Alfredo Alberti

NS5A is the major phosphoprotein of HCV (13) and it can be phosphorylated by CK2, a pleiotropic cellular kinase constitutively active. CK2 plays a key role in the life cycle of many viruses being the biological activity of several viral proteins modulated by CK2-mediated phosphorylation. In a previous study, we have shown that CK2 recognition sites are differently present in the diverse HCV genotypes being more frequent and highly conserved in HCV-3 as compared to other genotypes. These findings were supported by the in vitro phosphorylation experiments using synthetic peptides that showed a correlation between the predicted number of CK2 sites and the phosphorylation level of the peptide.

Perspective

The good correlation between predicted and observed phosphorylation, suggests that similar CK2 dependent NS5A phosphorylation events may takes place within cells infected with different HCV sequences. Therefore, in collaboration with the group of Prof. Lorenzo Pinna, we have designed a series of experiments to further investigate the ability of CK2 to phosphorylate NS5A variants. In particular, we have selected and expressed a number of NS5A sequences of HCV genotypes 1a, 1b and 3 having a diverse predicted number of CK2 sites. The principal aim of this project is to verify whether these NS5A are differently phosphorylated according to their number of predicted CK2 sites, by using in vitro phosphorylation assays. To this purpose, NS5A recombinant proteins will be used as substrate for purified CK2 in presence of γ³³-ATP. Lambda phosphatase treatment of the same substrate will be then performed to prove the specificity of the previous reaction. Furthermore, site directed mutagenesis will be used to disrupt CK2 predicted sites in the different cloned NS5A and thus to confirm the phosphorylatability of each site.

Relevance

This part of the study will give us a better insight of the role of CK2 in the modulation of NS5A phosphorylation in vivo. Within this context, its is known that changes in the degree of phosphorylation may have a regulatory role in determining NS5A functions by addressing the protein towards different biological activities in association with distinct cellular-viral proteins. As a consequence, variation in CK2 phosphorylation extent may ultimately generate differences in the overall NS5A phosphorylation leading to profound changes in mechanisms associated with viral infection, pathogenesis, persistence, and viral phenotype. If confirmed in in vivo experiments, differences in the CK2 mediated phosphorylation of NS5A might be at the basis of diverse protein properties and can eventually explain some of the heterogeneous pathobiological features of HCV.

MTP/HCV proteins interaction and liver steatosis

Hepatic steatosis is a frequent histopathological finding in chronic hepatitis C, being more common in patients infected with HCV-3. Recently we have demonstrated, that liver steatosis in HCV infected patients correlate with a reduced gene expression and activity of the microsomal triglycerides transfer protein (MTP), responsible for hepatic VLDL assembly and secretion. These results indicate that MTP has a major role in HCV induced steatosis and led us to propose a new pathogenetic hypothesis to explain this histological feature.

Perspective

The relationship between MTP and the intracellular expression of genotype-specific HCV proteins is currently under investigation.

In particular, we are performing in vitro experiments using human hepatocarcinoma cell lines (Huh7) transfected with the two HCV proteins, NS5A and Core, both of genotype 1 and genotype 3. In order to study the subcellular localization of the viral proteins, transfected cells were permeabilized and treated with specific antibody for immunofluorescence assays. Major findings, although preliminary, show that: 1) the Core protein is mainly localized on lipid droplets (with a macrovescicular pattern more frequently observed with Core of genotype 3); 2) NS5A mainly localizes in the perinuclear space and exhibits a net-like staining. Interestingly, NS5A of genotype 3 localizes also on mitochondria.

Next, we will measure the MTP gene expression and MTP transport activity in NS5A and/or Core transfected cells to evaluate the possible effects of these proteins on MTP when different genotypes are considered. Furthermore, we will perform Western blot experiments to better characterize the interaction between the HCV protein and mitochondria using mitochondria isolated from NS5A transfected cells. Secondly, we will further investigate whether the viral protein is localized either on the external surface or in the internal surface as well as in the inter-membrane space of the mitochondria.

Relevance

It has been demonstrated that the expression of different HCV proteins, particularly Core and NS5A, can affect the host lipid metabolism both transcriptionally and post-transcriptionally. The study of the subcellular localization of the two HCV proteins will elucidate the role of the viral proteins both in the intrahepatic lipid metabolism impairment and their possible implications in the apoptotic mechanisms. The fact that NS5A of genotype 3 localizes on mitochondria, if confirmed, could be a new finding with regard to the role of this genotype in the re-arrangement of lipid metabolism and in the oxidative stress contributing to the severity of liver steatosis commonly observed during HCV-3 infection.

