Low oxygen tension enhances hepatitis C virus replication.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Detection of specific antibodies to HCV-ARF/CORE+1 protein in patients treated with pegylated interferon plus ribavirin.

Hepatitis C virus (HCV) infection is a major cause for chronic liver disease and hepatocellular carcinoma. The HCV-ARF/core+1 protein is an alternative product of HCV core-encoding sequence of unknown biological function. Highly purified HCV core and ARF/core+1 recombinant proteins from HCV genotype 1a and HCV-ARF/core+1 recombinant protein from HCV genotype 3a were expressed in Escherichia coli. Using an enzyme-linked immunosorbent assay, we assessed the prevalence of anti-ARF/core+1 antibodies in 90 chronic hepatitis C patients infected with HCV genotypes 1a/1b or 3a, treated with pegylated interferon (Peg-IFN-a-2a) plus ribavirin. Samples derived from 92 healthy blood donors were used as negative controls. All HCV-RNA-positive serum samples reacted with core 1a antigen, while 15 (37.5%) of 40 and 14 (28%) of 50 patients infected with HCV-1a/1b and HCV-3a, respectively, were found to have anti-ARF/core+1 antibodies into their serum before treatment initiation. These antibodies were persistently present during treatment follow-up and linked to elevated levels of HCV-RNA at baseline.

High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma.

The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11-160) and 1b (aa 11-144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85-144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.

A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera.

Hepatitis C virus (HCV) is a major disease agent affecting approximately 3% of the world's population. Expression in plant chloroplasts enables low-cost production of the conserved HCV core protein used in diagnostic tests to combat virus spread in developing countries with high infection rates. The bactericidal activity of the 21 kDa precore protein hinders cloning the core gene in plastid expression cassettes, which are active in bacteria due to the similarities between bacterial and plastid promoters and ribosome binding sites. This was overcome by using a topology-dependent expression cassette containing tandem rrn and psbA plastid promoters, whose activity was shown to be dependent on temperature. The viral core gene and a codon-optimised gene encoding a C-terminal truncated 16 kDa core polypeptide were expressed in tobacco chloroplasts. The codon-optimised gene increased monocistronic core mRNA levels by at least 2-fold and core polypeptides by over 5-fold, relative to the native viral gene. Expression of the 16 kDa core polypeptide was stable in leaves of different ages. Anti-core antibodies in HCV-infected human sera were detected by the 16 kDa core polypeptide in total leaf protein fractionated on Western blots providing a first step towards developing a chloroplast-based HCV diagnostic method.

Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins.

We report the construction of two HSV-1 recombinants encoding chimeric forms of the E2 glycoprotein of HCV-1a composed of the ectodomain of E2 (aa384-611 or 384-711) fused to different parts of the transmembrane and cytoplasmic domain of the HSV-1 gC glycoprotein (gC). The parental HSV-1, known as KgBpK(-)gC(-), is deleted for gC and the main heparan sulphate (HS) binding domain of gB, and it exhibits impaired binding (ca. 80%) to HS compared to the wild type virus KOS [Laquerre, S., Argnani, R., Anderson, D.B., Zucchini, S., Manservigi, R., Glorioso, J.C., 1998. Heparan sulphate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J. Virol. 72, 6119-6130]. We show that gC:E2 proteins are efficiently expressed and transported to the cell surface. We also demonstrate that HSV-1 can incorporate both gC:E2 chimeric proteins into particles and show that incorporation of both chimeric molecules in the viral envelope partially restored binding (ca. 20%) of the HSV-1 recombinants to heparan sulphate. Finally, we showed that the gC:E2ScaI chimeric glycoprotein was able to bind a recombinant form of hCD81 and virion-expressed gC:E2ScaI permitted the binding of the HSV-1 recombinant virus to the hCD81 molecule.

Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein.

The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.

Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus.

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of non-aggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

Suppression of the ERK1/2 signaling pathway from HCV NS5A protein expressed by herpes simplex recombinant viruses.

