Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.     

Subgenomic replicon derived from a cell line infected with the hepatitis C virus

Recently, cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. To see whether a HCV sequence derived from HCV-infected cultured cell sequence can be used for the construction of a functional replicon, a HCV subgenomic RNA carrying a neomycin-resistant gene was constructed using the HCV genome RNA obtained from cultured cells infected with HCV. After transfection, G418-resistant Huh-7 cells were selected and subcloned. Finally, the production of HCV proteins and de novo synthesis of subgenomic RNA were confirmed in the selected cell clone, indicating that this subgenomic RNA replicated in cultured cells and functioned as a replicon. These results suggest that the HCV genome obtained from an in vitro HCV infection system with cultured cells can be used to develop a subgenomic replicon system with diverse HCV sequences.     

嵌合中国分离株基因的丙型肝炎病毒（1b／2a）细胞培养模型的建立

目的：建立嵌合中国分离株基因的丙型肝炎病毒（HCV）细胞培养模型。方法：利用3片段融合PCR的方法将中国HCV河北分离株（1b）的全长包膜蛋白基因引入JFH1（2a）株基因骨架,构建包膜蛋白基因区相互置换的嵌合HCV（1b／2a）全长基因组,经线性化后体外转录获得全长RNA,转染Huh7．5．1细胞系,用免疫荧光及蛋白印迹实验检测。结果：该RNA可以产生具有体外感染活性的嵌合HCV,且感染性可在共同培养的细胞间传播。结论：首次在国内建立了嵌合中国HCV分离株基因的HCV细胞培养体系。     

HCV 1a／1b型嵌合体能在HepG2细胞内复制与表达

采用HCV 1a／1b嵌合体cDNA构建表达质粒转染HepG2细胞,以免疫组化和Westem blotting检测HCV蛋白表达,RT-PCR检测HCV正、负链RNA,研究丙型肝炎病毒(HCV) 1a和1b型嵌合体全长cDNA在HepG2细胞中的复制和表达。结果证明,转染细胞中检测到分子量约70kDa的HCV NS3蛋白,转染细胞连续传20代,仍能检测到HCV正、负链RNA。表明该HCV嵌合体可以在细胞中复制和表达,HCV1b型的RNA依赖的RNA聚合酶(RdRp)可以起始含1a型非编码区的病毒复制。HCV5′端非翻译区第11、12、13、34和35位核苷酸改变可不影响其与核糖体结合。3′非翻译区9400,9403和9407位核苷酸改变,9435位缺失“A”,9409,9410位及9495,9496,9497位分别插入“TT”和“AAT”可不影响RdRp的生物活性。本研究对阐明HCV复制和翻译机制有重要意义。     

嵌合型HCV全长cDNA的构建及体外转录制备感染性RNA的研究

构建HCV la/1b嵌合型全长cDNA克隆,进行体外转录,脂质体法转染HepG2细胞,以RT-PCR法检测HCV正、负链RNA,Western印迹检测HCV蛋白表达.结果表明,细胞在转染后8代(约35d)内,能间断检测到HCV正、负链RNA以及相对分子质量约70000的HCV NS3蛋白,证明该HCV嵌合体可以在细胞中复制和表达.本研究表明含有该嵌合型全长cDNA的质粒可以为后续HCV的研究提供大量可重复的性质均一的病毒模板,有助于深入了解HCV的复制机制.     

Molecular Genetic Study of the Interaction of Sindbis Virus E2 with Ross River Virus E1 for Virus Budding

Glycoprotein PE2 of Sindbis virus will form a heterodimer with glycoprotein E1 of Ross River virus that is cleaved to an E2/E1 heterodimer and transported to the cell plasma membrane, but this chimeric heterodimer fails to interact with Sindbis virus nucleocapsids, and very little budding to produce mature virus occurs upon infection with chimeric viruses. We have isolated in both Sindbis virus E2 and in Ross River virus E1 a series of suppressing mutations that adapt these two proteins to one another and allow increased levels of chimeric virus production. Two adaptive E1 changes in an ectodomain immediately adjacent to the membrane anchor and five adaptive E2 changes in a 12-residue ectodomain centered on Asp-242 have been identified. One change in Ross River virus E1 (Gln-411→Leu) and one change in Sindbis virus E2 (Asp-248→Tyr) were investigated in detail. Each change individually leads to about a 10-fold increase in virus production, and combined the two changes lead to a 100-fold increase in virus. During passage of a chimeric virus containing Ross River virus E1 and Sindbis virus E2, the E2 change was first selected, followed by the E1 change. Heterodimers containing these two adaptive mutations have a demonstrably increased degree of interaction with Sindbis virus nucleocapsids. In the parental chimera, no interaction between heterodimers and capsids was visible at the plasma membrane in electron microscopic studies, whereas alignment of nucleocapsids along the plasma membrane, indicating interaction of heterodimers with nucleocapsids, was readily seen in the adapted chimera. The significance of these findings in light of our current understanding of alphavirus budding is discussed.     

Cell clones selected from the Huh7 human hepatoma cell line support efficient replication of a subgenomic GB virus B replicon

Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.     

肝癌细胞系（HepG2）感染HCV RNA的研究

为建立丙型肝炎病毒（HCV）体外感染和细胞培养系统,用定量的HCV RNA阳性血清感染人肝癌细胞系（HepG2细胞系）,应用地高辛标记HCV RNA探针原位杂交技术和RT－PCR方法对感染后的细胞和上清液听 HCV RNA进行了检测。在感染后的第一代至第七代的细胞中出现特异性杂交阳性信号,第一代、第二代和第六代检测出HCV RNA正链,并在感染后第一、二代检测出HCV RNA负链。显示HCV不仅能在体外感染HepG2细胞系,而且在基因的复制,证明HepG2细胞能作为HCV的体外细胞培育系。     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

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.     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.     

Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice

A chimeric flavivirus infectious cDNA was constructed by exchanging the premembrane (prM) and envelope (E) genes of the yellow fever virus vaccine strain 17D (YF17D) with the corresponding genes of Modoc virus (MOD). This latter virus belongs to the cluster of the "not-known vector" flaviviruses and is, unlike YF17D, neuroinvasive in SCID mice. Replication of in vitro-transcribed RNA from this chimeric flavivirus was shown by [(3)H]uridine labeling and RNA analysis. Expression of the MOD prM and E proteins was monitored by radioimmunoprecipitation and revealed that the MOD proteins were correctly and efficiently produced from the chimeric precursor protein. The MOD E protein was shown to be N-linked glycosylated, whereas prM, as predicted from the genome sequence, did not contain N-linked carbohydrates. In Vero cells, the chimeric virus replicated with a similar efficiency as the parental viruses, although it formed smaller plaques than YF17D and MOD. In SCID mice that had been infected intraperitoneally with the chimeric virus, the viral load increased steadily until death. The MOD/YF virus, like MOD from which it had acquired the prM and E structural proteins, but unlike YF, proved neuroinvasive in SCID mice. Animals developed neurological symptoms about 15 days after inoculation and died shortly thereafter. The distribution of MOD/YF RNA in the brain of infected mice was similar to that observed in MOD-infected mice. The observations provide compelling evidence that the determinants of neuroinvasiveness of flaviviruses are entirely located in the envelope proteins prM and E.     

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.     

Characterization of hepatitis C virus replication in cloned cells obtained from a human T-cell leukemia virus type 1-infected cell line, MT-2.

We recently found that a human T-cell leukemia virus type 1-infected cell line, MT-2, could support the replication of hepatitis C virus (HCV) (N. Kato, T. Nakazawa, T. Mizutani, and K. Shimotohno, Biochem. Biophys. Res. Commun. 206:863-869, 1995). In order to develop a culture system in which HCV replicates more efficiently, we examined the efficiency of HCV replication in cloned MT-2 cell lines by the limiting dilution method. Consequently, we obtained five clones in which intracellular positive-stranded HCV RNA could be detected until at least 21 days postinoculation (p.i.), as opposed to 15 days p.i. in uncloned MT-2 cells. MT-2C, one of the five clones which supported HCV replication up to 30 days p.i., was used for further characterization of HCV replication. Semiquantitative analysis of HCV by PCR revealed that RNA synthesis in infected cells increased after inoculation, reached a maximum level at 4 days p.i., and maintained this level until at least 11 days p.i. The 5' untranslated region of negative-stranded HCV RNA was also detected in the infected cells by two different methods with strand specificity. These results suggest that HCV replicated and multiplied in the MT-2C cells. HCV-infected MT-2C cells that were treated with antibiotics, such as G418 and hygromycin B, sustained HCV RNA for a longer period than did untreated cells. We demonstrated inhibitory effects on HCV replication by an antisense oligonucleotide complementary to the HCV core encoding region and by interferon-alpha. Furthermore, cell-free viral transmission was demonstrated by this culture system. These results suggest that our cell culture system will be useful for studying the mechanism of HCV replication, for screening antiviral agents, and for developing HCV vaccines.     

Expression and purification of E2/NS1 protein of hepatitis C virus and detection of anti-E2/NS1 antibodies in chronic liver disease patients

Glycoproteins on the surface of viral particles present the main target of neutralizing antibodies. The structural proteins of most Flaviviruses are known to elicit neutralizing antibodies and, thus, to help in both the natural resolution of the infection and the protection from challenge with homologous hepatitis C virus (HCV). Because such antigens are associated with the viral clearance in both humans and chimpanzees, we aimed to express the E2/NS1 protein of HCV and to study the role of anti-E2/NS1 antibodies in the natural resolution of HCV infection. The prevalence of anti-E2/NS1 antibodies to recombinant E2/NS1 protein was seen by Western blot in chronic liver disease patients (15 chronic hepatitis and 12 cirrhotic patients), who were positive for anti-HCV and negative for HBV infection. The study also included 2 negative controls (positive for HBV infection and negative for anti-HCV antibodies) and 2 healthy controls (negative for both HBV and HCV infection). Anti-E2/NS1 was present in 20% of the chronic hepatitis and 16% of the cirrhosis patients. None of the controls were positive for anti-E2/NS1 antibodies. Serum samples positive for anti-E2/NS1 antibodies were also positive for HCV RNA by RT/PCR. Accordingly, the presence of anti-E2/NS1 may have very little or no role in the natural resolution of HCV infection.     

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.     

Coexpression of hepatitis C virus E1 and E2 chimeric envelope glycoproteins displays separable ligand sensitivity and increases pseudotype infectious titer

We have previously reported that a pseudotype virus generated by reconstitution of hepatitis C virus (HCV) chimeric envelope glycoprotein E1-G or E2-G on the surface of a temperature-sensitive mutant of vesicular stomatitis virus (VSVts045) interacts independently with mammalian cells to initiate infection. Here, we examined whether coexpression of both of the envelope glycoproteins on pseudotype particles would augment virus infectivity and/or alter the functional properties of the individual subunits. Stable transfectants of baby hamster kidney (BHK) epithelial cells expressing either one or both of the chimeric envelope glycoproteins of HCV on the cell surface were generated. The infectious titer of the VSV pseudotype, derived from a stable cell line incorporating both of the chimeric glycoproteins of HCV, was approximately 4- to 5-fold higher than that of a pseudotype bearing E1-G alone or approximately 25- to 30-fold higher than that of E2-G alone when assayed with a number of mammalian cell lines. Further studies suggested that that the E1-G/E2-G or E2-G pseudotype was more sensitive to the inhibitory effect of heparin than the E1-G pseudotype. Treatment of the E1-G/E2-G pseudotype with a negatively charged sulfated sialyl lipid (NMSO3) displayed a approximately 4-fold-higher sensitivity to neutralization than pseudotypes with either of the two individual glycoproteins. In contrast, VSVts045, used as a backbone for the generation of pseudotypes, displayed at least 20-fold-higher sensitivity to NMSO3-mediated inhibition of virus plaque formation. The effect of low-density lipoprotein on the E1-G pseudotype was greater than that apparent for the E1-G/E2-G pseudotype. The treatment of cells with monoclonal antibodies to CD81 displayed an inhibitory effect upon the pseudotype with E1-G/E2-G or with E2-G alone. Taken together, our results indicate that the HCV E1 and E2 glycoproteins have separable functional properties and that the presence of these two envelope glycoproteins on VSV/HCV pseudotype particles increases infectious titer.     

我国西部1a型丙肝病毒结构区的cDNA克隆和序列分析

克隆我国西部丙型肝炎病毒全部结构区基因,为探讨HCV的变异和致癌作用,研制丙肝疫苗作准各。从1名西安市丙肝感染血液中,用Trizol试剂裂解HCV病毒颗粒,用糖原与RNA共沉淀提取HCVRNA,用AMV逆转录酶和随机引物逆转录为eDNA,用RT-PCR方法扩增目的片段并转化到pGEM-T质粒,得到pGEM-T-HCVc、pGEM-T-HCVel和pGEM-T-HCVe2克隆。将以上3个克隆用特定的酶降解,用连结酶进行连接,构建了HCV结构区的完整克隆。将克隆好的质粒pGEM-T-HCVjg从两端双向测广手,得到完整的HCV结构基因eDNA核苷酸序列,总长度为2238bp,与已发表的HCV序列长度一致,未发现缺失或移码突变,序列中间亦未发现转录终止子。本序列与HCVla、lb序列核苷酸的同源性分别为91．8％、84．5％;氨基酸的同源性分别为94.2％、86．3％;应为HCVla亚型。以上结果说明我国西部地区存在HCVla亚型,所克隆HCV结构区eDNA克隆可以用于基因工程表达。