Microsomal triglyceride transfer protein (MTP) gene polymorphism and lipid metabolism in HCV-infected patients

Genetic variability can modulate the concentration of MTP in the ER, which affect the secretion pattern of lipoproteins. Mutations in the coding region of MTP inhibit its transport activity leading to abetalipoproteinemia and liver steatosis. A common polymorphism in the promoter region of MTP gene, -493G/T, has been studied and the T allele seems associated to increased MTP transcriptional activity and to low serum levels of LDL cholesterol in healthy subjects. Moreover the -493 G/T polymorphism seems to be implicated in the susceptibility for the development of steatohepatitis in patients with type II diabetes. In a recent study, Namikawa has demonstrated that the G allele frequency is higher in patients with NASH compared to controls and that the grade of steatosis is more severe in NASH patients with genotype G/G compared to patients with G/T genotype.

Perspective

The aim of our study is to examine the functional -493 G/T polymorphism roles in the alteration of lipid serum levels and in the susceptibility for the development of liver steatosis in patients with chronic hepatitis C infection.

Insulin's role on the interferon (IFN) signaling

Hepatitis C is progressive in 30-40% of the patients and the pathogenesis of the liver injury is only in part known and included toxic direct effects of the virus on the infected cells by oxidative stress, intrahepatic store of fat and cellular death. These cellular modifications, HCV mediated, have an important role not only in disease progression but also to reduce the antiviral response. Treatment failure could be due to different and not completely characterized factors such as viral genotype, viral load and direct viral mechanisms that interfere with the cellular signal and gene expression that are induced by IFN stimulation. Recently the interest is focused on some host factors that could be likely to determine the failure or the reduction of the response to the pharmacological treatment, in particular the presence of steatosis in the liver and the insulin resistance (IR). In a recent study we have shown the presence of a direct correlation between liver steatosis and the MTP's liver expression and activity, suggesting a direct mechanism of interference of the HCV proteins with the MTP activity at least in the genotype 3 while, in the patients infected by the other genotypes, the IR seems to be the main pathogenetic factor for the development of hepatic steatosis. Importantly, the presence of IR, due to viral or metabolic factors, seems to be a common feature among non -responders patients.

Though the IR could correlate with different host factors (i.e., Body Mass Index, genetic predisposition, etc), recent data have suggested a direct role of the virus in the interference with the insulin signaling in HCV-infected patients.

Perspective

We plan to set up experimental models, using hepatic cellular lines (Huh7 and HepG2) and primary hepatocytes (rat hepatocytes, human primary hepatocytes from transplanted livers, but inadequate to transplant) transfected with HCV viral protein, to study the molecular and cellular mechanisms leading to disease progression and resistance to IFN therapy, with particular attention to the relationship between IR and cellular resistance to IFN.

In particular, we will analyze the effect of insulin on some genes involved in the IFN signaling (PKR, MxA and, 2'-5'OAS) in cells pre-treated with IFN. In parallel, we'll analyze the effects of insulin on SOCS1 and SOCS3 genes expression as well as on adiponectin and some cytokines that can modulate the insulin signaling (i.e: TNF, IL-6). Gene expression will be measured by RealTime PCR. We will also study the potential influence of the HCV NS5A and core proteins in the mechanisms at the basis of IR and cellular resistance to IFN. To this purpose, cells will be transfected in transient manner with viral proteins CORE and NS5A, and treated with IFN and/or insulin to assess, by RealTime PCR, the effect of these viral proteins on the expression of genes involved in the IFN and insulin signaling.

Relevance

The results of these experiments should clarify the molecular mechanisms at the basis of the development of the liver damage and of the antiviral resistance therapy in the chronic hepatitis HCV, and in particular the relationship between cellular resistance to the insulin and cellular resistance to the IFN. The possibility to use in the our experimental model of viral proteins derived from different clinical isolates, it will allow us to correlate the experimental data with clinical phenotypes (higher or lower disease activity, liver damage, alteration of the lipid metabolism and antiviral treatment response), with possible implication in the development of new diagnostic and prognostic tools and improvement of therapeutic strategies.

Viral markers of clinical relevance

As part of our research and laboratory activities, we recently introduced some innovative diagnostic tools to assist physicians in the clinical management of patients with HCV or HBV infection.

