Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES.

Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

Characterisation and chromosomal localisation of the rat alpha- and beta-adducin-encoding genes.

A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.

Genomic organisation and chromosomal localisation of the gene encoding human beta adducin.

Adducin (ADD) is a heterodimeric protein of the membrane skeleton with subunits of 103 (alpha) and 97 kDa (beta). It promotes the assembly of the spectrin-actin network. We have previously shown that one point mutation in each of the alpha and beta rat ADD-encoding genes is associated with blood pressure variation in an animal model for hypertension, the Milan hypertensive strain of rats, probably due to a change in the phosphorylation pattern. In fact, the rat mutations, Y to F for alpha and R to Q for beta, are located, respectively, in a tyrosine kinase and a protein kinase A phosphorylation site. We have now determined, for the human beta-ADD-encoding gene, its chromosomal localisation, exon-intron organisation and alternative splicing patterns. We report here that human beta-ADD is localised on chromosome 2 and we also show a characteristic 3' end alternative splicing of the beta-ADD RNA that generates two distinct beta-ADD families, namely ADD 63 and 97; both of them in turn present a very complex differential splicing pattern in the internal exons.

In vivo translational efficiency of different hepatitis C virus 5'-UTRs.

Initiation of translation in hepatitis C virus (HCV) is dependent on the presence of an internal ribosome entry site (IRES) contained in its 341-nt-long 5'-untranslated region (UTR). This region is very conserved among different isolates and has been used to classify HCV isolates in six different genotypes. These genotypes differ in nucleotide sequence that generally preserve the IRES structure. However, the small differences seen may be biologically and clinically significant as the HCV strains seem to differ from each other in several important ways, such as the behaviour of the viral infection and the response to interferon therapy. Therefore, differences in translational initiation efficiency amongst the various genotypes could provide an explanation for these phenomena. Using a bicistronic expression system we have compared the in vivo translational ability of the three most common European genotypes of HCV (1, 2, and 3). The results show that genotype 3 is less able than 1 and 2 to promote translation initiation. In addition, by site-directed mutagenesis of the sequence of the IRES domain III apical stem loop structure, we have shown that the conservation of the primary nucleotide sequence and not only the structure, is important for the conservation of IRES function.

Immunoreactivity of chimeric proteins carrying the HIV-1 epitope IGPGRAF. Correlation between predicted conformation and antigenicity.

Sera from HIV-1 infected individuals were examined for their reactivity to the principal neutralizing domain, IGPGRAF sequence, of the V3-loop of HIV-1. Four hybrid proteins carrying this sequence inserted in four different outer loops of a protein that makes up the capsid of an insect virus were used as antigen in a Western blot assay for this survey. All the four antigens showed different activity: sera that recognise all antigens to sera that reacted with only one of them. Competition experiments indicated that the antibodies recognised these proteins with different affinity. Molecular modelling of the hybrid proteins predicted that the inserted sequence adopted different conformations in each position. Comparison of predicted most stable conformations for IGPGRAF indicated that there is a close relationship between conformational similarity to a V3-loop reference structure and the degree of reactivity with sera.

Conformational display of two neutralizing epitopes of HIV-1 gp41 on the Flock House virus capsid protein.

We have developed an antigen presenting system based on the capsid protein of the Flock House virus (FHV) and used it to display, in different positions on its external surface, two neutralizing epitopes found at residues 735-752 of HIV-1 gp160. We have compared the immunoreactivity of these FHV chimeric proteins and of the corresponding synthetic peptide using a panel of neutralizing mouse monoclonal antibodies (mAbs) directed against two distinct sequences (IEEE and ERDRD) contained in this epitope of the gp41 region. We have observed that both the FHV chimeric protein and the synthetic peptide are clearly detected in ELISA procedures by the mAbs recognizing the sequence IEEE. The denaturation of these recombinant proteins had little effect on the recognition pattern of this group of monoclonals, suggesting minor conformational requirements for the display of this epitope. The FHV chimeric proteins were also recognized by the mAbs directed against the ERDRD epitope, whereas the corresponding synthetic peptide was not recognized. In this case, denaturation of these recombinant proteins completely abolished the reactivity of the second group of mAbs, arguing for the existence of strong conformational constraints. Additionally, we have investigated whether an isolated loop structure from the FHV protein was sufficient to provide the conformational requirements for the presentation of these epitopes. These experiments have shown that the stabilized loop structure, although improving the presentation of both epitopes, is not as efficient as the native loop in the intact FHV protein. The data obtained with these mAbs support the recently observed limitations in the use of synthetic peptides for the screening of the immune response against conformational epitopes. The establishment of appropriate tools able to present epitope sequences in a structure resembling the native conformation will be useful for accurate epidemiological studies and for the design of new epitope-specific vaccines.

