Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity.     

Prevalence of amino acid mutation in hepatitis C virus core region among Japanese volunteer blood donors

It is not known whether there is a trend of increasing or decreasing incidence of new hepatitis C virus (HCV) infections in Japan. From the treatment point of view, it is important to verify HCV genotypes and the prevalence of treatment-resistant clones of HCV. At the Japanese Red Cross blood centers, all blood samples obtained from blood donation have been screened using serological methods and the minipool nucleic acid amplification testing. One hundred and fourteen donors have been identified over the past 10 years to be HCV RNA-only positive without detectable anti-HCV and were considered to be in the acute phase of HCV infection. There was a trend of decreasing incidence of such new infections among the blood donors. HCV RNA-only-positive samples were examined further for genotyping and HCV RNA quantitation. Genotype 2 (2a plus 2b) was predominant (78.2%) among them followed by genotype 1b (21.2%). Direct sequencing was carried out to detect the possible treatment-resistant mutant clones 70Q and 91M, clones with amino acid substitutions at positions 70 and 91 of the HCV core protein, respectively. 70Q and 91M were found regularly in donors with genotype 1b, but not in those with other genotypes. No particular endemic areas for the mutant clones were identified. There was no significant difference in the mean viral titer between the 70Q mutant type and the non-70Q wild-type. Even in newly infected people, the mutant clone 70Q was detected frequently.     

The effect of arginine deprivation on the cytopathogenic effect and replication of human cytomegalovirus

Arginine is necessary for the development of the cytopathogenic effect of human cytomegalovirus in human embryonic fibroblasts. It is also required, though in greater concentrations, for the production of infective virions, the requirement being at an early stage of replication. Inhibitor studies suggested that this block in replication caused by arginine deficiency was prior to the formation of viral DNA. Withdrawal of arginine from the medium 24 or 48 hours after infection resulted in a decline in virus production indicating that the continued presence of the amino acid is necessary for constant virus production. Infected cultures deprived of arginine could be stimulated to produce cytopathic effects and infective virions by replacement of the amino acid even eight days after inoculation, demonstrating that the information for cytomegalovirus replication remains intact within the cell. This establishment of latency in vitro may be related to the ability of the virus to establish a similar state in vivo.     

HCV 1b亚型ISDR变异影响PEG-IFN/RBV抗病毒疗效的新特点分析

目的探讨HCV干扰素敏感决定区（ISDR）氨基酸（aa）序列变异度对聚乙二醇干扰素（PEG-IFN）联合利巴韦林（RBV）治疗1b亚型慢性丙型肝炎（CHC）疗效的影响。方法 58例HCV1b亚型慢性感染者采用PEG-IFN/RBV联合方案治疗48周,并随访24周。定量检测血清HCV RNA,逆转录-聚合酶链反应扩增治疗前血标本中HCV ISDR片段并测序,MEGA4分析氨基酸序列变异度;二分类Logistic回归分析各变量与持续病毒学应答（SVR）之间的关系。结果治疗前血清HCV的ISDR氨基酸序列与HCVJ株比较,15例为野生型（未突变）,42例为中间型（1-3个突变）,1例为突变型（≥4个突变）。其中2218位点突变最多,约为60.3%（35/58）。ISDRaa突变数目与SVR关系密切（P=0.000）,ISDRaa突变数≥2的CHC组所获得的EVR和SVR明显高于aa突变数〈2的CHC组（P=0.041/P=0.012）。结论华南HCV1b亚型ISDR突变型（氨基酸变异≥4）极少。ISDRaa变异能够预测SVR;采用PEG-IFN/RBV联合方案治疗,对SVR有预测价值的ISDRaa突变数可由4个减少为2个。     

Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity

Remarkable advances have been made through recent demonstrations of hepatitis C virus (HCV) replication in cultured cells transfected with in vitro synthesized viral genome RNA. From the HCV JFH1 (genotype 2a) subcultured successively in Huh-7 cells we have identified several missense mutations near the junction of NS5A and NS5B genes. Reverse genetic analysis indicated that two mutations in the N-terminal region of NS5B replicase caused delayed viral RNA replication and protein expression in the early stage of infection. However, the mutant viruses showed significantly alleviated effects on cell growth inhibition, proteolysis of viral proteins, apoptotic DNA cleavage, and induction of antiviral responses, giving rise to a 100-fold higher titer compared to the parental JFH1 virus in a more extended time period. These results suggested that delayed replication and reduced cytotoxicity can be characteristic features of cell culture-adaptive mutants with enhanced infectivity.     

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).     

Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.

BACKGROUND: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. OBJECTIVE: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). STUDY DESIGN: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. RESULTS: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC(50) values 2-3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. CONCLUSION: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1.     

Hepatitis C virus E2 and NS5A region variability during sequential treatment with two interferon-alpha preparations

To determine the pattern and significance of the HCV genetic heterogeneity before and during treatment with recombinant-2b or lymphoblastoid alpha-interferon, hypervariable region 1 (HVR-1) and NS5A quasispecies were characterised by cloning and sequencing in 12 HCV-1b-infected subjects. Patients were either responder-relapsers or non-responders to treatment. Extensive amino acid sequence analysis was applied to reveal the significance of HCV variation at key sites within HVR-1 and NS5A regions. Genetic complexity, genetic diversity, and the non-synonymous to synonymous substitution ratios of HVR-1 quasispecies decreased during treatment in responder-relapser patients only, and more markedly so following lymphoblastoid alpha-interferon. In non-responders, the HVR-1 quasispecies broadened. Amino acids G406 and Q409, which represent a major viral epitope, were highly conserved throughout treatment. Responder-relapser patients had a higher mutation frequency in NS5A than non-responders. Lymphoblastoid alpha-interferon promoted the selection of intermediate Interferon Sensitivity Determining Region (ISDR) sequences, whereas recombinant-2b alpha-interferon favoured maintenance or selection of conserved ISDR sequences. Variability upstream of the ISDR was associated with treatment response, but the amino acid substitutions conferring higher replicative ability to in vitro HCV replicons were absent in in vivo isolates. In conclusion, the pattern of HVR-1 quasispecies evolution correlates with the clinical response, and the conservation of specific amino acids may be useful for immune targeting in vivo. In responder-relapser patients, the initial HVR-1 evolution resembles that found in sustained responders. Variability within the entire NS5A, as opposed to a single region (ISDR), may have a role in influencing alpha-interferon treatment outcome. A differential effect of different alpha-interferon preparations on HCV quasispecies kinetics may exist.     

Fluctuations of hepatitis C virus load are not related to amino acid substitutions in hypervariable region 1 and interferon sensitivity determining region.

Hepatitis C virus (HCV) load is one of the most important predictive factors of response to interferon treatment. However, little is known about the mode and determinants of viremia. The mode of viremia was investigated in 78 patients with chronic HCV genotype 1b infection during 1-2 years follow up. Virus load, determined by a branched chain DNA amplification assay, was stable in 73 of 78 (93.6%) patients, whereas 5 (6.4%) showed marked fluctuation (from undetectable level to more than 10 Meq/ml) in viral titer. To study the mechanisms mediating fluctuations in viral titer, amino acid sequences of two regions were examined; hypervariable region (HVR) 1 and the interferon sensitivity determining region (ISDR). Multiple amino acid substitutions were observed in HVR 1 but no relationship was evident between substitutions and virus titers. In contrast, no amino acid substitutions were observed in the ISDR in any patients with stable virus titer during a follow-up period of 12-24 months (7-24 samples) or in one patient who was observed for 15 years. Interestingly, multiple amino acid substitutions in the ISDR appeared in only two of the five patients with marked titer fluctuation, when the virus decreased markedly. Alanine aminotransferase levels in these five patients correlated with viral load. The data suggest that amino acid substitutions in HVR1 and ISDR are not essential for changes in viral titer. Possible mechanisms of fluctuations of viral titer and amino acid substitutions in the ISDR accompanying marked reductions in viral load are discussed.     

Separation of the functions controlled by adenovirus 2 lp locus

The adenovirus lp+ locus is located within early region E1b (map position 4.5-11.2) and codes for a 19-kDa tumor antigen (175R). Genetic analysis of the viral mutants that map within this region indicates that the lp+ locus controls multiple functions in cell transformation and in productive viral infection. Viral mutants mapping within the lp+ locus produce wt-like cytopathic effect (cyt+) or a cytocidal (cyt) effect. Earlier results have shown that many of the viral mutants that produce cyt phenotype in infected cells are transformation defective. In the present studies we show that one of the cyt mutants, cyt 106 which has a single amino acid substitution at position 20 transforms the established rat embryo cell line, CREF, at somewhat reduced frequency. Nonetheless, the cyt106-transformed cells appear to be fully transformed when compared with Ad2 wild type transformed cells. Unlike most other cyt mutants, cyt 106 is dominant over Ad2 wild type in mixed infections as judged by the plaque morphology in infective center assays and by the cytopathic effect. The dominant nature of the mutation may contribute to the observed transformation characteristics. Earlier we have shown that a cytocidal mutant, dl250 is partially defective in viral DNA synthesis in human KB cells. Now we show that two other cyt mutants cyt 5 (with a chain termination mutation near the C terminus) and cyt 6 (with a single amino acid substitution at position 44) are also partially defective in viral DNA synthesis in human KB cells. In contrast to these mutants, mutant cyt 106 induces normal replication of viral DNA. The DNA replication defect in mutants cyt 5 and 6 can be complemented in trans in 293 cells that constitutively express the 175R T antigen. Our results also indicate that a domain of this protein around the 44th amino acid is important for efficient viral DNA synthesis in KB cells.     

Contribution of the Gag-Pol transframe domain p6* and its coding sequence to morphogenesis and replication of human immunodeficiency virus type 1

The human immunodeficiency virus type-1 (HIV-1) transframe domain p6* is located between the nucleocapsid protein (NC) and the protease (PR) within the Gag-Pol precursor. This flexible, 68-amino-acid HIV-1 p6* domain has been suggested to negatively interfere with HIV PR activity in vitro proposing a contribution of either the C-terminal p6* tetrapeptide, internal cryptic PR cleavage sites, or a zymogen-related mechanism to a regulated PR activation. To assess these hypotheses in the viral context, a series of recombinant HX10-based provirus constructs has been established with clustered amino acid substitutions throughout the entire p6* coding sequence. Comparative analysis of the mutant proviral clones in different cell culture systems revealed that mutations within the well-conserved amino-terminal p6* region modified the Gag/Gag-Pol ratio and thus resulted in the release of viruses with impaired infectivity. Clustered amino acid substitutions destroying (i) the predicted cryptic PR cleavage sites or (ii) homologies to the pepsinogen propeptide did not influence viral replication in cell culture, whereas substitutions of the carboxyl-terminal p6* residues 62 to 68 altering proper release of the mature PR from the Gag-Pol precursor drastically reduced viral infectivity. Thus, the critical contribution of p6* and overlapping cis-acting sequence elements to timely regulated virus maturation and infectivity is closely linked to precise ribosomal frameshifting and proper N-terminal release of the viral PR from the Gag-Pol precursor, clearly disproving the hypothesis that sequence motifs in the central part of p6* modulate PR activation and viral infectivity.     

短双链RNA对柯萨奇B组3型病毒体外感染的抑制作用研究

目的利用RNA干扰技术，在细胞、蛋白和基因水平上观察短双链RNA对柯萨奇病毒体外感染Hela细胞的增殖抑制作用、对病毒RNA复制和蛋白合成的影响。方法应用病毒致细胞病变作用保护实验、空斑形成减少实验、Western Blot、RT-PCR等方法，在体外检测其抗CVB3病毒作用。结果设计、合成的8条SiRNA中，针对基因组2B、VP4、2A、3C区的SiRNA-2、SiRNA-3、SiRNA-6、SiRNA-7具有不同程度的抑制病毒作用。其中，病毒基因组2B的SiRN．2作用效果最好，对Hela细胞CVB3感染72h后致细胞病变和空斑形成的抑制率为95％，抑制病毒蛋白的合成，病毒基因的复制水平也显著降低，在病毒0．1、0．01、0．001、0．0001 MOI感染水平上对CVB3致细胞病变作用的抑制率分别为30％、70％、88％和99％（48h）。结论针对CVB3基因组2B的SiRNA-2具有较强的抑制柯萨奇B3型病毒感染的作用。     

The methyltransferase domain of the 1a protein of cowpea chlorotic mottle virus controls local and systemic accumulation in cowpea

The type strain of cowpea chlorotic mottle virus (CCMV-T) induces a local and systemic infection in California Blackeye cowpea (Vigna unguiculata (L.) Walp. subs. unguiculata cv. California Blackeye), but accumulates to low levels in inoculated leaves and fails to accumulate systemically in the cowpea plant introduction (PI) 186465. CCMV-R, a mutant strain derived from CCMV-T, accumulates to higher levels than CCMV-T in inoculated leaves and systemically infects PI 186465 plants. The phenotypic determinant of CCMV-R was previously mapped to viral RNA1, but the location of the determinant within RNA1 was not identified. Pseudorecombinants generated from genomic cDNA clones of CCMV-T and CCMV-R indicated that the phenotypic differences on PI 186465 were independent of replication. Through the use of chimeric RNA1 cDNA clones containing portions of CCMV-T and CCMV-R and site-directed mutagenesis, two nucleotides, 299 (amino acid residue 77) and 951 (amino acid residue 294), were identified as being independently critical for the local and systemic accumulation patterns of CCMV-R in PI 186465 plants. A second independently derived CCMV-R-like mutant, identified nucleotide 216 (amino acid residue 49) as being critical for induction of the CCMV-R infection phenotype. Amino acid residues 49, 77, and 294 are within the methytransferase domain of the CCMV 1a protein, suggesting that the methytransferase domain has a role in cell-to-cell and systemic accumulation of the virus that is independent of replication.     

Characterization of hepatitis C virus quasispecies by matrix-assisted laser desorption ionization-time of flight (mass spectrometry) mutation detection

Hepatitis C virus (HCV), the causative agent of hepatitis C, frequently causes chronic infection. The mechanisms of viral persistence continue to be the object of investigation. An important aspect of HCV chronic infection is the quasispecies nature of the viral population, which has been particularly well documented in the hypervariable region 1 of the E2 glycoprotein. Recent studies show that characterization of the quasispecies diversity at the amino acid level can help to predict the outcome of HCV infection. Currently the accurate characterization of HCV quasispecies requires the cloning of PCR products, followed by the sequencing of many clones. In this study we present a new method to characterize HCV quasispecies, based on in vitro translation of the amplicons, followed by mass spectrometry analysis of the resulting peptide mix. The assay was used on reference HCV samples and on clinical samples. In principle, this method could be applied to other chronic viral infections in which quasispecies play a role.     

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation.     

HtrA homologue of Legionella pneumophila: an indispensable element for intracellular infection of mammalian but not protozoan cells

Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103mapstoR and S212mapstoA) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrA mutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrA promoter to probe the phagosomal microenvironment harboring L. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotA mutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.     

Cell proliferative response to vaccinia virus is mediated by VGF

VGF, a polypeptide encoded by vaccinia virus, shares amino acid sequence homology and functional properties with cellular growth factors EGF and TGF-alpha. The availability of a VGF minus (VGF-) virus mutant has enabled us to examine the role of VGF in the replication of virus in vitro and in vivo. Studies in vitro with A431 cells (high EGF receptor density) showed that VGF+ wild-type virus induced the rapid formation of a focus of infection (not a plaque) which could be blocked by a monoclonal antibody to the EGF receptor. In vivo experiments with chicken embryos indicated that VGF+ virus stimulated the growth of ectodermal and entodermal cells of the chorioallantoic membrane. At early times, the majority of proliferating cells contained no detectable virus antigen, indicating that cell growth preceded infection and was a consequence of VGF secretion. Relative to VGF- virus, VGF+ virus produced lesions which contained more proliferating cells, more virus antigen, and increased amounts of infectious progeny. Secretion of VGF thus explains the conundrum of a nontransforming, strongly cytopathic virus inducing a hyperplastic cell response.     

In vitro selected Con1 subgenomic replicons resistant to 2'-C-methyl-cytidine or to R1479 show lack of cross resistance

The HCV polymerase is an attractive target for the development of new and specific anti-HCV drugs. Herein, the characterization of the inhibitory effect of 2'-C-Methyl-Cytidine shows that it is a potent inhibitor of both genotype 1b and 1a HCV replicon replication, both of laboratory-optimized as well as of NS5B clinical isolates-chimera replicons. The corresponding 5'-triphosphate derivative is a potent inhibitor of native HCV replicase isolated from replicon cells and of the recombinant genotype 1b and 1a HCV polymerase-mediated RNA synthesis. Resistance to 2'-C-Methyl-Cytidine was mapped to amino acid substitution S282T in the NS5B coding region. Cross-resistance was observed to 2'-C-Methyl-Adenosine but not to interferon alpha-2a, to non-nucleoside HCV polymerase inhibitors or to R1479, a new and potent nucleoside inhibitor of NS5B polymerase. In vitro studies mapped resistance to R1479 to amino acid substitutions S96T and S96T/N142T of the NS5B polymerase. These mutations did not confer resistance to 2-C-Methyl-Cytidine, thus confirming the lack of cross-resistance between these two HCV inhibitors. These data will allow the optimization of new polymerase inhibitors and their use in combination therapy.