Two herpes simplex type 1 (HSV-1) recombinant viruses carrying the hepatitis C virus (HCV) NS5A open reading frame under the control of the cytomegalovirus immediate early (IE) or a herpes simplex chimeric promoter (alpha 4 gamma 1UL19) were constructed and characterized. Expression studies showed that both HSV-NS5A recombinant viruses were able to express high levels of the NS5A protein in infected cells. Most importantly, using this system, we demonstrated that the NS5A protein interacts with the growth receptor-bound protein 2 (Grb2) and inhibits the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HeLa, NIH3T3 or liver infected cells. Thus, our studies confirm the ability of the NS5A protein to perturb the extracellular signal-regulated kinase (ERK) pathway in HeLa cells by the use of an alternative system for NS5A expression and extend this observation to additional cell lines. We conclude that HSV-based viral vectors may provide a useful system for studying the expression and selected functional properties of the HCV NS5A protein.

Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays.

In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV.

The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins.

Herpes simplex virus 1 glycoprotein E (gE-1) is highly phosphorylated in culture cells during infection. In this report, it is shown that phosphorylation is mediated by host enzymes in human cells stably transfected with gE, in the absence of other herpesvirus products. In contrast, a tailless gE product (C terminus deletion mutant) is not phosphorylated. By using an in vitro kinase assay combined with linker-insertion mutagenesis, it is shown that casein kinase II catalyses the phosphorylation of the C-terminal domain of the protein. Also, it is demonstrated that the serine residues at positions 476 and/or 477 in the cytoplasmic portion of the protein are the major acceptors for the phosphate groups.

Mutational analysis of a conserved tetraloop in the 5' untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function.

The 5' untranslated region of hepatitis C virus RNA forms an extensive secondary structure including several hairpin motifs and mediates translation initiation by an internal ribosome entry site-dependent pathway. We report, here, an extensive mutagenesis analysis of a highly conserved tetraloop in the 5' untranslated region of hepatitis C virus, namely hairpin IIIe (295'-GAUA-298'). Our results demonstrate that hairpin IIIe is essential for the internal ribosome entry site function. Moreover, they indicate the importance of the primary structure of this motif because mutations in all four nucleotides of the loop caused a severe loss of internal ribosome entry site activity. These data represent the first experimental evidence for the functional significance of tetraloops in internal ribosome entry site-driven translation of hepatitis C virus.

Detection of human papillomaviruses in squamous cell carcinomas of the lung.

The aim of this study was to evaluate the possible association of human papillomaviruses (HPV) with the development of squamous cell lung carcinomas (SqCLCs). Tissue material from 52 cases of SqCLCs were studied, and the data were evaluated according to the degree of differentiation, HPV presence and type. Analysis was performed by polymerase chain reaction (PCR) method using consensus primers, and the results were confirmed by subsequent Southern blot hybridization. Overall, the results showed 69% positivity (n=32). Forty-one cases were examined for the presence of specific HPV types (6/11 and 16/18) by hybridization of the PCR products with 32P-labelled probes. HPV 6/11 types were detected in 6 of the 29 positive cases (20.6%). HPV 16/18 types were the most prevalent types, and were detected in 11/29 cases (37.9%: 4/10 of well-differentiated cases, 6/25 of moderately and 1/6 of poorly differentiated carcinomas). Our results confirm the possibility that HPV might play a role in the development of SqCLCs and suggest a possible relation of high-risk HPV16/18 types to tumour differentiation.

Mutational analysis of two unstructured domains of the 5' untranslated region of HCV RNA.

Translation initiation of hepatitis C virus (HCV) RNA genome is mediated by an internal ribosome entry site (IRES). To further comprehend the mechanism of translation initiation of HCV RNA, we investigated the importance of two unstructured, highly conserved, single-stranded pyrimidine-rich sequences located immediately upstream of domain II (nt38-43) and between domains II and III (nt120-125) in HCV translation. A series of defined mutations was engineered and introduced into a dicistronic vector in order to assess their impact on in vitro translation. Our data indicated that nucleotide sequence 38-43 is not essential for HCV translation. In contrast, mutational analysis of the second sequence motif (nt120-125) suggested that this region was important for maintaining the proper structure within the IRES element although the primary sequence itself was not critical for IRES function. More importantly, it appeared that mutations which allowed juxtaposition of neighboring bases (nt112-119) to the pseudoknot structure, were detrimental to translation initiation.

