Genetic heterogeneity of the hypervariable region I of Hepatitis C virus and lymphoproliferative disorders.

B-cell lymphoproliferative disorders (BCLD) have been associated with chronic hepatitis C virus (HCV) infection. The HCV glycoprotein E2 (gpE2) hypervariable region I (HVR-I) may be a potential antigenic candidate to promote B-cell proliferation. The purpose of this study was to analyze the influence of HVR-I sequence variability in the development of BCLD. HVR-I sequences were studied in 29 chronically HCV-infected patients with (n=15) or without (n=14) BCLD. After PCR amplification of the gpE2 region, analysis of the 81 bp HVR-I encoding fragment was performed on 7-18 clones per patient. HVR-I sequence complexity was slightly lower in patients with BCLD (mean 0.347) than without (0.468) (P=0.2), though, sequence diversities were similar (0.0370 vs 0.0954, P=0.239). Phylogenetic analysis did not reveal any BCLD-associated clustering. In our population, neither the recently described insertion between positions 1 and 2 of HVR-I nor residues at positions 4 and 13 were particularly linked to BCLD. As previously described, we confirm the high degree of conservation of HVR-I residues T-2, G-6 and G-23 in our patients. Contrary to recent findings, our analysis based on multiple clones per patient analysis did not reveal any particular motif associated with BCLD.

Similar increased serum dipeptidyl peptidase IV activity in chronic hepatitis C and other viral infections.

BACKGROUND: Dipeptidyl peptidase IV is a transmembrane enzyme widely expressed in many cell types, but also present as a soluble form in biological fluids. Its abnormal activity is sometimes associated with liver disease related pathologies. OBJECTIVES: The aim of this study was to evaluate the clinical relevance of changes in serum DPPIV activity in hepatitis C and other viral infections. STUDY DESIGN: DPPIV activity was assessed by using a microplate-based colorimetric assay on serum from 88 subjects: 12 healthy uninfected controls, 10 patients with primary biliary cirrhosis (PBC) as a reference group, 36 HCV-infected patients, and patients suffering from viral infections of different etiologies. Levels of DPPIV activity were compared with: (1) those of other serum biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyl transpeptidase (GGT), and bilirubin concentrations; and (2) criteria representative of liver histological status. RESULTS: Compared with healthy subjects, DPPIV activity was significantly increased during viral infections and in PBC (P<0.01). In HCV-infected patients, the median activity (interquartile range, IQR), 29.78 IU/l (24.66-35.95), differed significantly (P<0.05) from that of controls: 21.42 (19.76-24.93). No correlation was observed between DPPIV activity and either ALT, AST, bilirubin, or the stage of liver fibrosis and necroinflammatory activity, although GGT was moderately correlated (r=0.58, P<0.05). CONCLUSIONS: Although we confirmed an elevation of serum DPPIV activity in PBC, it seems to be a non-specific phenomenon common to viral infections. The absence of correlation between serum DPPIV and markers of liver disease in HCV-infected patients, suggests that this activity originates not only from the liver, but also from other sources such as peripheral blood cells involved in the control of viral infections.

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type.

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

Preferential associations of alleles of three distinct genes argue for the existence of two prototype variants of human herpesvirus 7.

We had previously described six distinct alleles of the glycoprotein B (gB) gene of human herpesvirus 7 (HHV-7). The genetic changes corresponding to these alleles did not affect gB gene transcription or translation in in vitro assays. The study of distinct HHV-7-positive human samples showed preferential associations of some gB alleles with some alleles of two other genes, distantly located on the HHV-7 genome, coding for the phosphoprotein p100 (p100) and the major capsid protein (MCP). Two allele combinations, corresponding to 44 and 31% of the samples studied, respectively, were interpreted as the genetic signatures of two major prototype HHV-7 variants.

Analysis of the glycosylation sites of hepatitis C virus (HCV) glycoprotein E1 and the influence of E1 glycans on the formation of the HCV glycoprotein complex.

The hepatitis C virus (HCV) genome encodes two membrane-associated envelope glycoproteins (E1 and E2), which are released from the viral polyprotein precursor by host signal peptidase cleavages. These glycoproteins interact to form a noncovalent heterodimeric complex, which is retained in the endoplasmic reticulum. HCV glycoproteins, E1 and E2, are heavily modified by N-linked glycosylation. A recent study has revealed that upon partial deglycosylation with endoglycosidase H only four of the five potential glycosylation sites of HCV glycoprotein E1 are utilized. In this work, the unused glycosylation site on the E1 glycoprotein was identified and the influence of N-linked glycosylation on the formation of the HCV glycoprotein complex was studied by expressing a panel of E1 glycosylation mutants in HepG2 cells. Each of the five potential N-linked glycosylation sites, located at amino acid positions 196, 209, 234, 305 and 325, respectively, on the HCV polyprotein, was mutated separately as well as in combination with the other sites. Expression of the mutated E1 proteins in HepG2 cells indicated that the fifth glycosylation site is not used for the addition of N-linked oligosaccharides and the Pro immediately following the sequon (Asn-Trp-Ser) precludes core glycosylation. The effect of each mutation on the formation of noncovalent E1E2 complexes was also analysed. As determined with the use of a conformation-sensitive monoclonal antibody, mutations at positions N2 and N3 had no, or only minor, effects on the assembly of the E1E2 complex, whereas a mutation at position N1 and predominantly at position N4 dramatically reduced the efficiency of the formation of noncovalent E1E2 complexes.

Evaluation of molecular strategies to develop a live dengue vaccine.

BACKGROUND: Millions of individuals are estimated to become infected with dengue virus each year, particularly in tropical and subtropical regions. Mortality is low but infection can lead to a severe form of dengue, characterised by haemorrhage and shock. A safe and effective vaccine against dengue is still not available. OBJECTIVE: To use the successful construction of dengue type 4 virus (DEN4) cDNA, which yields infectious RNA transcripts, to provide a new approach to the development of safe and effective dengue vaccines. STUDY DESIGN: The 3' and 5' noncoding (NC) regions of the genome were targeted to construct DEN4 deletion mutants, because the sequences in these regions are thought to play an important role in the regulation of viral replication. DEN4 cDNA was also employed to construct a viable chimeric virus with dengue type 1, 2 or 3 antigenicity, by substitution of heterotypic structural protein genes. RESULTS: Most viable mutants, recovered from the cDNA constructs, were partially restricted for growth in simian cells as analysed by plaque morphology assay and viral yield analysis. Several 3' NC deletion mutants which exhibited a range of growth restriction in cell culture were further evaluated for infectivity and immunogenicity in rhesus monkeys. Occurrence and duration of viraemia were reduced for these deletion mutants, compared to the wild type DEN4. Analysis of antibody response to infection in rhesus monkeys also indicated that some of these mutants were attenuated. These DEN4 deletion mutants represent promising live dengue vaccine candidates that merit further clinical evaluation. Chimera DEN1/DEN4 or DEN2/DEN4 which expresses DEN1 or DEN2 antigenicity were also used to infect monkeys. Most monkeys immunised with these chimeric viruses, singly or in combination, developed high titres of neutralising antibodies and were protected against homotypic wild type DEN1 or DEN2 challenge. CONCLUSIONS: DEN4 and its derived chimeric viruses of other three dengue serotype specificity, that contain appropriate attenuating mutations, have a potential use in a tetravalent live vaccine against dengue.

Yellow fever 5' noncoding region as a potential element to improve hepatitis C virus production through modification of translational control.

The lengthy 5' noncoding region (5' NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5' NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was compared in vitro in rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5' NCR. Surprisingly, the related distanced YFV 5' NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effective in vivo after transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5' NCR among the Flaviviridae family?

Antibody responses to hepatitis C envelope proteins in patients with acute or chronic hepatitis C.

