Guido Carloni

Ultrastructural observations of viral particles within hepatitis C virus-infected human B lymphoblastoid cell line

Hepatitis C virus (HCV)-infected TOFE cells, a human bone marrow-derived B-cell line, were studied by transmission electron microscopy (TEM). We reported the presence of virus-like particles, (VLPs) having a diameter of 45 nm on average, within the cytoplasm of HCV-infected cells and their absence in uninfected cells. The VLPs were mostly seen in dilated cisternae of endoplasmic reticulum (ER) and consisted of an electron-dense core and a membrane envelope with surface projections. In the HCV-infected TOFE cells, examination by TEM revealed tubular structures similar to those observed in liver cells of chimpanzees and humans infected by the virus. In some instances, the outer membrane of the particles was connected to the ER membrane, indicating possible virus budding into the cisternae. Rarely, coated vesicles containing a particle were seen. In most of the infected cells, enlarged cytoplasmic vacuoles filled with degenerative amorphous material were observed. The data suggest a flavivirus-like pattern of HCV morphogenesis in infected non-hepatic cells and identify lymphoblastoid TOFE cells as a valuable system for the study of the sequential steps of the infection process and the morphological effects produced by HCV infection.

Sequence analysis of the hepatitis B virus pre-C region in hepatocellular carcinoma [HCC] and nontumoral liver tissues from HCC patients

We investigated whether replication-competent pre-C/C defective mutants of hepatitis B virus (HBV) are detectable in primary human hepatocellular carcinoma (HCC) tissues from patients of a geographic area endemic for such mutants. DNAs extracted from formalin-fixed paraffin-embedded HCC samples were checked for the presence of specific HBV DNA sequences using the polymerase chain reaction (PCR). Amplified pre-C regions from nine HCC samples were directly sequenced as were samples of nontumoral liver tissues from five of these patients. The data show that hypervariable distal pre-C sequences were present in all nine HCC samples; this high variability was dependent on point mutations, which led to amino acid substitutions in nearly all cases. Interestingly, seven of the nine HBV DNA-positive samples from HCC tissues (but not samples from peritumoral liver tissue) showed mutations leading to amino acid substitution at the level of a distal cysteine residue. No mutation generating a translationally defective pre-C/C region was detectable in the tumor samples. Otherwise, in four of the six nontumoral liver tissues available from the same patients, a pre-C sequence with an in-frame TAG stop codon was detectable, although in three cases as a component of mixed population.

Identification of Six Putative Novel Human Papillomaviruses (HPV) and Characterization of Candidate HPV Type 87

ABSTRACT Six putative novel human papillomavirus (HPV) types were detected by using general primers for a conserved L1 HPV region in patients examined in gynecologic centers. One of the isolates, detected in samples from 4 patients with koilocytic atypia at cervical cytology (3 of whom were also infected with human immunodeficiency virus type 1), was completely sequenced, identified as a new HPV genotype, and designated candidate HPV87 (candHPV87) by the Reference Center for Human Papillomavirus. candHPV87 shows the classic HPV genome organization and the absence of a functional E5 coding region. Phylogenetic analysis documented that thecandHPV87 genome clusters within the A3 group of HPVs, together with HPV61, HPV72, HPV83, HPV84 and candHPV86, which have been completely sequenced, and a number of other putative novel genotypes (two of which are described in this work), which have been partially characterized. To address the growth-enhancing potential of candHPV87, the E6 and E7 putative coding regions were cloned and expressed in tissue cultures. The data indicate that both proteins stimulate cell division in tissue cultures more than those of low-risk HPVs, though not as much as those of HPV16. Taken together, the clinical, molecular, and biological data suggest that the novel papillomavirus characterized in the present study is a low- to intermediate-risk HPV.

