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

PROJECT

Romina Salpini, Tor Vergata University, Rome, Virology Group Arevir meeting Koln, 11/04/13

CDC Division of Viral Hepatitis. Chronic hepatitis B: Information on testing.

CHB Liver damage Cirrhosis Liver failure HCC

HBV HBV Asso Associated deat eaths

HCC 25%

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 N 19 48 Male, N (%) 19 (100) 48 (100) Genotype, N (%)

D

12 (57.9) 38 (79.2)

D1 3 (18.2) 9 (23.7) Not-D1 9 (81.8) 29 (76.3)

A

6 (36.8) 8 (16.7)

A1 2 (28.6) 5 (62.5) Not-A1 4 (71.4) 3 (37.5)

Others (F, G, E) 1 (5.3) 2 (4.1) Age (median, [IQR]) 66 (54-72) 50 (40-61) Serum HBV DNA (median, [IQR]) Log IU/ml 4.1 (3.2-6.0) 3.8 (2.5-5.4) Transaminases (median, [IQR])IU/ml

AST 131 (38.5-207) 34 (21-50) ALT 57 (39-11) 36 (28-66)

10 20 30 40 50 60 70 A49V L97I S98T F130L N40I K141N V177A P203Q S210R HCC patients Non-HCC patients

* ** ** * * * ** ** *** % of mutations

Nine novel genetic determinants in Pre-S1/S2 and S region significantly correlated with HCC in vivo

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.

Pre-S1 HBsAg Pre-S2

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.

Network of mutations in the surface glycoproteins involved in the onset of HCC

0.81 S210.R P203.Q 0.62 0.97 L97.I A49.V 0.58 K141.N N40.I 0.9 0.67 0.43 0.2

• 0.03

PreS1 S1 3 main clusters of mutations in preS-S region

Binding site to the hepatocytes (aa 12-50) Transactivation domain (aa 10-81) Binding site for Hsc70 (aa 63-107) CAD (aa 70-94) Binding site for capsid (aa 92-116) S Promotor (nt3045-3180) Viral Secretion (aa 109-113)

• A49VS1 is localized in the putative region of the large surface glycoprotein

involved in receptor recognition. Critical for entry phase.

• L97IS1 is localized in the domain of the large surface glycoprotein involved in the

interaction with the capsid. Critical for encapsidation.

(aa 1-108) (aa 109-164) (aa 109-162)

• 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)

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.

Network of mutations in the surface glycoproteins involved in the onset of HCC

0.81 S210.R P203.Q 0.62 0.97 L97.I A49.V 0.58 K141.N N40.I 0.9 0.67 0.43 0.2

• 0.03

HBs HBs

• Among all mutations identified, the N40I and K141N are the only

localized within specific HLA epitopes in the HBsAg

HLA Class I epitopes HLA Class II epitopes aa: 37-51 TSLNFLGGTTVCLGQ aa: 139-146 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

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.

Network of mutations in the surface glycoproteins involved in the onset of HCC

0.81 S210.R P203.Q 0.62 0.97 L97.I A49.V 0.58 K141.N N40.I 0.9 0.67 0.43 0.2

• 0.03

HBs HBs

Schematic representation of the Small Surface glycoprotein (HBsAg) encoded by the S region of HBV genome

P203Q S210R

Both P203QS and S210RS 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).

The intracellular retention of the HBsAg can induce an oxidative stress thus favoring the neoplastic transformation of the hepatocytes

Wang et al., Cancer Science 2006 HBsAg mutations affecting HBsAg secretion Hepato tocarcino nogene nesis

Five novel HBx genetic determinants significantly correlated with HCC in vivo

10 20 30 40 50 60 70 80 90 G22S F30V T47A F88I V102A Prevalence of HBx mutations * *** *** * *

HCC patients Non-HCC patients 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.

0.99 V131.I K130.M 0.85 0.67 F88.I V102.A 0.69 T47.A 0.88 F30.V G22.S 0.89 0.58 0.28

• 0.03
• 0.34

Two novel clusters

• G22S, F30V, and T47A are tightly

clustered with each other.

