Canine Hepacivirus NS3 Serine Protease Can Cleave the Human Adaptor Proteins MAVS and TRIF

Canine hepacivirus (CHV) was recently identified in domestic dogs and horses. The finding that CHV is genetically the virus most closely related to hepatitis C virus (HCV) has raised the question of whether HCV might have evolved as the result of close contact between dogs and/or horses and humans. The aim of this study was to investigate whether the NS3/4A serine protease of CHV specifically cleaves human mitochondrial antiviral signaling protein (MAVS) and Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF). The proteolytic activity of CHV NS3/4A was evaluated using a bacteriophage lambda genetic screen. Human MAVS- and TRIF-specific cleavage sites were engineered into the lambda cI repressor. Upon infection of Escherichia coli cells coexpressing these repressors and a CHV NS3/4A construct, lambda phage replicated up to 2000-fold more efficiently than in cells expressing a CHV protease variant carrying the inactivating substitution S139A. Comparable results were obtained when several HCV NS3/4A constructs of genotype 1b were assayed. This indicates that CHV can disrupt the human innate antiviral defense signaling pathway and suggests a possible evolutionary relationship between CHV and HCV.     

Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognized. We applied several computational methods to detect RNA secondary structure in the coding region of hepatitis C virus (HCV), including thermodynamic prediction, calculation of free energy on folding, and a newly developed method to scan sequences for covariant sites and associated secondary structures using a parsimony-based algorithm. Each of the prediction methods provided evidence for complex RNA folding in the core- and NS5B-encoding regions of the genome. The positioning of covariant sites and associated predicted stem-loop structures coincided with thermodynamic predictions of RNA base pairing, and localized precisely in parts of the genome with marked suppression of variability at synonymous sites. Combined, there was evidence for a total of six evolutionarily conserved stem-loop structures in the NS5B-encoding region and two in the core gene. The virus most closely related to HCV, GB virus-B (GBV-B) also showed evidence for similar internal base pairing in its coding region, although predictions of secondary structures were limited by the absence of comparative sequence data for this virus. While the role(s) of stem-loops in the coding region of HCV and GBV-B are currently unknown, the structure predictions in this study could provide the starting point for functional investigations using recently developed self-replicating clones of HCV.     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.

Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.     

Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.     

GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS

Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.     

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.     

Identification of a novel sequence at the 3' end of the GB virus B genome

GB virus B (GBV-B) is a virus of the family Flaviviridae that infects small primates (Saguinus sp. [tamarins]) and shows similarities to hepatitis C virus (HCV) in genome organization, protein function, tissue tropism, and pathogenicity. This suggests the possibility of using tamarins infected by GBV-B or GBV-B/HCV chimeric viruses as a surrogate animal model of HCV infection. To achieve the construction of such chimeric viruses, it is essential to produce a complete and infectious GBV-B genomic RNA. We have identified a novel sequence at the 3' end of the GBV-B genome and show that it can be arranged in a secondary structure resembling that of the 3' end of the HCV genome, which is known to be essential for infectivity.     

Identification of cellular factors associated with the 3'-nontranslated region of the hepatitis C virus genome

Chronic infection by hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remains largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3'-nontranslated region (3'-NTR) of the (+) strand HCV genome are essential for initiation of (-) strand synthesis. The RNA signals within the highly conserved 3'-NTR may be the site for recruiting cellular factors that mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our laboratory used in conjunction with LC/MS/MS allowed us to positively identify more than 70 cellular proteins that interact with the 3'-NTR (+) of HCV. Binding of these cellular proteins was not competed out by a 10-fold excess of nonspecific competitor RNA. With few exceptions, all of the identified cellular proteins are RNA-binding proteins whose reported cellular functions provide unique insights into host cell-virus interactions and possible mechanisms influencing HCV replication and HCV-associated pathogenesis. Small interfering RNA-mediated silencing of selected 3'-NTR-binding proteins in an HCV replicon cell line reduced replicon RNA to undetectable levels, suggesting important roles for these cellular factors in HCV replication.     

Structural map of a microRNA-122: hepatitis C virus complex

MicroRNA-122 (miR-122) enhances hepatitis C virus (HCV) fitness via targeting two sites in the 5'-untranslated region (UTR) of HCV. We used selective 2'-hydroxyl acylation analyzed by primer extension to resolve the HCV 5'-UTR's RNA secondary structure in the presence of miR-122. Nearly all nucleotides in miR-122 are involved in targeting the second site, beyond classic seed base pairings. These additional interactions enhance HCV replication in cell culture. To our knowledge, this is the first biophysical study of this complex to reveal the importance of 'tail' miR-122 nucleotide interactions.     

RNA binding protein 24 regulates the translation and replication of hepatitis C virus

The secondary structures of hepatitis C virus (HCV) RNA and the cellular proteins that bind to them are important for modulating both translation and RNA replication. However, the sets of RNA-binding proteins involved in the regulation of HCV translation, replication and encapsidation remain unknown. Here, we identified RNA binding motif protein 24 (RBM24) as a host factor participated in HCV translation and replication. Knockdown of RBM24 reduced HCV propagation in Huh7.5.1 cells. An enhanced translation and delayed RNA synthesis during the early phase of infection was observed in RBM24 silencing cells. However, both overexpression of RBM24 and recombinant human RBM24 protein suppressed HCV IRES-mediated translation. Further analysis revealed that the assembly of the 80S ribosome on the HCV IRES was interrupted by RBM24 protein through binding to the 5′-UTR. RBM24 could also interact with HCV Core and enhance the interaction of Core and 5′-UTR, which suppresses the expression of HCV. Moreover, RBM24 enhanced the interaction between the 5′- and 3′-UTRs in the HCV genome, which probably explained its requirement in HCV genome replication. Therefore, RBM24 is a novel host factor involved in HCV replication and may function at the switch from translation to replication.     

丙型肝炎病毒特异性核酶对HepG2.9706细胞HCV5′NCR基因表达的抑制作用

 为探讨锤头型核酶抗丙型肝炎病毒的作用 ,根据HCV 5′NCR及翻译起始区的二级结构 ,设计及合成了 1个锤头型核酶RzA .将其插入pCI neo表达载体的多克隆位点 ,构建了表达RzA的质粒pHCV RzA .采用转基因细胞模型HepG2 970 6细胞 ,评价了pHCV RzA质粒对HCV 5′NCR调控荧光素酶表达的抑制活性 .结果表明 ,在HepG2 970 6细胞中 ,pHCV RzA以序列特异性、剂量依赖性方式抑制荧光素酶基因的表达 ,抑制率达 80 % ,提示核酶有可能作为HCV感染基因治疗的一种有效途径 .     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.     

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.     

Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus

Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.     

Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.     

Detection of hepatitis C virus RNA in the hearts of patients with hepatogenic cardiomyopathy

We examined the Hepatitis C virus (HCV) genome in the myocardium and liver obtained at autopsy from seven patients with HCV-positive liver cirrhosis and hepatocellular carcinoma (HCC) by in situ hybridization and histopathological studies. The HCV virus genome was detected in the myocardium of one patient as well as in the liver in three out of seven patients. However, Epstein-Barr (EB) virus genome could not be detected in liver or myocardium. In the patient who showed positive reaction to HCV in myocardium, both serum HCV and Hepatitis B virus (HBV) antibodies were positive. It is unknown whether this was related to an immunological abnormality of the host or to an interaction between RNA and DNA viruses. In conclusion, we could identify the HCV genome in the myocardium of a patient with hepatogenic myocardosis.