Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.The flavivirus genus contains many medically important species, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). More than 2 billion people are at risk of infection by DENV alone, leading to an estimated 50 million cases annually, which may increase further as the range of the mosquito vector expands with urbanization (24). While disease from mosquito-borne flaviviruses is particularly common, there are other flaviviral human pathogens that exist with transmission cycles that do not involve mosquitoes. Tick-borne transmission is the other well-described route, but non-arthropod-borne routes also exist (for example, bats). It is likely that each transmission route has genetic adaptations that facilitate that route, but such changes are not yet understood (7).Serine/threonine phosphorylation is a conserved feature across all three genera of the family Flaviviridae, including the genus flavivirus (the others genera being pestivirus and hepacivirus). Among the features of Flaviviridae, the most-studied examples are the multiple phosphorylations of nonstructural protein 5A (NS5A) of hepatitis C virus, which exists in both basal (termed p56) and hyperphosphorylated (termed p58) states mediated by multiple kinases that both are necessary for and limit replication (14, 18, 23). Phosphorylation of NS5B, the RNA-dependent RNA polymerase (RdRP), has also been shown to affect replicon activity (10). In the genus flavivirus, several mosquito-borne viruses (DENV, WNV, and YFV) and at least one tick-borne encephalitis virus are known to have phosphorylated forms of nonstructural protein NS5 (2, 9, 11, 13, 19). In the genus flavivirus, NS5 is central to viral replication, as it possesses both RdRP and methyltransferase activities. DENV phosphorylation of NS5 correlates with the loss of NS5 interactions with the viral helicase NS3. A hyperphosphorylated form of NS5 was found to localize to the nucleus, away from the cytoplasmic sites of viral replication (6, 9). A nuclear localization sequence is present in DENV NS5 and is phosphorylated in vitro by host CKII, but the relationship between phosphorylation and nuclear localization has yet to be fully elucidated (17). Multiple different serine/threonine phosphorylation events likely occur in the flaviviral life cycle, potentially affecting various functions of NS5 (2), but the role of these events and identity of the kinase(s) responsible are largely unknown.In this report, we used mass spectrometry to identify serine/threonine phosphorylation sites in DENV. A single phosphoacceptor site, previously identified in YFV, is conserved specifically in the mosquito-borne flaviviruses but not the tick-borne flaviviruses. Furthermore, in vitro studies reveal that this site is phosphorylated by a cyclic-nucleotide-dependent kinase, protein kinase G (PKG), and a phosphoacceptor threonine/serine is required for replication. Taken together, these data implicate the PKG pathway in flaviviral replication for the first time and suggest a host cell pathway that could be targeted by antiviral therapy.     

Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core

The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy.     

甲型流行性感冒病毒NS蛋白研究进展

甲型流行性感冒(流感)病毒基因组由8个分节段的负链RNA组成,共编码10种蛋白,其中在病毒复制的早期即有NP蛋白和NS1蛋白的大量表达,提示这两种蛋白在病毒复制过程中及与细胞蛋白的相互作用中发挥着重要的功能.RNA第8节段编码两种蛋白,即非结构蛋白1(NS1)和2(NS2).     

HCV NS5A and NS5B enhance expression of human ceramide glucosyltransferase gene

Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection.Here we demonstrate that artificial up-regulation of fatty acid biosynth...     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.     

Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.     

庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880bp的庚型肝炎病毒cDNA在大肠杆菌BL21（DE3）菌株中得到表达。此cDNA被插入到表达质粒pGEX-5X-1中,位于编码日本血吸虫谷胱甘肽硫转移酶（GST）的DNA序列下游,并与GST处于同一阅读框。用乳糖在37℃下诱导表达出以体形式存在的GST－NS53融合蛋白,并用脲溶法提取了该蛋白;在20℃诱导时,表达出的蛋白大部分可溶,用谷胱甘肽Sepharose－4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明,此融合蛋白能这臾型肝炎病人的血清和自制的扩GST在汪有特异性地识别。用PCgene软件对NS53氨基酸序列的亲水性和抗原决定簇进行了分析,本研究为庚型肝炎ELISA诊断试剂研制打下了基础。     

庚型肝炎病毒NS5区蛋白鼠单克隆抗体的制备

庚型病毒性肝炎是近年来世界上才确认的一种新型肝炎［１～３］。庚型肝炎病毒（ＨＧＶ）呈世界性分布,经血液传播为主,也可母婴传播。ＨＧＶ容易形成持续性感染,类似ＨＩＶ和ＨＣＶ。据粗略估计,我国大约有１００万～１０００万ＨＧＶ携带者。因此,ＨＧＶ已成为继乙...     

庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880 bp的庚型肝炎病毒cDNA在大肠杆菌BL21(DE3)菌株中得到表达。此cDNA被插入到表达质粒pGEX-5X-1中,位于编码日本血吸虫谷胱甘肽硫转移酶（GST）的DNA序列下游,并与GST处于同一阅读框。用乳糖在37℃下诱导表达出以包涵体形式存在的GST-NS53融合蛋白,并用脲溶法提取了该蛋白;在20℃诱导时,表达出的蛋白大部分可溶,用谷胱甘肽Sepharose-4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明,此融合蛋白能被庚型肝炎病人的血清和自制的抗GST血清特异性地识别。用PCgene软件对NS53氨基酸序列的亲水性和抗原决定簇进行了分析。本研究为庚型肝炎ELISA诊断试剂研制打下了基础。     

丙型肝炎病毒NS5A基因在昆虫细胞中的表达及其分布研究

应用PCR方法从含有丙肝病毒全部非结构蛋白基因的质粒pBAC25中扩增出全长的NS5A基因DNA片段(约1.34kb),PCR扩增NS5A基因片段克隆到转移载体pBlueBacHisA中.重组转移质粒pBlueBacHis5A DNA与野生型杆状病毒(AcNPV)DNA共转染SF-9昆虫细胞,通过空斑纯化获得带有NS5A基因的重组病AcNS5A.对重组病毒基因组DNA进行酶切和PCR鉴定,证实HCV NS5A基因已插入重组病毒基因组中.AcNS5A感染SF-9细胞后,在细胞中表达出一条64kD的蛋白,用Western-blot分析,结果表明这种蛋白与抗HCV HS5A特异性抗体发生强烈反应,说明NS5A基因已在细胞中得到表达,应用免疫荧光技术与免疫组化技术进一步研究NS5A蛋白在昆虫细胞中不同时间的表达情况及其分布,结果表明,NS5A蛋白在AcNS5A重组病毒感染细胞24h后主要分布在细胞质膜上,而在48h后则同时分布于细胞质膜和细胞核内,在72h则完全布满整个细胞,我们认为NS5A蛋白定位于质膜和细胞核中,暗示着在病毒复制过程中NS5A蛋白可能参与病毒RNA在质膜上复制和细胞基因表达的调控.     

丙型肝炎病毒NS5A基因在昆虫细胞中的表达及其分布研究

应用PCR方法从含有丙肝病毒全部非结构蛋白基因的质粒pBAC25中扩增出全长的NS5A基因DNA片段（约1.34kb),PCR扩增NS5A基因片段克隆到转移载体plueBacHisA中。重组转移质粒pBlueBacHis5ADNA与野生型杆状病（AcNPV)DNA共转染SF-9昆虫细胞,通过空斑纯化获得带有NS5A基因的重组病AcNS5A,对重组病毒基因组DNA进行酶切和PCR鉴定,证实HCV NS5A基因已插入重组病毒基因组中,AcNS5A感染SF-9细胞后,在细胞中表达出一条64kD的蛋白,用Western-blot分析,结果表明这种蛋白与抗HVCHS5A特异性抗体发生强烈反应,说明NS5A基因已在细胞中得到表达,应用免疫荧光技术与免疫组化技术进一步研究NS5A蛋白在昆虫细胞中不同时间的表达情况及其分布,结果表明,NS5A蛋白在AcNS5A重组病毒感染细胞24h后主要分布在细胞质膜上,而在48h后则同时分布于细胞质膜和细胞核内,在72h则完全布满整个细胞,我们认为NS5A蛋白定位于质膜和细胞核中,暗示着在病毒复制过程中NS5A蛋白可能参与病毒RNA在质膜上复制和细胞基因表达的调控。     

NS1 Protein of Influenza A Virus Down-Regulates Apoptosis

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.     