Factors associated with serum HCV RNA positivity in anti-HCV antibody positive intravenous drug users.

Serum hepatitis C virus (HCV) RNA, HCV genotypes and liver function tests were evaluated in a series of 189 unselected, consecutive anti-HCV positive intravenous drug users (IVDUs). Serum HCV RNA was detected in 106/189 patients. Abnormal liver function tests were associated with alcohol abuse, but not with the presence of serum HCV RNA. Among 109 patients retested after a mean follow-up of 21 months, 41 were intermittently serum HCV RNA positive. Patients persistently negative had more commonly a past history of acute hepatitis. A history of prostitution and/or a pattern of abuse involving >30 injections per week were related to infection by genotype 3a. In conclusion, serum HCV RNA is either transiently or persistently detectable in most anti-HCV positive IVDUs, but bears no association with abnormal liver biochemistry. Infection by HCV-3a is more common in IVDUs with more deviant life styles. In those cases where serum HCV RNA is found repeatedly negative, HCV infection may have been cleared, possibly through an episode of acute hepatitis.

Cohort effect of HCV infection in liver cirrhosis assessed by a 25 year study.

BACKGROUND: The role of HCV infection in the development of chronic liver disease is still unclear. OBJECTIVES: Assess the presence of HCV infection in patients with liver cirrhosis. STUDY DESIGN: 123 cases of cirrhotic liver randomly selected over a 25 years period (1969-1994) from the autopsy archives of the Pathology Department of the University of Trieste, Italy, were analyzed for the presence of HCV viral genome. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissues of the cirrhotic liver. Genotype analysis for HCV was performed after RT-PCR by dot-blot hybridization with the three major genotype-specific probes (G1, G2 and G3). RESULTS: The overall HCV genome frequency was 50.4% (62/123). The positivity was quite constant in the 1969-1979 period (35-38%), rose to 65% in 1984, peaked to 77% in 1989 (P<0.005 vs. the previous decade), and decreased to 50% in 1994. HCV genotype G1 was found in 89% of the 62 positive samples. The mean age of death of HCV-positive and HCV-negative patients was comparable (69+/-12 vs. 67+/-16 years, NS). CONCLUSIONS: These data show an increasing frequency of HCV infection in cirrhotic liver tissues from 1969 to 1994, which peaked in 1989. The genotype G1 was the almost uniquely associated with cirrhosis. These findings indicate that the HCV infection occurred around the late 1950s-early 1960s, thus supporting the hypothesis of a cohort effect. HCV infection seems not to alter the natural history of liver cirrhosis as indicated by the comparable age at death of HCV positive and HCV negative patients.

IgM antibody response to the hepatitis C virus core protein in intravenous drug users.

To verify whether a solid-phase enzyme immunoassay for serum IgM antibodies to the hepatitis C virus (HCV) core protein (IgM anti-HCVcore) might be proposed as a surrogate test for serum HCV RNA, we studied 86 anti-HCV antibody-positive intravenous drug users. Serum HCV RNA was demonstrated by RT-PCR with primers derived from the 5' non-coding and the core region. IgM anti-HCVcore antibodies were found in 62/86 (72%) subjects; circulating HCV RNA was detected by the 5' noncoding assay in 53/86 samples (62%) and by the core region assay in 35/86 samples (41%). IgM anti-HCVcore reactivity was associated with core HCV RNA seropositivity (p < 0.05) but not with 5' noncoding HCV RNA seropositivity (p = NS). Patients infected by HCV type 1a were more-often positive for IgM anti-HCVcore (p < 0.05) and for core HCV RNA (p = 0.005) than patients infected by other HCV genotypes. IgM anti-HCVcore reactivity was significantly more common in subjects positive for core HCV RNA (p < 0.005) and in subjects aged > 30 years (p < 0.05). In conclusion, the IgM anti-HCVcore assay frequently tests positive in intravenous drug users, particularly when infected by HCV 1a, but is not a surrogate of testing for serum HCV RNA.

Comparison of genotyping and serotyping methods for the identification of hepatitis C virus types.

The usefulness of identification of hepatitis C virus (HCV) genotype has recently been investigated for the clinical management of patients infected by HCV. In the present study, the HCV genotype infecting 127 patients was determined by two different methods: HCV genotyping using a dot-blot assay with type-specific probes derived from the 5'-UTR of HCV genome and HCV serotyping using an ELISA system in which type-specific antibodies against the NS4 region were detected. Overall, a good correlation of the two methods was observed, the main discrepancy being 4 patients with sequence-confirmed HCV-2 (2 cases) and HCV-3 (2 cases) genotypes recognized as HCV-1 by serotyping. Mixed infections were not detected by either method. In 19 PCR negative sera, in which the HCV genotype could not be evaluated, no particular serotype profile was observed. In conclusion, the molecular and serological techniques are almost equivalent in determining the viral type, although in individual cases, especially in PCR negative patients, the clinical meaning of the serotyping result remains to be determined.

