Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.Hepatitis C virus (HCV) infection is a major global health problem. More than 170 million people worldwide are infected with HCV. HCV causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (18). A member of the family Flaviviridae, HCV has a positive-sense, single-stranded RNA genome that is packaged into an enveloped viral particle. The genome encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, envelope protein 1 (E1), E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (12, 13). Core associates with the lipid droplet (LD). The role of this association remained elusive until robust HCV replication systems became available (32). We previously showed that the LD is an important organelle for HCV production (23). In hepatocytes, the LD is physiologically important as a lipid source for the production of lipoproteins such as very low density lipoprotein (VLDL) (11). VLDL is synthesized in the liver as a triglyceride/cholesterol ester-rich particle (diameter, 30 to 100 nm) surrounded by apolipoproteins such as apolipoprotein B100 (abbreviated as ApoB throughout), ApoC''s, and ApoE. VLDL is released into blood vessels to be delivered as a lipid source to peripheral cells, and it is also readsorbed by liver cells after processing (5).HCV particles circulating in the blood of HCV carriers associate with lipoproteins, such as low-density lipoprotein (LDL), VLDL, and chylomicrons; thus, these are termed lipo-viro particles (LVPs) (1, 26). Purified LVPs from circulating blood contain triglyceride, ApoB, ApoB48, ApoCII, ApoCIII, ApoE, and virus components such as HCV RNA and core (8), indicating that the LVP has dual viral and lipoprotein characteristics. The HCVcc strain, which contains a chimeric HCV-2a genome with a structural region from HCV-J6 and nonstructural/noncoding regions from an infectious JFH1 virus, can establish long-term infection in chimpanzees. Viruses recovered from the chimpanzee contain infectious virus particles with a slightly low density, suggesting that an in vivo association with low-density factors influences infectivity (19). However, the role of a lipoprotein-like component of LVPs in virus replication is not clear. Moreover, the mechanism by which LVPs are generated during HCV production is unknown.When HCV-producing cells are treated with an inhibitor of microsomal triglyceride transfer protein (MTP) or with ApoB-specific small interfering RNA (siRNA), the production of HCV particles is suppressed (10, 14, 25). Therefore, lipoprotein biosynthesis appears to play an important role in the production of infectious HCV and its egress from infected cells. ApoB, ApoC1, and ApoE associate with infectious virus particles in the HCVcc infection/replication system (4, 6, 15, 22, 27). Furthermore, ApoE depletion suppresses the production of infectious HCV (4, 6, 15, 27). These reports strongly suggest the importance of lipoprotein function to the HCV life cycle. However, the precise roles of lipoproteins and apolipoproteins in virus production and infectivity are not fully understood.We analyzed the production of HCV from cells in which apolipoprotein production was knocked down with siRNA. We found that ApoE is required for the infectivity of HCV, a finding consistent with other reports (4, 6, 15). ApoE is a polymorphic protein with three major isoforms: ApoE2, ApoE3, and ApoE4. The three isoforms differ by amino acid substitutions at one or two sites (residues 130 and 176) on the 317-amino-acid chain of the ApoE molecule. The polymorphism of ApoE influences its multiple functions due to isoform-dependent differences in receptor-binding activity and lipoprotein association preference. For example, ApoE2 has drastically lower LDL receptor (LDLR) binding activity than ApoE3 and ApoE4 (7). In the present study, we investigated the role of ApoE isoforms in virus production and infectivity.(Part of this study was presented at the 16th International Symposium on Hepatitis C Virus and Related Viruses, Nice, France, 3 to 7 October 2009.)     

分子克隆化禽网状内皮组织增生症病毒传染性及其前病毒全基因组序列研究

利用脂质体转染技术,将含有SNV株禽网状内皮组织增生症病毒 （REV）前病毒全基因组cDNA克隆质粒转染鸡胚成纤维细胞（CEF）.用对REV的单克隆抗体和抗REV env-gp90的鼠血清作间接免疫荧光反应,在原始的转染细胞及随后传代的细胞中均显示病毒特异性抗原.而且,在连续传代细胞中的阳性率明显升高.用REV特异性引物对进一步传代后的细胞基因组作PCR,也检测出REV基因组.这些结果均表明所得到的分子克隆化病毒具有传染性,因而也进一步证明所用的质粒克隆包含有具感染性的全病毒基因组.对该全基因组cDNA克隆进行酶切所获得的数个亚克隆进行测序,并将序列进行拼接,完成了REV全基因组序列.REV的这个传染性克隆将有助于进一步研究REV的分子生物学特性.     

A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background

Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings

In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10⁻⁹ IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion

This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.     

Genetic Diversity and Tissue Compartmentalization of the Hepatitis C Virus Genome in Blood Mononuclear Cells, Liver, and Serum from Chronic Hepatitis C Patients

The degree of genetic variability in the hypervariable region 1 of hepatitis C virus (HCV) was analyzed by cloning and sequencing HCV genomes obtained in paired samples of serum, liver tissue, and peripheral blood mononuclear cells (PBMC) from four chronic hepatitis C patients. Genetic variability in serum was higher than in liver tissue or PBMC at the level of complexity (the number of different sequences obtained from each type of tissue) as well as at the level of genetic distance between all pairs of sequences within each tissue (compared by the Student t test; P < 0.001 for two patients and P < 0.01 for another). The spectrum of viral genomes differed among the three types of tissue, as shown by segregation of sequences according to their tissue of origin in phylogenetic analysis and by statistical analysis of mean genetic distances observed between sequences obtained from different tissues (P < 0.001), but sequences from liver tissue and PBMC were more closely related to each other than to those from serum.     

Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.     

丙型肝炎病毒基因变异及候选保守表位分析

HCV分离株主要分为4个基因型(HCV Ⅰ～Ⅳ),各型间的氨基酸及核苷酸组成同源性均小于80％, 氨基酸变异率分别为C 8%,E1 35%,E2/NS1 53%,NS3 27%,NS4 35%,NS5 39%.不同型别的HCV有不同的地区分布特征.根据HCV表达产物多肽的保守性、亲水性、抗原性及空间构型等特性,已在HCV表达产物中鉴定出一些高度保守的候选B细胞表位及T细胞表位, 其中B细胞表位一般为12～40肽, T细胞表位一般为7～9肽, 这些B/T细胞保守表位的鉴定, 将有助于推动HCV的免疫治疗及疫苗研究的发展.     

丙型肝炎病毒体外培养系统研究进展

由于缺少丙型肝炎病毒（HCV）的细胞培养系统和小动物模型，因此对其生活周期、作用机制至今仍不是很清楚，从而严重阻碍了丙型肝炎疫苗及相关治疗药物的开发与研制。一直以来，人们研究的HCV体外培养细胞模型包括感染模型和转染模型两种，感染模型由于原代肝细胞培养问题未能解决而难以成功，而转染模型的发展可喜。但是HCV复制子只能在极少数细胞中短暂复制，且产生的病毒量很低。1999年建立了亚基因组复制子，使人们有机会对其进行深入研究，但须人为引入碱基突变。最近建立的全基因复制子不需要引入突变即形成病毒粒子，是一项重大突破。概述了HCV体外培养系统的研究进展。     

丙型肝炎病毒高变区基因变异特点初探

对两例ＨＣＶＲＮＡ持续阳性者分三个时间点随访五年,测定了ＨＣＶ的高变区基因序列,每个时间点平均测定２８个克隆,总共测定了１６８个克隆。研究发现ＨＣＶ高变区基因变异有五个明显特点：（１）变异程度大,高变区至少有８９％的核苷酸位点都可能发生变异;（２）变异形式多样,除常见的替代突变外,缺失突变（缺失１、２或３个核苷酸）的克隆数占总克隆数的２２．５％;（３）优势克隆明显,即有若干个克隆高变区的核苷酸和氨基酸序列相同;（４）类似株现象严重;（５）变异幅度大。该研究说明ＨＣＶ高变区基因变异具有高度复杂多样性。     

Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.Hepatitis C virus (HCV) is a major contributor to the development of human liver diseases, infecting approximately 2% of the population, or 130 million people, worldwide (2). Up to 80% of HCV infections progress to chronic hepatitis and can lead to cirrhosis and hepatocellular carcinoma (38). No vaccine exists to prevent HCV infection, and current treatments are frequently inadequate.HCV is an enveloped virus of the genus Hepacivirus in the family Flaviviridae (30). The single-stranded, positive-sense RNA genome encodes a polyprotein of about 3,000 amino acids, which is processed by viral and host proteases into three structural proteins (the core protein, E1, and E2) and seven presumed nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The core protein is thought to encapsidate the RNA genome within the virion, forming a complex that is surrounded by a host cell-derived lipid bilayer displaying the envelope glycoproteins, E1 and E2. Although not thought to be components of the virion, p7 and NS2 have recently been implicated in the production of infectious virus (5, 12, 29, 33, 39). The remaining nonstructural proteins, NS3 to NS5B, are essential for genome replication and have additional emerging roles in virus assembly. NS3 possesses RNA helicase/NTPase activities and together with its cofactor, NS4A, forms the major viral protease. NS5B is the RNA-dependent RNA polymerase (reviewed in references 16 and 27).The core protein is the first protein produced during translation of the incoming viral genome. A signal sequence in its C-terminal region targets the nascent E1 glycoprotein to the endoplasmic reticulum (ER) membrane and is the substrate for processing by two host proteases. Cleavage by signal peptidase (SP) following core amino acid 191 (31) is thought to precede processing by signal peptide peptidase (SPP) (20, 26), an integral membrane aspartyl protease that cleaves within transmembrane segments (37). The C terminus of the mature, infectious-virion-associated core protein has not been determined, but it is speculated to lie between amino acids 173 and 182 (24, 31). SPP processing has been shown to mobilize the core from the ER membrane and enable it to traffic to lipid droplets (20). These triglyceride-rich storage organelles have recently been shown to be the sites of HCV particle assembly (21). Consistent with this finding, impaired SPP activity leads to decreased HCV infectious titers (34). Dual processing of the core proteins is a common feature of the Flaviviridae family. GB virus B, a hepacivirus, and classical swine fever virus, a related pestivirus, encode core proteins that undergo SP and SPP processing during maturation (8, 35). In the genus Flavivirus, the capsid protein undergoes regulated cleavage by the viral NS2B-3 protease and SP; this stepwise processing has been shown to be essential for proper encapsidation of genomes into infectious particles (3).The development of an infectious cell culture system for HCV has been a major breakthrough in the field (7). Many details of virus morphogenesis and infectivity, however, are still unknown. In this study, we examined the role of the C-terminal portion of the HCV core protein and identified individual amino acids that are essential for infectious virus assembly and core protein stability. Findings from alanine-scanning and transcomplementation studies suggest that at least 177 residues of the core protein are needed to produce infectious particles.     

Elucidation of Hepatitis C Virus Transmission and Early Diversification by Single Genome Sequencing

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures.     

Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

Hepatitis C Virus (HCV) NS4B protein has many roles in HCV genome replication. Recently, our laboratory (Q. Han, J. Aligo, D. Manna, K. Belton, S. V. Chintapalli, Y. Hong, R. L. Patterson, D. B. van Rossum, and K. V. Konan, J. Virol. 85:6464–6479, 2011) and others (D. M. Jones, A. H. Patel, P. Targett-Adams, and J. McLauchlan, J. Virol. 83:2163–2177, 2009; D. Paul, I. Romero-Brey, J. Gouttenoire, S. Stoitsova, J. Krijnse-Locker, D. Moradpour, and R. Bartenschlager, J. Virol. 85:6963–6976, 2011) have also reported NS4B''s function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B''s function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state.     

Spatiotemporal Analysis of Hepatitis C Virus Infection

Hepatitis C virus (HCV) entry, translation, replication, and assembly occur with defined kinetics in distinct subcellular compartments. It is unclear how HCV spatially and temporally regulates these events within the host cell to coordinate its infection. We have developed a single molecule RNA detection assay that facilitates the simultaneous visualization of HCV (+) and (−) RNA strands at the single cell level using high-resolution confocal microscopy. We detect (+) strand RNAs as early as 2 hours post-infection and (−) strand RNAs as early as 4 hours post-infection. Single cell levels of (+) and (−) RNA vary considerably with an average (+):(−) RNA ratio of 10 and a range from 1–35. We next developed microscopic assays to identify HCV (+) and (−) RNAs associated with actively translating ribosomes, replication, virion assembly and intracellular virions. (+) RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a shift to replication and then virion assembly. (−) RNAs have a strong colocalization with NS5A, but not NS3, at early time points that correlate with replication compartment formation. At later times, only ~30% of the replication complexes appear to be active at a given time, as defined by (−) strand colocalization with either (+) RNA, NS3, or NS5A. While both (+) and (−) RNAs colocalize with the viral proteins NS3 and NS5A, only the plus strand preferentially colocalizes with the viral envelope E2 protein. These results suggest a defined spatiotemporal regulation of HCV infection with highly varied replication efficiencies at the single cell level. This approach can be applicable to all plus strand RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle.     

Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.Hepatitis C virus (HCV) is an enveloped virus belonging to the family Flaviviridae. The virus genome is a positive-stranded RNA of about 9,600 nucleotides, which contains a single open reading frame (ORF) encoding both structural (core, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (47). Two highly conserved untranslated regions (UTRs) are found at the 5′ and 3′ ends, which play critical roles in both viral translation and replication (13, 14, 23).HCV is estimated to infect 170 million people worldwide (1, 43) and in a high percentage of individuals causes a chronic liver infection that frequently evolves into an array of diseases, including cirrhosis (12, 15, 38) and hepatocellular carcinoma (4, 7, 17, 30, 38, 49).The HCV RNA-dependent RNA polymerase (NS5B) has a high frequency of incorrect nucleotide insertions, in the range of 10⁻⁴ to 10⁻⁵ base substitutions per site, which can result in the rapid generation of HCV quasispecies (37, 45). Because of this huge genetic diversity, HCV is currently classified into six major genotypes and more than 80 subtypes (44). Recombination may be another mechanism exploited by HCV to increase genetic diversity: naturally occurring intergenotypic recombinant viruses that often have their recombination points in the trans-membrane domains of NS2 were recently identified (20, 21, 26, 27, 33, 35).Recent publications have reported the presence of natural HCV subgenomic RNAs in serum and liver of infected patients, mostly containing large in-frame deletions from E1 up to NS2, always found together with the full-length wild-type (wt) RNAs (5, 16, 36, 54). These mutant viral genomes persist for a long time (at least 2 years), and sequence analysis suggests that subgenomic (the predominant species during this period) and full-length HCV evolve independently (54). The relative abundance and persistence of such subgenomic RNAs in vivo suggests that (i) they are capable of autonomous replication and (ii) they can be packaged and secreted in infectious viral particles, presumably harboring the envelope proteins from wt virus coinfecting the same cell.Analysis of the genetic structure of 18 independent subgenomic deletion-containing RNAs (5, 16, 36, 54) strongly suggests that the possibility of recombination and/or deletion is restricted to specific regions.As expected, the 5′ UTR, the 3′ UTR, and the region coding from NS3 to NS5B are always conserved, in line with the notion that these regions are the minimal requirements for RNA replication. In addition to the regions required for RNA replication, however, naturally occurring subgenomic HCV RNA invariably contains an intact core region and the protease domain of NS2. In fact, the 5′ deletion boundary falls between the codons for amino acid 189 of core and amino acid 4 of E1 in 33% of cases and between the codons for amino acids 21 and 29 of E1 in 39% of cases, such that in 72% of cases overall, the 5′ boundary is between the codons for amino acid 189 of core and amino acid 29 of E1 (see Fig. S1 in the supplemental material). Likewise, the 3′ boundary shows a distinct localization, occurring between the codons for amino acids 51 and 79 of NS2 in 56% of cases.In the present study, we modified the infectious isolate JFH-1 in order to recapitulate in vitro the genetic structure of two of the most representative in-frame natural subgenomic deletion-containing RNAs found circulating in patients (28, 51, 57). Using this system, we analyzed natural subgenomic variants for their ability to replicate autonomously and demonstrated that the natural subgenomic deletion mutants are replication competent and are trans-packaged into infectious virions when coexpressed together with wt virus. Furthermore, our data suggest that the presence of the NS2 protease domain is required in order to generate the correct NS3 N terminus, required for RNA replication. Unexpectedly, the presence of NS2 generates, in turn, a strict cis requirement for the core region in order to allow efficient trans-packaging of the subgenomic RNA, revealing a complex interplay between the NS2 and the core viral genes. Finally, we performed trans-complementation studies of the subgenomic deletions with different HCV structural proteins to gain insight into the minimal requirements for the assembly and release of HCV virions.     

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.