al., 2006; Sheng et al., 2010), thus, ANXA2 might participate in the CSFV production process by binding to NS5A protein. In fact, ANXA2 has been found to be involved in the formation of HCV replication complex on the lipid raft (Saxena et al., 2012). Mutation analysis revealed that amino acids 335–389 of NS5A might be responsible for ANXA2 binding (Fig. 3c, d). NS5A is a multifunctional protein. The C terminus of CSFV NS5A contains several binding sites, such as a NS5B binding site and a 39UTR binding site (Fig. 3a) (Chen et al., 2012). Recently, we have found that the C-terminal sequence from amino acids 478–487 of NS5A is important for the interaction between CSFV NS5A and Core protein, which also might be related to the production of infectious CSFV particles (Sheng et al., 2014). The amino acid fragment responsible for ANXA2 binding is adjacent to the NS5B binding site at the C terminus of CSFV NS5A (Fig. 3a) (Chen et al., 2012). Furthermore, R338, N359 and G378 of CSFV NS5A were observed be pivotal for ANXA2–NS5A interactions (Fig. 3c, e). Substitution of these amino acids had no effect on viral RNA replication but substantially reduced CSFV production (Fig. 3f, g), which might partly be due to this substitution destroying ANXA2–NS5A interactions. These results suggested that ANXA2 might participate in the CSFV production process by binding NS5A. This is http://vir.sgmjournals.org attractive for further investigation of the role of ANXA2 in the life cycle of pestivirus and HCV. Acknowledgements This work was supported by the National Natural Science Foundation of China (30870492, 31070671), the Natural Science Foundation of Shanghai (12ZR1422100), the Shanghai Municipal Science and Technology Commission (11JC1409300) and the Shanghai Municipal Education Commission (13ZZ102, 11YZ87, 14ZZ123). References Appel, N., Zayas, M., Miller, S., Krijnse-Locker, J., Schaller, T., Friebe, P., Kallis, S., Engel, U. & Bartenschlager, R. (2008). Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4, e1000035. Backes, P., Quinkert, D., Reiss, S., Binder, M., Zayas, M., Rescher, U., Gerke, V., Bartenschlager, R. & Lohmann, V. (2010). Role of annexin A2 in the production of infectious hepatitis C virus particles. J Virol 84, 5775–5789. Beaton, A. R., Rodriguez, J., Reddy, Y. K. & Roy, P. (2002). The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release. Proc Natl Acad Sci U S A 99, 13154–13159. Becher, P. & Thiel, H. J. (2002). Genus Pestivirus (Flaviviridae). In The Springer Index of Viruses, pp. 327–331. Edited by C. A. Tidona & G. Darai. Heidelberg, Germany: Springer-Verlag. Chen, Y., Xiao, J., Xiao, J., Sheng, C., Wang, J., Jia, L., Zhi, Y., Li, G., Chen, J. & Xiao, M. (2012). Classical swine fever virus NS5A regulates viral RNA replication through binding to NS5B and 3’UTR. Virology 432, 376–388. Drust, D. S. & Creutz, C. E. (1988). Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature 331, 88–91. Fletcher, S. P. & Jackson, R. J. (2002). Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 59 untranslated region important for IRES function. J Virol 76, 5024– 5033. Gao, L., Aizaki, H., He, J. W. & Lai, M. M. C. (2004). Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J Virol 78, 3480–3488. González-Reyes, S., Garcı́a-Manso, A., del Barrio, G., Dalton, K. P., González-Molleda, L., Arrojo-Fernández, J., Nicieza, I. & Parra, F. (2009). Role of annexin A2 in cellular entry of rabbit vesivirus. J Gen Virol 90, 2724–2730. Heinz, F. X., Collett, M. S., Purcell, R. H., Gould, E. A., Howard, C. R., Houghton, M., Moormann, R. J. M., Rice, C. M. & Thiel, H.-J. (2000). Family Flaviviridae. In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses, pp. 859–878. Edited by C. M. Fauquet, M. H. V. van Regenmortel, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch & other authors. San Diego: Academic Press. Isken, O., Grassmann, C. W., Sarisky, R. T., Kann, M., Zhang, S., Grosse, F., Kao, P. N. & Behrens, S. E. (2003). Members of the NF90/ NFAR protein group are involved in the life cycle of a positive-strand RNA virus. EMBO J 22, 5655–5665. Isken, O., Grassmann, C. W., Yu, H. & Behrens, S. E. (2004). Complex signals in the genomic 39 nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA. RNA 10, 1637–1652. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 1031 C. Sheng and others LeBouder, F., Morello, E., Rimmelzwaan, G. F., Bosse, F., Péchoux, C., Delmas, B. & Riteau, B. (2008). Annexin II incorporated into influenza virus particles supports virus replication by converting plasminogen into plasmin. J Virol 82, 6820–6828. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., Date, T., Matsuura, Y., Miyamura, T. & other authors (2008). Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J Virol 82, 7964–7976. Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M., Bartenschlager, R., Wakita, T., Hijikata, M. & Shimotohno, K. (2007). The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9, 1089–1097. Moennig, V. & Plagemann, P. G. W. (1992). The pestiviruses. Adv Virus Res 41, 53–98. Pankraz, A., Thiel, H. J. & Becher, P. (2005). Essential and Shi, Z., Sun, J., Guo, H. & Tu, C. (2009). Genomic expression profiling of peripheral blood leukocytes of pigs infected with highly virulent classical swine fever virus strain Shimen. J Gen Virol 90, 1670–1680. Steffens, S., Thiel, H. J. & Behrens, S. E. (1999). The RNA- dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro. J Gen Virol 80, 2583– 2590. Sun, J., Jiang, Y., Shi, Z., Yan, Y., Guo, H., He, F. & Tu, C. (2008). Proteomic alteration of PK-15 cells after infection by classical swine fever virus. J Proteome Res 7, 5263–5269. Sun, J., Shi, Z., Guo, H. & Tu, C. (2010). Changes in the porcine peripheral blood mononuclear cell proteome induced by infection with highly virulent classical swine fever virus. J Gen Virol 91, 2254– 2262. Tellinghuisen, T. L., Paulson, M. S. & Rice, C. M. (2006). The NS5A nonessential elements in the 39 nontranslated region of bovine viral diarrhea virus. J Virol 79, 9119–9127. protein of bovine viral diarrhea virus contains an essential zincbinding site similar to that of the hepatitis C virus NS5A protein. J Virol 80, 7450–7458. Ryzhova, E. V., Vos, R. M., Albright, A. V., Harrist, A. V., Harvey, T. & González-Scarano, F. (2006). Annexin 2: a novel human immuno- Wang, Y., Zhu, Z., Wang, P., Yu, J., Wan, L., Chen, J. & Xiao, M. (2011). deficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages. J Virol 80, 2694–2704. Saxena, V., Lai, C. K., Chao, T. C., Jeng, K. S. & Lai, M. M. C. (2012). Annexin A2 is involved in the formation of hepatitis C virus replication complex on the lipid raft. J Virol 86, 4139–4150. Sheng, C., Zhu, Z., Yu, J., Wan, L., Wang, Y., Chen, J., Gu, F. & Xiao, M. (2010). Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis. Vet Microbiol 140, 72–80. Sheng, C., Wang, J., Xiao, J., Xiao, J., Chen, Y., Jia, L., Zhi, Y., Li, G. & Xiao, M. (2012). Classical swine fever virus NS5B protein suppresses Characterisation of interaction between NS3 and NS5B protein of classical swine fever virus by deletion of terminal sequences of NS5B. Virus Res 156, 98–106. Wright, J. F., Kurosky, A., Pryzdial, E. L. & Wasi, S. (1995). Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts. J Virol 69, 4784–4791. Xiao, M., Gao, J., Wang, W., Wang, Y., Chen, J., Chen, J. & Li, B. (2004). Specific interaction between the classical swine fever virus NS5B protein and the viral genome. Eur J Biochem 271, 3888–3896. Xiao, M., Wang, Y., Zhu, Z., Yu, J., Wan, L. & Chen, J. (2009). Influence the inhibitory effect of NS5A on viral translation by binding to NS5A. Journal of General Virology 93, 939–950. of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation. J Gen Virol 90, 2923–2928. Sheng, C., Yao, Y., Chen, B., Wang, Y., Chen, J. & Xiao, M. (2013). Xiao, M., Wang, Y., Zhu, Z., Ding, C., Yu, J., Wan, L. & Chen, J. (2011). RNA helicase is involved in the expression and replication of classical swine fever virus and interacts with untranslated region. Virus Res 171, 257–261. Influence of the 59-proximal elements of the 59-untranslated region of classical swine fever virus on translation and replication. J Gen Virol 92, 1087–1096. Sheng, C., Kou, S., Jiang, Q., Zhou, C., Xiao, J., Li, J., Chen, B., Zhao, Y., Wang, Y. & Xiao, M. (2014). Characterization of the C-terminal Xu, X., Guo, H., Xiao, C., Zha, Y., Shi, Z., Xia, X. & Tu, C. (2008). In sequence of NS5A necessary for the assembly and production of classical swine fever virus infectious particles. Res Vet Sci 97, 449– 454. 1032 vitro inhibition of classical swine fever virus replication by siRNAs targeting Npro and NS5B genes. Antiviral Res 78, 188–193. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 04:42:49 Journal of General Virology 96