V3 loop core region serotyping of HIV-1 infected patients using the FHV epitope presenting system.

We have reported recently a new epitope presenting system based on the Flock House Virus (FHV) capsid protein. The HIV-1 V3 loop core sequence IGPGRAF was inserted in different sites of this carrier molecule. Immunoreactivity experiments and molecular modelling consistently showed that the most reactive recombinant protein displayed the IGPGRAF sequence in a conformation which is most similar to that of a V3 loop reference structure. The same insertion site was then used to display the V3 loop apex sequences of six different HIV-1 isolates. Sera from 32 HIV-1 infected patients were examined for their reactivity to our chimeric proteins and the results were compared with those obtained using synthetic V3 loop peptides. The data obtained were confirmed by nested PCR amplification and direct sequencing of the patient's V3 loops. The results showed that the V3 loop serotyping using the FHV hybrid proteins, was more specific than that obtained using synthetic peptides. This system will therefore be a useful tool for the correct evaluation of the immune response against different V3 loop core sequences.

Modulation of the immune response to DNA vaccine by co-delivery of costimulatory molecules.

We have investigated methods for modulating immune responses, against herpes simplex virus (HSV), generated from DNA vaccination by co-delivery of genes encoding costimulatory molecules. A strong delayed-type hypersensitivity (DTH) reaction was induced in mice co-injected via the intradermal (i.d.) route with a eukaryotic expression plasmid encoding the CD80 molecule (pCD80) and a plasmid encoding the glycoprotein D of the HSV-2 (pgD). Furthermore, when spleen cells from these mice were cultured in the presence of inactivated HSV, a significant increase in the expression of interleukin-2 receptor (IL-2R) was observed in the CD4 subset compared with mice immunized only with pgD. Analysis of cytokine synthesis at the single-cell level indicated that CD80 genes induce a significant increase in the number of interferon-gamma (IFN-gamma)-, IL-2- and IL-4-secreting cells in the spleen. On the other hand, co-administration of the CD80 gene via the intramuscular (i.m.) route did not induce an increase in the cell-mediated immune response. When a plasmid carrying the CD86 gene (pCD86) was co-injected via the i.m. route with the pgD plasmid, a small decrease in the number of IFN-gamma-secreting cells was observed. This down-regulation of the immune response was also observed when eukaryotic expression cassettes for CD80 and for CD86 were co-administered with the pgD plasmid via the i.d. route. However, co-injection of pCD86 via the i.m. route produced a small increase in the number of IL-4-secreting cells. When immunized mice were challenged intravaginally with 100 plaque-forming units of virus, only co-injection of the CD80 gene by the i.d. route provoked an adjuvant effect compared with mice immunized with pgD alone. A reduction in the titres of HSV in vaginal washings was observed together with a decrease in the lesion score.

Histone H3 modification in BHK cells infected with foot-and-mouth disease virus.

Infection of BHK cells with foot-and-mouth disease virus (FMDV) causes a thorough change in the electrophoretic profile of whole nuclear histones. It consists in the disappearance of histone H3 and the appearance of a new polypeptide (Pi) which migrates between histones H2A and H4 on SDS-polyacrylamide gels. Protein Pi is detected at 2 hr postinfection (pi), the time in which viral RNA synthesis begins to increase, and reaches equimolecular amounts with the remaining core histones 1 hr later, when the disappearance of histone H3 is almost complete. Labeling of cells prior to infection demonstrates that Pi is not a novo product but the result of a viral-induced processing of a host precursor synthetized beforehand. Protein Pi comigrates with histone H2A/B in acetic acid/urea polyacrylamide gels and it shares common major peptides with histone H3 under controlled proteolysis with protease V8 or trypsin. The mononucleosomal and nucleosomal DNA pattern analysis after micrococcal nuclease treatment of nuclei from infected and mock-infected cells did not show any significant differences even though after 3 hr (p.i.), protein Pi replaces histone H3 in the nucleosomal structure. It was concluded that FMDV infection is responsible for a specific modification in the nucleus of infected cells which leads, after 3 hr (p.i.), to a complete histone H3 protein Pi transition in the nucleosomes.

Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice.

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral genetic immunization. The eukaryotic expression vector pCMV containing the gene of the glycoprotein D (gD) of the herpes simplex virus 2 was used to transform Salmonellae. The oral immunization with the transformed salmonellae elicited a strong cellular immune response in both, the mucosal and systemic compartments (spleen, ileal lymph nodes and Peyer patches). The immune response mainly consisted in a dramatic activation of IFN-gamma-secreting cells. Twenty hours following the challenge with five lethal doses of virus, mRNA for IFN-gamma was observed in vaginal tissues from mice immunized with salmonella harboring the plasmid pgD but not in tissues from mice immunized by the intramuscular route with pgD. After an intravaginal challenge all immunized mice survived without developing symptoms. Furthermore, the immunization with Salmonella resulted in a more effective control of viral shedding than intramuscular immunization. We have unequivocally demonstrated by the introduction of an intron in the green fluorescent protein that the expression of the plasmid was due to the transcription of the protein by an eukaryotic nuclear process and not as a result of expression of the protein by the bacteria. Macrophages and dendritic cells were found expressing the protein in systemic and mucosal compartments of the immune system.

Codelivery of DNA coding for the soluble form of CD86 results in the down-regulation of the immune response to DNA vaccines.

The costimulatory pathway that includes CD80, CD86, CD28, and CTLA-4 plays a key role in regulating T cell activation and tolerance and is a promising therapeutic target. We have studied the possibility of down-regulating the immune response to DNA vaccine by codelivery of a plasmid coding for the extracellular domains of CD86 (pDelta86). We found that DeltaCD86 was able to inhibit the engagement of FcCTLA-4 but not of FcCD28 to CD80 and CD86 expressed on COS cells. Coadministration of plasmid pDelta86 encoding for the extracellular domains of CD86 along with a plasmid encoding for the glycoprotein D (pgD) of herpes simplex virus-2 (a membrane-bound protein) by the im route in mice resulted in a strong inhibition of the cell-mediated immune response in the spleen and in draining lymph nodes. In addition, when pDelta86 was coadministered together with a plasmid encoding for the ovalbumin (pOVA) (a soluble protein), a strong inhibition of the cell-mediated immune response was observed in draining lymph nodes and only a partial inhibition was found in the spleen. Furthermore, only a partial down-regulation of the humoral immune response was observed. The mechanism involved could be a preferential engagement of DeltaCD86 to CTLA-4 leading to the transmission of a negative signal to T lymphocytes.

Superiority of intramuscular route and full length glycoprotein D for DNA vaccination against herpes simplex 2. Enhancement of protection by the co-delivery of the GM-CSF gene.

Immunization with naked DNA has been analyzed in two critical variables: the site of injection and the cellular compartment to which the coded protein is directed. The gene for the full length of the glycoprotein D (gD) of HSV-2 under the control of the citomegalovirus (CMV) promoter was injected via the intradermal (i.d.) or the intramuscular (i.m.) routes in mice. Immunization in the quadricep muscle was superior to the intradermal immunization in the footpads. A stronger activation of IFN-gamma-secreting cells in the spleen and draining lymph nodes (DLN) was induced, resulting in a more efficient protection against an intravaginal challenge. In order to analyze the effect of the cellular localizations of the coded protein, the DNA for the truncated form of the gD (DeltagD) was injected via the i.m. route. Immunization with a vector encoding for DeltagD resulted in higher antibody levels in serum and vaginal washes than immunization with the gene for the full length gD. However, immunization with the DeltagD DNA elicited a much weaker cell-mediated immune response and was inferior to gD DNA in providing protection against a lethal intravaginal challenge with HSV. Co-injection of an expression cassette for the granulocyte-macrophage colony-stimulating factor (GM-CSF) increased both the humoral and cell-mediated immune response with both gD and DeltagD. A strong activation of IL-4-secreting cells was observed in the spleen and DLN together with an increase in the number of IFN-gamma-secreting cells. In addition, a reduction in the vaginal virus titers after an intravaginal challenge was observed in mice co-injected with the GM-CSF gene as compared to those immunized with pCDNAgD only.

Localization of the different hepatitis C virus core gene products expressed in COS-1 cells.

In this study we have investigated the subcellular localization in transfected COS-1 cells of the two major forms of the hepatitis C virus core protein: the immature protein of 191 residues (p21) and its proteolytically cleaved product of 173 residues (p19). In this study, and unlike previous investigations, we have been able to distinguish separately the localization of p21 from p19. This was achieved by the addition of a C-terminal HSV Tag to the p21 full coding sequence, and exploiting the fact that it is subsequently lost in the p19 product. In order to obtain an accurate localization of both p21 and p19 we used a mouse anti-HSV Tag MAb together with a human anti-core MAb (B12.F8) to perform double immunofluorescence studies. The results have shown that p21 is always localized around the nuclear envelope. On the other hand, p19 can be found diffused in the cytoplasm to different degrees. These in vivo results reinforce the proposed links between the regulated processing of the hepatitis C virus core protein and the possibility that this may contribute towards the regulation of its diverse biological functions.