Association between sex hormones and anti-S/RBD antibody responses to COVID-19 vaccines in healthcare workers.

Healthcare workers (HCWs) are the target population for vaccination against coronavirus disease (COVID-19) as they are at a high risk of exposure and transmission of pathogens to patients. Neutralizing antibodies developed after COVID-19 vaccination decline within few months of vaccination. Several factors, including age and sex, can affect the intensity, efficacy, and duration of immune response to vaccines. However, sex-specific analyses of humoral responses to COVID-19 vaccines are lacking. This study aimed to evaluate sex-based differences in anti-S/RBD (Receptor Binding Domain) responses at three different time points after the second dose of mRNA COVID-19 vaccine in HCWs in relation to age, and to investigate the role of sex hormones as potential markers of response. Anti-S/RBD levels after two doses of the mRNA vaccine were collected from 521 HCWs naïve to COVID-19, working at two Italian Clinical Centers. Multiple regression analysis was applied to evaluate the association between anti-S levels and sex, age, and plasma levels of sex hormones. Significantly higher anti-S/RBD response to the COVID-19 vaccination was found in female HCWs, and a significant and more abrupt decline in response with time was observed in women than that in men. A novel, positive association of testosterone plasma levels and higher anti-S levels in male HCWs was found, suggesting its potential role as sex specific marker in males. In conclusion, understanding the sex-based differences in humoral immune responses to vaccines may potentially improve vaccination strategies and optimize surveillance programs for HCWs.

Coronavirus Interplay With Lipid Rafts and Autophagy Unveils Promising Therapeutic Targets.

Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses that can infect animal and human hosts. The infection induces mild or sometimes severe acute respiratory diseases. Nowadays, the appearance of a new, highly pathogenic and lethal coronavirus variant, SARS-CoV-2, responsible for a pandemic (COVID-19), represents a global problem for human health. Unfortunately, only limited approaches are available to treat coronavirus infections and a vaccine against this new coronavirus variant is not yet available. The plasma membrane microdomain lipid rafts have been found by researchers to be involved in the replication cycle of numerous viruses, including coronaviruses. Indeed, some pathogen recognition receptors for coronaviruses as for other viruses cluster into lipid rafts, and it is therefore conceivable that the first contact between virus and host cells occurs into these specialized regions, representing a port of cell entry for viruses. Recent data highlighted the peculiar pro-viral or anti-viral role played by autophagy in the host immune responses to viral infections. Coronaviruses, like other viruses, were reported to be able to exploit the autophagic machinery to increase their replication or to inhibit the degradation of viral products. Agents known to disrupt lipid rafts, such as metil-ß-cyclodextrins or statins, as well as autophagy inhibitor agents, were shown to have an anti-viral role. In this review, we briefly describe the involvement of lipid rafts and autophagy in coronavirus infection and replication. We also hint how lipid rafts and autophagy may represent a potential therapeutic target to be investigated for the treatment of coronavirus infections.

Sex Disparity in Response to Hepatitis B Vaccine Related to the Age of Vaccination.

Hepatitis B virus (HBV) infection is one of the major infectious hazards for health-care workers (HCWs) because of the frequency of percutaneous exposures to blood or body fluids. For this reason, all HCWs should be vaccinated, including students in medicine and health professional degree programs. The aim of this study was to assess the immune coverage to anti-HBV vaccine and long-lasting protective titres of anti-HBs antibodies in female and male students to evaluate gender-related differences in response to HBV vaccination. Data relative to anti-HBs antibody titre, sex, age, and age at vaccination were collected and analyzed from 5291 Italian students (1812 males and 3479 females) of the graduate courses at the School of Medicine, who underwent the mandatory health surveillance of workers exposed to biological risk. The results indicated that gender affects the immune response to HBV vaccine, particularly evident in the case of females vaccinated after one year of age who exhibited a statistically significant (p = 0.0023) 1.21-fold increase in median antibody titre with respect to males. Our findings could contribute to the optimization of HBV vaccination schedules in health surveillance of HCWs.

Correction: X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

X-chromosome-linked miR548am-5p is a key regulator of sex disparity in the susceptibility to mitochondria-mediated apoptosis.

Sex dimorphism in cell response to stress has previously been investigated by different research groups. This dimorphism could be at least in part accounted for by sex-biased expression of regulatory elements such as microRNAs (miRs). In order to spot previously unknown miR expression differences we took advantage of prior knowledge on specialized databases to identify X chromosome-encoded miRs potentially escaping X chromosome inactivation (XCI). MiR-548am-5p emerged as potentially XCI escaper and was experimentally verified to be significantly up-regulated in human XX primary dermal fibroblasts (DFs) compared to XY ones. Accordingly, miR-548am-5p target mRNAs, e.g. the transcript for Bax, was differently modulated in XX and XY DFs. Functional analyses indicated that XY DFs were more prone to mitochondria-mediated apoptosis than XX ones. Experimentally induced overexpression of miR548am-5p in XY cells by lentivirus vector transduction decreased apoptosis susceptibility, whereas its down-regulation in XX cells enhanced apoptosis susceptibility. These data indicate that this approach could be used to identify previously unreported sex-biased differences in miR expression and that a miR identified with this approach, miR548am-5p, can account for sex-dependent differences observed in the susceptibility to mitochondrial apoptosis of human DFs.

Erratum to "Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures".

[This corrects the article DOI: 10.1155/2019/6452390.].

Counteraction of HCV-Induced Oxidative Stress Concurs to Establish Chronic Infection in Liver Cell Cultures.

Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an in vitro system able to recapitulate the stages of infection in vivo. Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an in vitro system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection.

Sex-Dependent Outcome of Hepatitis B and C Viruses Infections: Synergy of Sex Hormones and Immune Responses?

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are hepatotropic viruses that differ in their genomic content, life cycle and molecular prognosis. HBV and HCV establish chronic lifespan infections that can evolve to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). This malignant liver cancer affects more commonly male patients than females, with a male-to-female incidence ratio of 2:1 up to 7:1. Sex significantly contributes to shape the immune responses, contributing to differences in the pathogenesis of infectious diseases, in males and females patients. Females usually develop more intense innate, humoral and cellular immune responses to viral infections and to vaccination compared to male subjects. Sex hormones, in turn, differentially affect the immune responses to viruses, by specific binding to the hormone receptors expressed on the immune cells. In general, estrogens have immune-stimulating effect, while androgens are immune-suppressing. However, sex hormones, such as androgen, can also directly interact with HBV genome integrated into the cell nucleus and activate transcription of HBV oncoproteins. On the other side, estradiol and estrogen receptors protect liver cells from inflammatory damage, apoptosis and oxidative stress, which contribute to fibrosis and malignant transformation preceding HCC. In HCV-associated cirrhosis and HCC the decreased expression of estrogen receptor alfa (ERα) in male patients may explain the worse outcome of HCV infection in men than in women. The synergistic action of male and female sex hormones and of immune responses, together with viral factors contribute to the mechanism of sex/gender disparity in the outcome and progression of hepatitis viruses infection.

The Natural Agonist of Estrogen Receptor ß Silibinin Plays an Immunosuppressive Role Representing a Potential Therapeutic Tool in Rheumatoid Arthritis.

Estrogens, in particular 17ß-estradiol (E2), have a strong influence on the immune system and also affect pathological conditions such as autoimmune diseases. The biological effects of E2 are mediated by two intracellular receptors, i.e., estrogen receptor (ER)α and ERß, which function as ligand-activated nuclear transcription factors producing genomic effects. Immune cells express both ERα and ERß that play a complex role in modulating inflammation. Phytoestrogens display estrogen-like effects. Among them, silibinin, the major active constituent of silymarin extracted by the milk thistle (Silybum marianum), has been suggested to have an ERß selective binding. Silibinin is known to have anti-inflammatory, hepatoprotective, and anticarcinogenic effects; however, the role of silibinin in modulating human immune responses and its impact on autoimmunity remains unclear. Aim of this study was to dissect the ability of the ERß natural ligand silibinin to modulate T cell immunity, taking into account possible differences between females and males, and to define its possible role as therapeutic tool in immune-mediated diseases. To this purpose, female and age-matched male healthy subjects and patients with active rheumatoid arthritis (RA) were recruited. We evaluated the ability of silibinin to modulate ERß expression in T lymphocytes and its effects on T cell functions (i.e., apoptosis, proliferation, and cytokine production). We also analyzed whether silibinin was able to modulate the expression of microRNA-155 (miR-155), which strongly contributes to the pathogenesis of RA driving aberrant activation of the immune system. We demonstrated that silibinin upregulated ERß expression, induced apoptosis, inhibited proliferation, and reduced expression of the pro-inflammatory cytokines IL-17 and TNF-α, through ERß binding, in T lymphocytes from female and male healthy donors. We obtained similar results in T lymphocytes from patients with active RA in term of apoptosis, proliferation, and cytokine production. In addition, we found that silibinin acted as an epigenetic modifier, down-modulating the expression of miR-155. In conclusion, our data demonstrated an immunosuppressive role of silibinin, supporting its application in the treatment of autoimmune diseases as drug, but also as dietary nutritional supplement, opening new perspective in the field of autoimmune disease management.

DNA Vectors Generating Engineered Exosomes Potential CTL Vaccine Candidates Against AIDS, Hepatitis B, and Tumors.

Eukaryotic cells constitutively produce nanovesicles of 50-150 nm of diameter, referred to as exosomes, upon release of the contents of multivesicular bodies (MVBs). We recently characterized a novel, exosome-based way to induce cytotoxic T lymphocyte (CTL) immunization against full-length antigens. It is based on DNA vectors expressing products of fusion between the exosome-anchoring protein Nef mutant (Nefmut) with the antigen of interest. The strong efficiency of Nefmut to accumulate in MVBs results in the production of exosomes incorporating huge amounts of the desired antigen. When translated in animals, the injection of Nefmut-based DNA vectors generates engineered exosomes whose internalization in antigen-presenting cells induces cross-priming and antigen-specific CTL immunity. Here, we describe the molecular strategies we followed to produce DNA vectors aimed at generating immunogenic exosomes potentially useful to elicit a CTL immune response against antigens expressed by the etiologic agents of major chronic viral infections, i.e., HIV-1, HBV, and the novel tumor-associated antigen HOXB7. Unique methods intended to counteract intrinsic RNA instability and nuclear localization of the antigens have been developed. The success we met with the production of these engineered exosomes opens the way towards pre-clinic experimentations devoted to the optimization of new vaccine candidates against major infectious and tumor pathologies.

Engineered exosomes emerging from muscle cells break immune tolerance to HER2 in transgenic mice and induce antigen-specific CTLs upon challenge by human dendritic cells.

We recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e., nanovesicles constitutively released by all cells, with full-length antigens of choice upon fusion with an exosome-anchoring protein referred to as Nefmut. They are produced upon intramuscular injection of a DNA vector and, when uploaded with a viral tumor antigen, were found to elicit an immune response inhibiting the tumor growth in a model of transplantable tumors. However, for a possible application in cancer immunotherapy, a number of key issues remained unmet. Among these, we investigated: (i) whether the immunogenic stimulus induced by the engineered exosomes can break immune tolerance, and (ii) their effectiveness when applied in human system. As a model of immune tolerance, we considered mice transgenic for the expression of activated rat HER2/neu which spontaneously develop adenocarcinomas in all mammary glands. When these mice were injected with a DNA vector expressing the product of fusion between Nefmut and the extracellular domain of HER2/neu, antigen-specific CD8+ T lymphocytes became readily detectable. This immune response associated with a HER2-directed CTL activity and a significant delay in tumor development. On the other hand, through cross-priming experiments, we demonstrated the effectiveness of the engineered exosomes emerging from transfected human primary muscle cells in inducing antigen-specific CTLs. We propose our CTL vaccine platform as part of new immunotherapy strategies against tumors expressing self-antigens, i.e., products highly expressed in oncologic lesions but tolerated by the immune system. KEY MESSAGES: We established a novel, exosome-based method to produce unrestricted CTL vaccines. This strategy is effective in breaking the tolerance towards tumor self-antigens. Our method is also useful to elicit antigen-specific CTL immunity in humans. These findings open the way towards the use of this antitumor strategy in clinic.

An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nefmut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nefmut , and are uploaded in exosomes at levels comparable to Nefmut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8+ T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform.

Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nefmut exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nefmut fused with HPV E7. In this way, we predicted that the expression of the Nefmut/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nefmut/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nefmut/E7 developed a CD8+ T-cell immune response against both Nef and E7. Conversely, no CD8+ T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8+ T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nefmut/E7 vector. Finally, we provide evidence that the injection of Nefmut/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we established a novel method to generate immunogenic exosomes in vivo by the intramuscular inoculation of DNA vectors expressing the exosome-anchoring protein Nefmut and its derivatives.

Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

The CD8⁺ T Cell-Mediated Immunity Induced by HPV-E6 Uploaded in Engineered Exosomes Is Improved by ISCOMATRIXTM Adjuvant.

We recently described the induction of an efficient CD8⁺ T cell-mediated immune response against a tumor-associated antigen (TAA) uploaded in engineered exosomes used as an immunogen delivery tool. This immune response cleared tumor cells inoculated after immunization, and controlled the growth of tumors implanted before immunization. We looked for new protocols aimed at increasing the CD8⁺ T cell specific response to the antigen uploaded in engineered exosomes, assuming that an optimized CD8⁺ T cell immune response would correlate with a more effective depletion of tumor cells in the therapeutic setting. By considering HPV-E6 as a model of TAA, we found that the in vitro co-administration of engineered exosomes and ISCOMATRIXTM adjuvant, i.e., an adjuvant composed of purified ISCOPREPTM saponin, cholesterol, and phospholipids, led to a stronger antigen cross-presentation in both B- lymphoblastoid cell lines ( and monocyte-derived immature dendritic cells compared with that induced by the exosomes alone. Consistently, the co-inoculation in mice of ISCOMATRIXTM adjuvant and engineered exosomes induced a significant increase of TAA-specific CD8⁺ T cells compared to mice immunized with the exosomes alone. This result holds promise for effective usage of exosomes as well as alternative nanovesicles in anti-tumor therapeutic approaches.

The influence of sex and gender on immunity, infection and vaccination.

Sex/gender significantly contribute to shape the immune responses, contributing to differences in the pathogenesis of infectious diseases in males and females, the response to viral vaccines and the prevalence of autoimmune diseases. Females typically develop higher innate, humoral and cellular immune responses to viral infections and in response to vaccine. At the same time, women are more prone to autoimmune diseases and experience more adverse reactions to vaccination. Hormonal, genetic and environmental factors between males and females may affect the immune responses and the sex-related outcome of vaccination. Knowledge of the mechanisms involved in sex disparity in immune responses will contribute to identify the ways to reduce adverse reactions in females and to improve the immune responses in males. This is necessary to adequately protect both sexes against the immune-mediated and infectious diseases with the long-term goal of personalizing the therapies for males and females.

Incorporation of Heterologous Proteins in Engineered Exosomes.

Engineering exosomes to upload heterologous proteins represents the last frontier in terms of nanoparticle-based technology. A limited number of methods suitable to associate proteins to exosome membrane has been described so far, and very little is known regarding the possibility to upload proteins inside exosomes. We optimized a method of protein incorporation in exosomes by exploiting the unique properties of a nonfunctional mutant of the HIV-1 Nef protein referred to as Nef(mut). It incorporates at high extents in exosomes meanwhile acting as carrier of protein antigens fused at its C-terminus. Manipulating Nef(mut) allows the incorporation into exosomes of high amounts of heterologous proteins which thus remain protected from external neutralization/degradation factors. These features, together with flexibility in terms of incorporation of foreign antigens and ease of production, make Nef(mut)-based exosomes a convenient vehicle for different applications (e.g., protein transduction, immunization) whose performances are comparable with those of alternative, more complex nanoparticle-based delivery systems.

The Contribution of Extracellular Nef to HIV-Induced Pathogenesis.

Nef is an accessory protein expressed exclusively in primate lentiviruses. It is devoid of enzymatic activities while interacting with several cell proteins as an adaptor/scaffold protein. Intracellular functions of Nef largely account for many pathogenic effects observed in AIDS disease. Nef, despite lacking known secretory pathways, can be detected in plasma of HIV-1-infected patients at the concentration varing from 5 to 10 ng/ml. Remarkably, the levels of Nef in plasma of HIV patients do not correlate with viral load or number of CD4(+) T lymphocytes, and persist during antiretroviral therapy. Here, we review literature data describing how Nef can be transmitted from HIV-1- infected cells to bystander ones, and the effects of extracellular Nef in different cell types. Overall, large part of experimental evidences supports the idea that extracellular Nef plays a relevant role in AIDS pathogenesis. Hence, efforts focused on the identification of Nef-inhibiting drugs would be of relevance to establish new therapeutic approaches supporting current antiretroviral therapies.

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.

BACKGROUND: Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130. METHODS: We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated. RESULTS: We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected. CONCLUSIONS: HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.