Antibody responses to the hepatitis C virus (HCV) envelope proteins E1 and E2 were analyzed using two original assays in sera from 86 patients in different stages of disease. A Western blot assay and an immunofluorescence assay (IFA) were developed using envelope proteins produced, respectively, in Escherichia coli and in CV1 cells infected with a recombinant SV40. As a third method, the INNO-LIA HCV Ab III assay including E2 synthetic peptides was used. Of 38 chronically infected patients positive for anti-E2 antibodies by IFA, 26 were positive in the Western blot assay (68%) and 25 in the INNO-LIA test (66%). Thus, the detection of anti-envelope antibodies is highly dependent on the antigen formulation, and a native glycosylated form of the proteins is probably needed for their efficient detection. This study shows that the antibody response to HCV envelope proteins depends on the phase of infection. A few acutely infected patients displayed a response to E1 or E2 (36% by Western blot, 7% by IFA), and these antibodies seem to develop in patients evolving toward chronicity. The high prevalence in chronically infected subjects (62% to E2 by Western blot, 90% by IFA), particularly in subjects with essential mixed cryoglobulinemia (68% and 100%), confirms that the resolution of infection involves more than these antibodies. The antienvelope response in patients treated with interferon was investigated, but no significant relationship was found between antibody level prior to treatment and the evolution of hepatitis. The detection of anti-envelope antibodies, therefore, is not predictive of the response to antiviral therapy.

Processing of the E1 glycoprotein of hepatitis C virus expressed in mammalian cells.

The structural part of the hepatitis C virus (HCV) genome encodes a capsid protein, C and two envelope glycoproteins, E1 and E2, released from the virus polyprotein precursor by signalase(s) cleavage(s). The processing of E1 was investigated by infecting simian cells with recombinant vaccinia viruses expressing parts of the HCV structural proteins. When the predicted E1 sequence was expressed alone (amino acid residues 174-370 of the polyprotein) or with the capsid protein gene (residues 1-370). it showed an apparent molecular mass of 35 kDa as measured by SDS-PAGE analysis. However, when E1 was expressed as part of a truncated C-E1-truncated E2 polypeptide (residues 132-383), the processed E1 product had the expected apparent molecular mass of 31 kDa, suggesting that flanking sequences are necessary for the generation of the mature 31 kDa El form. The N-terminal sequence of the two E1 forms was found to be the same. Analysis of the glycosylation pattern showed that, in both species, only four of the five potential N-linked glycosylation sites were recognized, indicating that glycosylation was not involved in the molecular mass difference. We showed that expression of E1 with or without the hydrophobic stretch of amino acids residues 371-383, defined as the E2 signal sequence, may be responsible for the difference in electrophoretic mobility of the two E1 species. In vitro translation assays and site-directed mutagenesis experiments suggest that this sequence remains part of the 31 kDa E1 mature protein.

Growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the RNA genome.

The dengue type 4 virus (DEN4) RNA genome contains a 101-nt 5' noncoding (NC) sequence which is predicted to form a stable secondary structure. DEN4 cDNA from which infectious RNA can be transcribed was used to engineer deletions in the 5' NC region for functional analysis of RNA structure and for isolation of DEN4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. Eleven distinct deletions in the region of the DEN4 genome between nts 18 and 98 were constructed; each mutation was predicted to alter or disrupt the local base-parings in the 5' NC RNA structure. An infectious virus was not recovered from the RNA transcripts of five of these deletion mutants. Significantly, four of the five apparently lethal deletions were located in a 5- to 6-nt base-paired region of a predicted long stem or adjacent to it. In contrast, with one exception, mutants which yielded infectious virus had deletions which were located in a loop or short stem region. The effect of the deletions on the efficiency of translation of viral RNA transcripts was examined in vitro. The RNA transcripts of deletion constructs which did not yield viable virus were translated at an efficiency ranging from 40 to 160% that of wild-type virus transcripts. The translation efficiency of infectious RNA transcripts also varied. Deletion mutants recovered from RNA transcripts that exhibited low to moderate efficiency of translation had a small plaque morphology and exhibited reduced growth in simian LLC-MK2 and mosquito C6/36 cells compared to the wild-type virus. Among the 11 mutant constructs, deletion of nts 82-87 caused the greatest reduction in translation efficiency. Nevertheless, an infectious virus was recovered from LLC-MK2 cells transfected with the RNA transcripts of mutant d(82-87). The progeny of this mutant produced small plaques on LLC-MK2 cells and grew to low titer in these cells. Unlike wild-type DEN4 or other DEN4 deletion mutants tested, mutant d(82-87) failed to produce plaques on C6/36 cells and was also replication-defective in Aedes aegypti and Aedes albopictus following intrathoracic inoculation.