Type-specific prevalence of genital human papillomaviruses in benign, premalignant, and malignant biopsies in patients from Greece.

BACKGROUND AND OBJECTIVES: More than 30 different human papillomavirus (HPV) types infect the anogenital mucosa and are responsible for a variety of benign, premalignant, and malignant lesions including cervical cancer. The goal of this study was to determine the distribution of individual HPV types in various grads of cervical precancerous lesions and cervical carcinoma in patients from Greece. STUDY DESIGN: Specimens were analyzed for HPV-DNA sequences by polymerase chain reaction and Southern blot hybridization. Polymerase chain reaction analysis was performed with consensus and type-specific primers. Restriction length fragment polymorphism analysis and/or hybridization of the general primer polymerase chain reaction product were used for HPV typing. RESULTS: In cervical carcinomas HPV-16 was found in 56%, HPV-18 in 23%, and HPV-31 in 6% of the HPV-positive patients. In precancerous lesions HPV-16 was found in 13% of low-grade squamous intraepithelial lesions (LG-SIL) as compared with 41% of high-grade squamous intraepithelial lesions (HG-SIL) patients. HPV-18 was found at similar frequency both in LG-SIL (13%) and HG-SIL (14%). HPV-31 and HPV-33 were detected at moderate levels both in LG-SIL (11%) and in HG-SIL (14%). In addition, HPV-53 and HPV-66 were detected at low frequency in LG-SIL (2%), whereas HPV-51 was found only in HG-SIL (4%). Finally, HPV-6 was associated with 13% of LG-SIL. CONCLUSIONS: Overall, the prevalence rate of the genital HPV types was in the range previously described for many western countries but the HPV-18 positivity was higher than that reported for most European countries.

Genital papillomavirus in Greek women with high-grade cervical intraepithelial neoplasia and cervical carcinoma.

Fifty biopsies from high-grade squamous intraepithelial lesions (HG-SIL) and 14 cervical carcinoma biopsies from Greek women were screened for the presence of human papillomavirus (HPV) DNA sequences by Southern blot hybridization and by the polymerase chain reaction (PCR) for the presence of different HPV types. In high-grade SIL, HPV DNA sequences were detected in 44 of 50 biopsies with the following distribution: 36% HPV 16, 12% HPV 18, 6% HPV 31, 6% HPV 33, 4% HPV 51, and 24% unclassified HPV types. In cervical carcinoma biopsies, 13 of 14 specimens were positive for HPV DNA sequences. Six biopsies were positive for HPV 16, three were positive for HPV 18, and four contained unclassified HPV types. Overall, of the total 64 biopsies, 57 (89%) were positive for HPV DNA sequences. Of these, Southern blot hybridization alone detected HPV DNA sequences in 39 cases, whereas by PCR 18 additional specimens were found to be positive for HPV. Among the HPV 16-positive biopsies, two samples produced a Pstl banding pattern very similar but not identical to that of HPV 16 prototype and were referred to as HPV 16 isolates. One HPV 16 isolate appears to carry a mutation within the carboxy-terminal half of the L2 gene that results in the loss of a Pstl site. The other HPV 16 isolate had a similar Pstl banding pattern to that previously reported as HPV 16 "variant" found in Cape Town [Williamson et al., 1989, Journal of Medical Virology 28: 146-149, 1994, Journal of Medical Virology 43: 231-237.] and in Italy [Li Vigni et al., 1994, 2nd International Congress of Papillomavirus in Human Pathology (Abstracts), p 100.].

Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice.

The objective of this study was to examine the protective efficacy of purified recombinant herpes simplex virus type 1 (HSV-1) glycoprotein E (gE-1) in the mouse lethal challenge model. A secreted form of gE-1 (hgE-1s) protein, containing amino acids 1-406, was produced in human cells by using the episomal replicating pRP-RSV expression vector. In addition, a portion of the gE-1 (bgE-1t) protein corresponding to amino acids 90-406, was expressed in Escherichia coli as a fusion protein with maltose binding protein using the pMAL-c2 expression vector. Mice vaccinated with hgE-1s developed high serum titres of HSV-1-neutralizing antibodies and were significantly protected from intraperitoneal lethal HSV-1 challenge, whereas mice vaccinated with bgE-1t exhibited only moderate levels of protective immunity. These results demonstrate that the expression of gE-1 in human cells has a strong impact on its protective efficacy and that most importantly the hgE-1s protein could be of values as a component of an HSV multi-subunit vaccine.

Characterization of the US8.5 protein of herpes simplex virus.

In a previous study a novel gene designated as US8.5 was identified in the unique short component of the herpes simplex virus type 1 (HSV1) genome. Epitope tagging experiments suggested the existence of a protein encoded by this gene in HSV1 infected cells. To further analyze the US8.5 gene product and function, a rabbit polyclonal antiserum was raised against a recombinant beta-galactosidase-US8.5 fusion protein expressed in E. coli. This antiserum reacted specifically with a 19 kDa protein in HSV1(F) infected cells as shown by immunoblotting and immunoprecipitation experiments. In addition, using the same antiserum a 21 kDa protein was detected in lysates from cells infected with HSV2(G), which was most likely the HSV2 homolog of US8.5. Kinetic studies indicated that US8.5 is expressed as gamma 1 gene. In addition, the US8.5 protein was immunoprecipitated with the anti-US8.5 serum from 32P-labeled lysates of Vero cells infected with HSV1, demonstrating that the protein is phosphorylated. Immunofluorescence studies localized the US8.5 protein to the nucleoli of HSV1 infected cells.

Escherichia coli expressed herpes simplex virus gG1 and gG2 proteins in ELISA and immunoblotting assays.

The type 1 and type 2 glycoprotein G (gG1 and gG2) of herpes simplex virus (HSV) were expressed in Escherichia coli as fusion proteins with the maltose binding protein (MBP) using the pMAL-c2 expression vector. The MBP-gG1 fusion protein contains all but the four amino acids from the amino-terminus of gG1, whereas the MBP-gG2 fusion protein was missing the first 30 amino acids that comprise the signal peptide of the protein. The diagnostic value of these antigens was examined by two methods: (1) immunoblot assay based on MBP-gG1 and MBP-gG2 fusion proteins present in crude E. coli cell extracts and (2) enzyme-linked immunosorbent assay (ELISA) of immunoaffinity-purified recombinant MBP-gG1 and MBP-gG2 fusion proteins. Of 28 serum samples known to have antibody to HSV-1 (10 specimens positive for HSV-1 alone and 18 specimens positive for mixed antibody to HSV-1/HSV-2), 27 were reactive to the MBP-gG1 recombinant protein both in ELISA and in immunoblotting. In addition, of 20 serum samples known to have antibody to HSV-2 (2 specimens positive for HSV-2 alone and 18 samples positive for mixed antibody to HSV-1/HSV-2), 15 were found to be reactive to the MBP-gG2 recombinant protein by ELISA and 16 by immunoblotting. None of the 13 HSV-antibody-negative serum samples showed reactivity to the MBP-gG1 or MBP-gG2 antigens by either assay. Moreover, none of the serum samples known to have antibody to HSV-1 alone showed reactivity to the MBP-gG2 recombinant antigen. This study verified the potential application of the E. coli-expressed recombinant gG1 and gG2 proteins as diagnostic antigens and demonstrated the MBP fusion system to be a simple and effective method of producing adequate amounts of low-cost, easily purified gG antigens.

Regulation of high-affinity leucine transport in Escherichia coli.

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in livR and 1stR, genes for negatively acting regulatory elements, and by mutations in rho, the gene for transcription termination. All four genes for high-affinity leucine transport (livJ, livK, livH, and livG) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ-livK-livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is activated by mutation or deletion. The results support the existence of separate promotors for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucine-specific transport systems by increasing the expression of livJ and livK, genes for the LIV-specific and leucine-specific binding proteins, respectively.