A single-step DNA extraction procedure for the detection of serum hepatitis B virus sequences by the polymerase chain reaction

A rapid single-step procedure for the isolation of low molecular weight DNA using guanidinium thiocyanate and phenol as protein denaturants is described and applied for the detection of specific hepatitis B virus (HBV) DNA sequences from serum samples by the polymerase chain reaction (PCR). The novel technique is efficient and, when compared to the standard proteinase K/phenol/chloroform method has the advantage of being faster and easily adaptable to the routine processing of a high number of clinical samples by PCR and spot hybridization techniques.

Hepatitis C virus infection of a Vero cell clone displaying efficient virus-cell binding

The susceptibility of Vero cells and derivative cell clones to hepatitis C virus (HCV) infection was assayed by qualitative and quantitative polymerase chain reaction (PCR)-based methods. Cell extracts from Vero cells inoculated with HCV were tested for the presence of both positive and negative strands of HCV RNA; in parallel, cell-free HCV genomes were assayed in culture supernatant fluids. Quantitation of genomic HCV RNA molecules in infected cells by competitive reverse transcription PCR (cRT-PCR) indicated that HCV replication was more efficient in a derivative clone (named clone 10) than in parental Vero cells or other clones under study. Analysis of HCV-binding to cell receptors, performed by cRT-PCR quantitation of viral particles adsorbed to the cell surface, demonstrated a 10-fold higher virus-binding level of clone 10 than that of parental Vero cells. The results shown here indicate that the Vero clone 10 may constitute an efficient model system for analysing early events in HCV infection as well as a source of virus for diagnostic and biotechnological applications.

Expression of high‐ and low‐affinity epidermal growth factor receptors in human hepatoma cell lines

Data are presented from a comparative research on expression of epidermal growth factor (EGF) receptors and response to EGF of six independently established cell lines derived from human hepatoma. These lines differ in terms of the degree of differentiation, presence of hepatitis B virus (HBV) DNA copies in integrated form and expression of HBV genes. Our results indicate differential expression of membrane EGF receptors and differential response to EGF under serum‐ and hormone‐free culture conditions. Furthermore, a significant difference in affinity could be detected between EGF receptors of the two highly dedifferentiated cell lines (HA22T/VGH and Li7A) whose replication is inhibited by EGF concentrations capable of stimulating more differentiated phenotypes.

HCV Infection by Cell-to-Cell Transmission: Choice or Necessity?

In vitro models of HCV infection have allowed for the clarifying of molecules and mechanisms involved in the main steps of virus cell-entry. HCV entry and neutralization appear to be closely related. Neutralizing antibodies inhibit the E2-CD81 binding, therefore CD81 is considered to be a major target of immune response. The tight-junction proteins are also implicated in E2-binding to CD81 and successive steps of virus entry, in cooperation with several co-receptors, whose involvement has still to be elucidated. Increasing evidence has emphasized the importance of cell-to-cell HCV-transmission in chronic infection. This route for infection could favour virus-escape from host-neutralization though its CD81-dependency is still debated. The main reasons which have delayed our understanding of HCV-infection are here critically reviewed, as are the challenges faced by investigators in the field. A deeper insight into the different pathways involved could help to elucidate some crucial features of HCV infection mechanisms and disclose important implications in its pathogenesis, which could help in suggesting new targets for successful immune-prophylactic/therapeutic strategies.

Morphological modifications induced by HCV infection in the TOFE human lymphoblastoid cell line

In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.

Differential response of the human hepatoma-derived cell line HA22T/VGH to polypeptide mitogens

Several human cell lines derived from primary cancer of the liver are able to grow under serum‐free conditions and produce spreading and growth factors which are released into the culture medium. Since this autocrine growth under hormone‐free conditions might play a basic role in malignant transformation, we studied the effect on cell replication and the presence of specific membrane receptors of epidermal growth factor (EGF) and insulin on a dedifferentiated human hepatoma cell line, named HA22T/VGH. Our results point to a similar inhibitory effect on cell replication in the presence of both EGF and insulin, in spite of detecting different affinities of binding.

Integration and loss of a single ν‐Ki‐ras gene affects tumorigenic potential of human osteosarcoma cells

The human osteosarcoma cell line Te85 clone F‐5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non‐tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c‐ras products and the expression of v‐ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes.