Key patterns of mutations in HBx and in Pre-S1/2-S regions involved - PowerPoint PPT Presentation

Key patterns of mutations in HBx and in Pre-S1/2-S regions involved in mechanisms underlying HBV- induced hepatocellular carcinoma in vivo Romina Salpini, Tor Vergata University, Rome, Virology Group Arevir meeting Koln, 11/04/13 PROJECT

HBV HBV Asso Associated deat eaths Liver damage Cirrhosis 25% CHB Liver failure HCC HCC CDC Division of Viral Hepatitis. Chronic hepatitis B: Information on testing.

Both the HBV X protein (HBx) and the surface glycoproteins (Large, medium and small) play a critical role in mediating HBV-induced tumorigenesis HBV genome Surface glycoproteins HBsAg HBx

Objective To define the correlation of mutations in the entire S open reading frame and in the X region with the onset of HBV- induced liver cancer

Methods This study included 67 HBV chronically-infected patients : - 19 with HBV-related HCC - 48 asymptomatic carriers used as control -The diagnosis of HCC was based on one of the following criteria: (1)positive histology or (2)elevated alpha-fetoprotein levels together with imaging features compatible with HCC • Association of mutations in HbX and in Pre-S1/S2-S regions with HCC was assessed by Fisher Exact test. • Interactions among mutations were assessed by hierarchical clustering analysis.

Patients’ Characteristics Char arac acteristic ics Pat atie ients wit ith HCC CC Pat atie ients wit ithout HCC CC 19 48 N 19 (100) 48 (100) Male, N (%) Genotype, N (%) 12 (57.9) 38 (79.2) D D1 3 (18.2) 9 (23.7) Not-D1 9 (81.8) 29 (76.3) 6 (36.8) 8 (16.7) A A1 2 (28.6) 5 (62.5) Not-A1 4 (71.4) 3 (37.5) 1 (5.3) 2 (4.1) Others (F, G, E) 66 (54-72) 50 (40-61) Age (median, [IQR]) Serum HBV DNA 3.8 (2.5-5.4) 4.1 (3.2-6.0) (median, [IQR]) Log IU/ml Transaminases (median, [IQR])IU/ml 131 (38.5-207) 34 (21-50) AST 57 (39-11) 36 (28-66) ALT

Nine novel genetic determinants in Pre-S1/S2 and S region significantly correlated with HCC in vivo * 70 Non-HCC patients 60 ** ** HCC patients % of mutations 50 *** 40 ** ** 30 * * * 20 10 0 A49V L97I S98T F130L N40I K141N V177A P203Q S210R HBsAg Pre-S1 Pre-S2 The histogram reports mutations in the pre-S1/S2 and S regions significantly correlated with HCC in vivo . Statistically significant differences were assessed by Fisher Exact Test. P values were corrected for multiple comparison by the Benjamini & Hochberg method using a False Positive Rate of 0.05 (Benjamini & Hochberg, 1995). * indicates P value <0.05, ** <0.01, *** <0.001 after Benjamini-Hochberg correction .

Network of mutations in the surface glycoproteins involved in the onset of HCC The dendrogram, obtained from average linkage hierarchical clustering, shows significant clusters involving mutations in pre-S and S regions. Length of branches reflects distances between mutations in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes. -0.03 0.62 0.2 0.81 0.43 0.58 0.67 0.97 3 main clusters of mutations in preS-S region 0.9 S210.R P203.Q K141.N A49.V N40.I L97.I PreS1 S1

• A49V S1 is localized in the putative region of the large surface glycoprotein involved in receptor recognition. Critical for entry phase. • L97I S1 is localized in the domain of the large surface glycoprotein involved in the interaction with the capsid. Critical for encapsidation. ( aa 109-164) ( aa 1-108) Binding site for Hsc70 (aa 63-107) CAD (aa 70-94) Binding site for capsid (aa 92-116) Viral Secretion (aa 109-113) Binding site to the hepatocytes (aa 12-50) Transactivation ( aa 109-162) domain (aa 10-81) S Promotor (nt3045-3180)

• The correlated pair A49V and L97I could contribute to HBV induced carcinogenesis by increasing HBV infectivity and replication capacity Higher replication capacity Higher viremia Increased risk of liver cancer (Mommeja-Marin H, et al. Hepatology 2003)

Network of mutations in the surface glycoproteins involved in the onset of HCC The dendrogram, obtained from average linkage hierarchical clustering, shows significant clusters involving mutations in pre-S and S regions. Length of branches reflects distances between mutations in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes. -0.03 0.62 0.2 0.81 0.43 0.58 0.67 0.97 0.9 S210.R P203.Q K141.N A49.V N40.I L97.I HBs HBs

• Among all mutations identified, the N40I and K141N are the only localized within specific HLA epitopes in the HBsAg HLA Class II epitopes HLA Class I epitopes aa: 37-51 aa: 139-146 TSLNFLGGTTVCLGQ CTKPTDGN The mapping of HLA epitopes in HBsAg is based on Desmond et al., Antiviral Ther 2008

• The correlated pair N40I and K141N could act as immune escape mutations …. …….thus increasing viral evasion from the immune system and consequently enhancing viral fitness and in turn the risk of liver cancer

Network of mutations in the surface glycoproteins involved in the onset of HCC The dendrogram, obtained from average linkage hierarchical clustering, shows significant clusters involving mutations in pre-S and S regions. Length of branches reflects distances between mutations in the original distance matrix. Bootstrap values, indicating the significance of clusters, are reported in the boxes. -0.03 0.62 0.2 0.81 0.43 0.58 0.67 0.97 0.9 S210.R P203.Q K141.N A49.V N40.I L97.I HBs HBs

Both P203Q S and S210R S reside in a region of the membrane- embedded C-terminal domain known to be crucial for HBsAg- secretion ( Jenna et al., Virology 1998; Jenna et al., J Virol 1999 ). S210R P203Q Schematic representation of the Small Surface glycoprotein (HBsAg) encoded by the S region of HBV genome

The intracellular retention of the HBsAg can induce an oxidative stress thus favoring the neoplastic transformation of the hepatocytes HBsAg mutations affecting HBsAg secretion Hepato tocarcino nogene nesis Wang et al., Cancer Science 2006

Five novel HBx genetic determinants significantly correlated with HCC in vivo * 90 Prevalence of HBx mutations 80 Non-HCC patients * 70 HCC patients *** 60 50 40 *** 30 * 20 10 0 G22S F30V T47A F88I V102A The histogram reports only HBx mutations significantly correlated with HCC in vivo. Mutations were defined according to the reference sequence of each specific sub-genotype identified. The analysis was performed on 60 HBx sequences derived from 42 chronically HBV-infected patients without HCC (control group) and from 18 HBV chronically infected patients with HCC.

Network of HBx mutations involved in the onset of HCC Clustering analysis supports the existence of 3 specific networks of HBx mutations involved in mechanisms underlying HCC in vivo -0.34 Two novel clusters -0.03 0.85 - G22S, F30V, and T47A are tightly clustered with each other. 0.28 0.99 - this cluster is linked with a second 0.69 0.67 cluster F88I and V102A 0.58 0.88 - K130M and V131I , correlated with K130.M V102.A HCC in previous studies, formed a V131.I G22.S T47.A F30.V 0.89 F88.I cluster apart

Localization of HBx mutations in the 3D structure of the HBx protein HBx mutations correlated with HCC are Trans-activation located in two distinct regions of the domain HBx negative HBx protein. regulatory domain F88I - Mutations at HBx positions 88 and 102 are localized in the tran- sactivation domain , known to interact F30V with several proteins involved in the control of cell proliferation. G22S V102 - Mutations at HBx positions 22, 30 T47A and 47 are localized in negative regulatory domain with anti-apoptotic activity located at the N-terminus part of HBx. G22S, F30V, and T47A are co- localized in a loop and are interfaced with each others. Ab initio putative structure of HBV X protein modified with USCF Chimera software HBx domains were defined according to Tang et al., Cancer Sci October 2006.

In order to study the impact of these mutations in preS-S and X region in HBV-induced hepatocellular carcinoma, in vitro studies have been performed and are still ongoing …

 The different mutations, identified in vivo as HCC related, have been inserted in a plasmid containing the full-lenght HBV genome, genotype D, by site-directed mutagenesis  Huh7 cells were transfected with the different plasmids containing the single or the double mutations  HBsAg level in the supernatants was measured at 3 days after transfections

PreS1-S98T significantly increases the amount of released HBsAg (both alone and in association with S-V177A) 200 - Ratio S98T/wt= 3.71 * * - Ratio S98T+V177A/wt= 3.18 150 HBsAg (IU/ml) 100 50 * 0 * p<0.05 preS1 mutants S mutants This confirms the potential involvement of preS1 mutations in increasing HBV replication capacity and transciptional activity, consequently enhancing the oncogenic potential of viral species carrying this mutation

Differently, the double mutant P203Q+S210R induce a statistically significant decrease in HBsAg released in supernatants. - Ratio P203Q+S210R/wt= 0.27 200 * * 150 HBsAg (IU/ml) 100 50 * 0 This datum is in line with the hypothesized involvement of C-terminal mutations in inducing HBsAg retention in hepatocytes, contributing to induce the oxidative stress observed in HCC

S region mutations were also tested transfecting Huh7 cells with a plasmid containing only the small HBsAg (226 aa)