Induction of Intrahepatic HCV NS4B,NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.     

Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B

NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities.     

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2 and NS5A-D3), CypA, and NS5B_Δ21. We show that three regions of NS5A-D2 (residues 250–262 (region A), 274–287 (region B), and 306–333 (region C)) interact with NS5B_Δ21, whereas NS5A-D3 does not. We show that both NS5B_Δ21 and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has been detected by NMR spectroscopy between HCV NS5B_Δ21 and host CypA. We show that cyclosporine A added to a sample containing NS5B_Δ21, NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B_Δ21. A high quality heteronuclear NMR spectrum of HCV NS5B_Δ21 has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight the potential of using NMR of NS5B_Δ21 as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins, inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and CypA, three essentials proteins for HCV replication.     

All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding

The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.Hepatitis C virus (HCV) is a human pathogen which chronically infects nearly 3% of the world''s population (36, 37). Persistent infection, in 80% of cases, leads to chronic hepatitis which can progress to liver cirrhosis and, in the worst cases, hepatocellular carcinoma (37). Current therapies lack specificity and efficacy due largely to an incomplete understanding of the complex molecular mechanisms of virus infectivity, RNA replication, and assembly (4, 36). HCV is a member of the Flaviviridae family of enveloped viruses (30), with a positive-sense RNA genome of ∼9.6 kb consisting of a single open reading frame (ORF) that encodes 10 structural and nonstructural viral proteins (3, 16, 25). Cap-independent translation of the ORF (29) yields a large polyprotein of approximately 3,000 amino acid residues that is cleaved co- and posttranslationally by host and viral proteases into 10 mature virus proteins; these cleavage products are ordered from the amino to the carboxy terminus as follows: core (C), envelope proteins 1 and 2 (E1 and E2), p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (3, 16, 25). At the flanking ends of the genome are two highly conserved untranslated regions (UTRs). The 5′ UTR is highly structured and consists of the internal ribosome entry site (IRES), which is important for the initiation of cap-independent translation of the polyprotein (29). The 3′ UTR consists of a short genotype-specific variable region, a tract of variable length comprising solely pyrimidine residues (predominantly U), and a conserved 98-nucleotide sequence, known as the X region, containing three stem-loops (13, 23) (Fig. ​(Fig.1A).1A). The 3′ UTR is the initiation site for the synthesis of the negative-strand RNA during viral replication (13) and is involved in translational regulation.Open in a separate windowFIG. 1.The HCV 3′ UTR RNA. (A) The positive-strand 3′ UTR consists of three distinct regions, i.e., a short genotype-specific variable region, a polypyrimidine tract [poly(U/UC)] of variable length, and a conserved 98-nucleotide sequence known as the X region containing three stable stem-loops. The predicted structure of the genotype 1b 3′ UTR is shown. (B) Left panel, the integrities of in vitro-transcribed radiolabeled full-length 3′ UTR RNAs of genotypes 1b (nucleotides 9375 to 9595) and 2a (nucleotides 9443 to 9678) and the poly(U/UC) (nucleotides 9406 to 9497) and X region (nucleotides 9498 to 9595) of genotype 1b are shown on denaturing polyacrylamide gels. Right panel, the integrities of in vitro-transcribed radiolabeled RNAs comprising the 3′-terminal NS5B-coding region plus the 3′ UTR RNAs of genotypes 1b (nucleotides 9136 to 9595) and 2a (nucleotides 9204 to 9678) (KL-3′ UTR) are shown on denaturing polyacrylamide gels.HCV RNA replication occurs on membranous structures derived from the endoplasmic reticulum (ER) in a complex that includes host cell factors as well as viral nonstructural proteins, including NS5B, the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome in vivo and in vitro (2, 25, 30). Initiation of the synthesis of the negative-strand RNA is thought to occur upon recognition and specific binding of the NS5B polymerase to the 3′ UTR of the genomic RNA (2, 16, 26). This replication activity and template specificity of NS5B in vivo are dependent, however, on the presence of the other nonstructural proteins, such as the proteases NS2 and NS3, which are required for polyprotein processing and helicase activity, and the multifunctional protein NS5A (16).NS5A is a proline-rich phosphoprotein that is absolutely required for viral replication and is also involved in virus particle assembly (9, 10, 20, 22, 35). Its specific function in the latter process is, however, still unknown. NS5A is membrane associated due to the presence of an N-terminal amphipathic helix that serves as a membrane anchor allowing association with ER-derived membranes (Fig. ​(Fig.2)2) (24, 27). The cytoplasmic portion of NS5A is organized into three domains that are separated by low-complexity sequences (Fig. ​(Fig.2A)2A) (20). The X-ray crystal structure of domain I has revealed that it is a zinc binding domain which forms a homodimer with contacts at the N-terminal ends of the molecules; the resultant large, basic groove at the dimeric interface has been proposed to be involved in RNA binding during viral replication (17, 33). NS5A has also been shown to interact with uridylate and guanylate-rich RNA and to bind to the 3′ ends of the HCV positive- and negative-strand RNAs (8). These observations suggest that NS5A may specifically interact with the large U/G stretches in the IRES of the 5′ UTR, implying a role in HCV translation and genome multiplication, while its interactions with the polypyrimidine tract of the 3′ UTR suggest that NS5A may affect the efficiency of RNA synthesis by NS5B (8, 28, 32). The reported interactions with both flanking regions of the HCV genome imply that NS5A may play a role in the switch between translation and replication that must occur during the viral life cycle (8).Open in a separate windowFIG. 2.Domain structure and expression of HCV NS5A. (A) Schematic diagram of the functional domains of NS5A and design of the constructs used in the study (genotype 1b NS5A protein numbering). The N-terminal amphipathic helix of NS5A (black box) is responsible for the interaction of NS5A with membranes. NS5A is organized into three domains that are separated by low-complexity sequences, indicated by black boxes. The NS5A constructs used all lacked the N-terminal amphipathic helix and were designed to include an N-terminal Strep tag and a C-terminal hexahistidine tag. (B and C) SDS-PAGE and Western blot analysis of the NS5A(ΔAH) and NS5A domain constructs purified by nickel affinity and Streptactin tag affinity chromatography. Coomassie brilliant blue-stained gels and Western blots (WB) using anti-NS5A antibodies for NS5A proteins of genotype 1b strain J4 (B) and genotype 2a strain JFH-1 (C) are shown.Among HCV genotypes, domains II and III are less well conserved than domain I (34). By mutational analysis, domain II, along with domain I, has been attributed to the replicase activity of NS5A (12). Contrastingly, domain III has been shown to be dispensable for RNA replication, and large heterologous insertions and deletions in this region can be tolerated, maintaining RNA replication (34). It has been shown, however, that these insertions and deletions within domain III do have an impact on virus particle assembly, highlighting the critical role of domain III NS5A in the viral life cycle (1, 10). Recent nuclear magnetic resonance (NMR) studies of domains II and III of NS5A revealed that they both adopt a natively unfolded state (6, 14, 15). The high degree of disorder and flexibility observed in these domains may contribute to the promiscuity of NS5A, which has been shown to interact with a variety of biological partners essential for NS5A function and virus persistence (11, 18, 19, 21, 31). In addition, regions within domains I and II of NS5A interact with NS5B, stimulating the in vitro activity of the polymerase and supporting the hypothesis that NS5A has a role in the modulation of RNA replication (28, 32).In this study, we have investigated in detail the RNA binding properties of NS5A. We have mapped the RNA binding regions of NS5A using bacterially expressed deletion constructs of NS5A and have assayed their binding affinity for HCV positive-strand 3′ UTR RNA. In addition, we provide evidence that the RNA binding activity of NS5A is specific and that NS5A interacts preferentially with the polypyrimidine region of the 3′ UTR.