Processing of dengue type 4 and other flavivirus nonstructural proteins.

Dengue type 4 (DEN4) and other flaviviruses employ host and viral proteases for polyprotein processing. Most proteolytic cleavages in the DEN4 nonstructural protein (NS) region are mediated by the viral NS2B-NS3 protease. The N-terminal third of NS3, containing sequences homologous to serine protease active sites, is the protease domain. To determine required sequences in NS2B, deletions were introduced into DEN4 NS2B-30% NS3 cDNA and the expressed polyproteins assayed for self-cleavage. A 40 amino acid segment within NS2B was essential. Sequence analysis of NS2B predicts that this segment constitutes a hydrophilic domain surrounded by hydrophobic regions. Hydrophobicity profiles of other flavivirus NS2Bs show similar patterns. Cleavage of DEN4 NS1-NS2A requires an octapeptide sequence at the NS1 C terminus and downstream NS2A. Comparison of the analogous octapeptide sequences among flaviviruses indicates a consensus cleavage sequence of (P8)/Met/Leu-Val-Xaa-Ser-Xaa-Val-Ala(P1), where Xaa are non-conserved amino acids. The effects on cleavage of amino acid substitutions in this consensus sequence were analyzed. Most substitutions of the conserved residues interfered with cleavage, whereas substitutions of non-conserved residues had little or no effect. These findings indicate that the responsible enzyme recognizes well-defined sequences at the cleavage site.

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.

The 15 amino acid residues preceding the amino terminus of the envelope protein in the yellow fever virus polyprotein precursor act as a signal peptide.

The 15 amino acids which precede the sequence of the envelope (E) protein in the yellow fever virus (YFV) polyprotein precursor have been proposed to function as a signal peptide for the E protein (P. Desprès A. Cahour, C. Wychowski, M. Girard and M. Bouloy; Ann. Inst. Pasteur/Virol., 139, 59-67, 1988). To confirm this hypothesis, recombinant SV40 genomes were constructed in which the sequence of the E protein, or that of the poliovirus VP0 capsid polypeptide were placed immediately downstream of and in frame with the sequence of the putative signal peptide, under the control of the late SV40 promoter. The E protein expressed by the hybrid virus SV-E was recognized by two neutralizing monoclonal antibodies directed against the YFV envelope protein. In this construct, the E protein was deleted of its C-terminal transmembrane zone. Therefore, as expected, the protein appeared to be efficiently transported along the exocytic pathway and excreted into the cell culture medium. In addition, when the putative signal peptide was fused in frame with poliovirus polypeptide VP0, the expressed chimeric polypeptide was targeted to the endoplasmic reticulum where it underwent glycosylation.

Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins.

The yellow fever virus (YFV) cDNA segment coding for the part of the precursor polyprotein generating the structural proteins C (capsid), prM (precursor to the membrane protein M), and E (envelope) was expressed in vitro by using the T7 promoter-polymerase transcription system coupled to translation in rabbit reticulocyte lysates. A polypeptide of the expected molecular weight was observed to accumulate in the assay and was processed into proteins C, prM, and E only when dog pancreas microsomal membranes were added to the translation system. Proteins prM and E were translocated inside the endoplasmic reticulum, where prM underwent glycosylation. Regions essential for translocation of these proteins were localized to the 18- and 15-amino-acid C-terminal hydrophobic regions of proteins C and prM, respectively. Translocation of protein prM appeared to be less efficient than that of protein E. Maturation of these proteins followed different kinetics, indicating that the prM signal is probably cleaved off more slowly. A polypeptide composed of proteins C and prM, similar to the NVx polypeptide described in yellow fever virus-infected cells, was also produced in the in vitro system in the presence of membranes. No mature protein M was detected, suggesting that the cleavage of prM to M is a late processing event mediated by a protease different from endoplasmic reticulum signalases.