Previous studies demonstrated that HCV virological response occurs early in the course of treatment with IFN where a rapid viral decline appears predictive of sustained response. On the other hand, as we shown in a previous study, the presence of MRV (detectable by HCV Qual TMA) at the end of therapy is highly predictive of relapse.

In patients infected with HBV, it has been recently suggested that genotypes could have clinical significance. Patients infected with HBV-C seem to have a more progressive course of liver disease compared to those infected with HBV-B. On the other hand, patients with HBV-D may also present a worse prognosis compared to those with HBV-A. On the other hand, patients treated with nuceos(t)ide analogs have been shown to develop specific mutations in the polymerase region that are associated with resistance. Many of these mutations have been shown to associate with clinical rebound of hepatitis B.

Perspective

For HCV infected patients undergoing antiviral therapy we provide assessment of the early kinetics of virological response that, integrated with management of minimal residual viremia, allows optimizing treatment schedule and duration. Early HCV kinetics will be determined by measuring HCV RNA levels by RealTime PCR in consecutive samples taken in the first month of therapy and minimal residual viremia will be checked for presence at the end of treatment using the HCV Qual TMA assay. For HBV infected patients treated with nucleos(t)ide analogs we provide genotyping and a "virtual phenotype" tests inclusive of virus sequence analysis and interpretation of its clinical consequences.

We will study the HBV polymerase full-length sequence derived from infected patients before starting and during the antiviral treatment to identify the selection of drug-resistant mutations that may lead to virological and biochemical breakthrough.

Relevance

Both, HCV kinetics during therapy and the identification of HBV resistant mutants are of paramount importance in order to optimized future patients management. The purpose of this project is, as a final point, to provide a tailored therapy for each patient taking into account duration/type of treatment.

Synoptic CV

2008–present	Professor of Gastroenterology, University of Padua
1990–2008	Associate Professor of Internal Medicine, University of Padua, Italy
1982	Postgraduate degree in Gastroenterology
1976–1979	Rusconi fellow, Liver Unit, King's College Hospital Medical School London, UK
1976	Postgraduate degree in Hematology and Laboratory Medicine
1972	MD, University of Padua Medical School, Italy

Honours

2006	Excellence award for medical education, Romanian Association of Hepatology
2005	WHO consultant for hepatitis B and C
2004	Hepatology award, Jordanian society of Internal Medicine and Gastroenterology NIH expert panel for viral hepatitis

^ TOP ^

Selected Publications (VIMM)

Gerotto M, Dal Pero F, Bortoletto G, Ferrari A, Pistis R, Sebastiani G, Fagiuoli S, Realdon S, Alberti A (2006) Hepatitis C minimal residual viremia (MRV) detected by TMA at the end of Peg-IFN plus ribavirin therapy predicts post-treatment relapse. J. Hepatol. 44:83-7.
Mirandola S, Realdon S, Iqbal J, Gerotto M, Dal Pero F, Bortoletto G, Marcolongo M, Vario A, Datz C, Hussain MM, Alberti A (2006) Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis. Gastroenterology 130:1661-9.
Alberti A (2005) Towards more individualised management of hepatitis C virus patients with initially or persistently normal alanineaminotransferase levels. J. Hepatol. 42:266-74.
Gerotto M, Dal Pero F, Bortoletto G, Realdon S, Ferrari A, Boccato S, Alberti A (2004) PKR gene expression and response to pegylated interferon plus ribavirin therapy in chronic hepatitis C. Antivir. Ther. (Lond.) 9:763-70.

Additional Publications

Ferri S, Dal Pero F, Bortoletto G, Bianchi FB, Lenzi M, Alberti A, Gerotto M (2006) Detailed analysis of the E2-IgM complex in hepatitis C-related type II mixed cryoglobulinaemia. J. Viral Hepat. 13:166-76.
Gerotto M, Resti M, Dal Pero F, Migliorato I, Alberti A, Bortolotti F (2006) Evolution of hepatitis C virus quasispecies in children with chronic hepatitis C. Infection 34:62-5.
Realdon S, Gerotto M, Dal Pero F, Marin O, Granato A, Basso G, Muraca M, Alberti A (2004) Proapoptotic effect of hepatitis C virus CORE protein in transiently transfected cells is enhanced by nuclear localization and is dependent on PKR activation. J. Hepatol. 40:77-85.
Tagariello G, Gerotto M, Pontisso P, Belvini D, Salviato R, Radossi P, Alberti A (2004) Hepatitis C virus quasispecies in the natural course of HCV-related disease in patients with haemophilia. Haemophilia 10:81-6.