Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner

Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.Hepatitis C virus (HCV) has a positive-stranded RNA genome and belongs to the family Flaviviridae (21). HCV chronically infects more than 130 million people worldwide (34), and HCV infection often induces liver cirrhosis and hepatocellular carcinoma (19, 28). To date, pegylated interferon (PEG-IFN) and ribavirin are the standard treatments for HCV infection. However, many patients cannot tolerate their serious side effects. Therefore, the development of new and safer therapeutic methods with better efficacy is urgently needed.Lipids play important roles in HCV infection and replication. For example, the HCV core associates with lipid droplets and recruits nonstructural proteins and replication complexes to lipid droplet-associated membranes which are involved in the production of infectious virus particles (24). HCV RNA replication depends on viral protein association with raft membranes (2, 30). The association of cholesterol and sphingolipid with HCV particles is also important for virion maturation and infectivity (3). The inhibitors of the sphingolipid biosynthetic pathway, ISP-1 and HPA-12, which specifically inhibit serine palmitoyltransferase (SPT) (23) and ceramide trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus (37), suppress HCV virus production in cell culture but not viral RNA replication by the JFH1 replicon (3). Other serine SPT inhibitors (myriocin and NA255) inhibit genotype 1b replication (4, 29, 33). Very-low-density lipoprotein (VLDL) also interacts with the HCV virion (15).Sakamoto et al. reported that sphingomyelin bound to HCV RNA-dependent polymerase (RdRp) at the sphingomyelin binding domain (SBD; amino acids 230 to 263 of RdRp) to recruit HCV RdRp on the lipid rafts, where the HCV complex assembles, and that NA255 suppressed HCV replication by releasing HCV RdRp from the lipid rafts (29). In the present study, we analyzed the effect of sphingomyelin on HCV RdRp activity in vitro and found that sphingomyelin activated HCV RdRp activity in a genotype-specific manner. We also determined the sphingomyelin activation domain and the activation mechanism. Finally, we confirmed our biochemical data by a HCV replicon system.     

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.     

Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.Human hepatitis delta virus (HDV) is an unusual subviral agent that increases the severity of acute and chronic liver disease in those infected with its helper, hepatitis B virus (23). The HDV genome is a 1,680-nucleotide (nt) single-stranded circular RNA that is replicated by a double-rolling-circle mechanism (reviewed in references 15 and 28). Both the genome and antigenome RNAs form a characteristic unbranched rod structure due to 70% sequence complementarity between the noncoding and coding regions of the RNA (10, 11, 31). HDV encodes just one protein, hepatitis delta antigen (HDAg), which forms ribonucleoprotein (RNP) complexes with both the genome and the antigenome in cells replicating HDV (3, 5, 30). These complexes play fundamental roles in viral RNA replication and packaging and their characterization is essential for understanding these processes, which are not well characterized.HDAg has been shown to form dimers and higher order multimers, even in the absence of HDV RNA (25, 30, 32). The multimerization activity has been localized to the amino-terminal third of the 195-amino-acid (aa) protein (12, 24, 30, 32). X-ray crystallographic analysis of a peptide comprised of aa 12 to 60 indicated that antiparallel dimers are stabilized by a coiled coil (aa 16 to 48), as well as a hydrophobic core region (aa 50 to 60) that also stabilizes interactions between dimers such that an octameric structure may form (35). Zuccola et al. found that bacterially expressed HDAg could be cross-linked in an octameric structure, and Cornillez-Ty et al. obtained evidence supporting such a structure in cells replicating HDV (7, 35). Site-directed mutations of HDAg amino acids critical for dimerization and/or multimerization abolish the ability of HDAg to support RNA replication (18, 32), indicating that the formation of HDAg multimers is essential for this process.We recently showed that bacterially expressed, C-terminally truncated HDAg forms stable RNP complexes in vitro with segments of HDV RNA that form unbranched rod structures (8). No particular sequences or structures in the RNA, other than the HDV unbranched rod, were essential for complex formation, but, remarkably, binding required that the RNA have a minimum length of at least about 300 nt. Overall, the results were consistent with the formation of a large RNP containing multiple copies of the 19-kDa protein that bound to the RNA either in a highly cooperative manner or as a preformed multimer. On the other hand, based on indirect measures of the RNA-binding activity of site-directed HDAg mutations in cells, others have found that HDAg multimerization might not be required for RNA-binding activity (18).Here, we directly analyze the role of HDAg multimerization in the formation of the HDV RNP complex. We find that HDAg binds to HDV unbranched rod RNA as a preformed multimer. Site-directed mutations that disrupted protein multimerization did not abolish binding but profoundly altered the nature of the RNA-protein complex. In particular, we found that multimerization is associated with RNA-binding specificity, including the RNA length requirement for binding. For the wild-type protein, RNP formation was found to be strongly temperature dependent, suggesting that conformational changes occur on binding, and providing a plausible explanation of the RNA length requirement for binding. Furthermore, we show that the protein binds as multiple multimeric units on longer RNAs, provided the length of the RNA is sufficient. We conclude that the HDAg multimer plays a critical role in the formation of properly structured HDV RNPs.     

pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用。通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况。试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平。在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%。初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达。本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持。     

pRNA介导的RNA干扰抑制HBV表达和复制的研究

为了研究由pRNA携带的siRNA(HBVsi18-42)所介导的RNAi过程能有效地抑制HBV的基因表达和病毒复制,我们利用细胞模型和高压注射小鼠模型评价HBVsi18-42对HBV复制和基因表达的抑制作用.通过Western印迹检测细胞内的HBsAg含量,用ELISA检测细胞培养上清和小鼠血清中的HBsAg水平,采用Southern印迹检测HBV的复制中间体,通过免疫组织化学检测肝组织切片中HBcAg的表达情况.试验结果显示,HBVsi18-42能以剂量依赖的方式在293T细胞中抑制HBsAg的表达以及在HepG2细胞中下调病毒HBsAg和HBeAg的表达和病毒复制中间体的水平.在小鼠模型中,注射后的3d内HBVsi18-42使小鼠血清中HBsAg的水平分别下降了98.98%、77.07%和60.73%,免疫组织化学检测显示,在注射后的第3天小鼠肝组织内HBcAg阳性细胞数减少了79.1%.初步结果显示HBVsi18-42无论是在细胞或是在小鼠模型中都能下调HBV的复制和基因的表达.本研究为我们下一步实现由pRNA介导的靶向RNAi及基因治疗提供了理论和技术支持.     

A Hepatitis C Virus NS5A Phosphorylation Site That Regulates RNA Replication

The hepatitis C virus NS5A protein is essential for RNA replication and virion assembly. NS5A is phosphorylated on multiple residues during infections, but these sites remain uncharacterized. Here we identify serine 222 of genotype 2a NS5A as a phosphorylation site that functions as a negative regulator of RNA replication. This site is a component of the hyperphosphorylated form of NS5A, which is in good agreement with previous observations that hyperphosphorylation negatively affects replication.