Modulations of the in vitro translational efficiencies of Yellow Fever virus mRNAs: interactions between coding and noncoding regions.

As an approach to define the structural features within the 5' noncoding region of Yellow Fever virus (YFV) that modulate mRNA translational efficiency, we have studied how minor changes in this region affect the translational capacity in vitro of the corresponding mRNAs. A cDNA sequence coding for part of the YFV structural proteins was inserted into the vector pGEM3 containing the bacteriophage T7 promoter. This vector was engineered by site-directed mutagenesis to permit in vitro synthesis of transcripts containing only 5 vector nucleotides at their 5' end. The sequence of the YFV 5' untranslated region was further modified in order to alter the secondary structure of resulting T7 transcripts. The efficiency of these messengers in programming cell-free translation systems varied from 1- to 15-fold, correlating inversely with the potential of the 5' untranslated sequences to form stable secondary structures. A chimaeric messenger containing the YFV 5' noncoding (5' NC) region linked to a heterologous mRNA derived from Germiston virus, was tested for its in vitro translatability. We found a translational efficiency about 2-fold higher than that obtained with homologous transcripts, suggesting that YFV 5' NC region can function as a potential enhancer for gene expression. Data obtained with a series of plasmids constructed by linking the native YFV 5'NC region to various coding regions of the YFV genome indicated that interactions between the untranslated sequence and protein coding regions influence mRNAs translational efficiency.

Expression of the yellow fever virus envelope protein using hybrid SV40/yellow fever viruses.

The cDNA coding for the yellow fever virus (YFV) envelope protein (E) was inserted into an SV40 vector under the control of the late promoter in place of the VP1 gene. The recombinant virus expressed a 52-Kd polypeptide which was detected by immunoprecipitation with a monoclonal antibody raised against the E protein. Surprisingly, this protein was visualized in the nucleus of the infected cells. The possible presence of a sequence involved in nuclear migration of the E protein and naturally ignored during virus infection is discussed. The sequence coding for the mature E protein is directly preceded in the YFV genome by a sequence of 45 nucleotides coding for a 15-amino-acid long hydrophobic oligopeptide. The sequence of this oligopeptide was inserted into the SV40 recombinant virus between the ATG codon and the first codon for the E protein. The E protein expressed by this new SV40 recombinant virus was found to be localized in the cytoplasm of the infected cells in association with intracellular membranes. These results strongly suggest that the 15-amino-acid long hydrophobic region naturally plays a role as a signal peptide for the E protein.

Natural antibodies against the carcinoembryonic antigen (CEA) and a related antigen, NCA, in human sera.

Natural antibodies directed against CEA and a related antigen, NCA, have been demonstrated in all normal and pathological sera using an EIA, although they have never been detected by RIA. These antibodies are not anti-blood group antibodies, as their titer was decreased only slightly by absorption with blood group substances. The study of their reactivity with deglycosylated antigens demonstrated that they were directed against peptidic epitopes. Antibodies against NCA or CEA, purified using specific immunosorbents, cross-reacted with all the antigens of the "CEA family" but not or only weakly with unrelated antigens.

High genetic stability of the region coding for the structural proteins of yellow fever virus strain 17D.

The genome of the Pasteur 17D-204 vaccine strain of yellow fever virus has been cloned into pBR327. The inserts of recombinant plasmids were analysed by restriction cleavage pattern and compared with that of the genome of another substrain previously cloned and sequenced. Ten of the overlapping inserts were found to contain the sequence of the complete genome. We have sequenced approximately 3680 bases of the 5' region which codes for the C, M and E structural proteins and the NS1 non-structural protein. This sequence is the same as that reported previously, indicating a remarkable stability of these two vaccine substrains.