• this cluster is linked with a second

cluster F88I and V102A

• K130M and V131I, correlated with

HCC in previous studies, formed a cluster apart

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

G22S F30V T47A F88I V102 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.

Localization of HBx mutations in the 3D structure of the HBx protein

HBx mutations correlated with HCC are located in two distinct regions of the HBx protein.

• Mutations at HBx positions 88 and

102 are localized in the tran- sactivation domain, known to interact with several proteins involved in the control of cell proliferation.

• Mutations at HBx positions 22, 30

and 47 are localized in negative regulatory domain with anti-apoptotic activity located at the N-terminus part

• f HBx. G22S, F30V, and T47A are co-

localized in a loop and are interfaced with each others. Trans-activation domain HBx negative regulatory domain

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

• ngoing …

 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

50 100 150 200

HBsAg (IU/ml)

* * *

* p<0.05

PreS1-S98T significantly increases the amount of released HBsAg (both alone and in association with S-V177A)

preS1 mutants S mutants

This confirms the potential involvement of preS1 mutations in increasing HBV replication capacity and transciptional activity, consequently enhancing the

• ncogenic potential of viral species carrying this mutation
• Ratio S98T/wt= 3.71
• Ratio S98T+V177A/wt= 3.18

Differently, the double mutant P203Q+S210R induce a statistically significant decrease in HBsAg released in supernatants.

50 100 150 200

HBsAg (IU/ml)

* * *

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

• Ratio P203Q+S210R/wt= 0.27

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

25 50 75 100

HBsAg (IU/ml)

* *

* p<0.05

The double mutant P203Q+S210R confirmed to significantly reduce the amount of HBsAg released in supernatant An additional couple of S mutations N40I+K141N were associated with a strong decrease of HBsAg released in supernatant.

C-terminal domain HBsAg

• Ratio K141N+N40I/wt= 0.17
• Ratio P203Q+S210R/wt= 0.12

The introduction of a new glycosilation site in S region (K141N), potentially modifying the processing of the protein endoplasmic reticulum, could explain the decreased release of the small glycoprotein

25 50 75 100

wt G22S F30V T47A F88I V102A K130M HBsAg (IU/ml)

* *

* p<0.05

The novel G22S as well as the known K130M in HBx double the amount of HBsAg released in supernatant

• Ratio G22S/wt= 2.04
• Ratio K130M/wt= 2.01

HBx mutants identified in vivo as associated with HCC

This increased viral activity could be play an important role in augmented progression to HCC of these HBV mutants

Conclusions

• Nine point mutations in the pre-S1/S2 and in S region, significantly

correlated with HCC in vivo, have been identified for the first time. They localize in domains critical for viral entry, packaging, intracellular signaling

• Preliminary in vitro studies seem to confirm the role of preS1-S98 mutation

in enhancing HBV activity. Conversely, S mutants particularly in C-terminal region, are associated with a decrease in HBsAg release, possibly related to an intracellular retention of

• HBsAg. This could explain the increased cancerogenic potential of viruses

carrying these mutations.

• Five novel mutations in HBx, significantly correlated with HCC in vivo, have

been identified. They tightly cluster with each other and localize in functional HBx domains playing a role in anti-apoptotic and proliferation signaling.

• The “strategical” position of these mutations and their involvement in

complex patterns underline their potential role as markers in predicting HCC development.

Thanks to……

The Clinicians Massimo Andreoni Università Tor Vergata Mario Angelico Università Tor Vergata Angelo Barlattani, Polimabulatorio San Giacomo, Roma Nerio Iapadre, Ospedale L'AQUILA Claudio Puoti,Ospedale di Marino Dante Di Giammartino, Presidio Ospedaliero "G. Mazzini" Teramo Gloria Taliani, Sapienza Università di Roma Maurizio Koch, Complesso ospedaliero San Filippo Neri, Roma Adriano Pellicelli, Azienda Ospedliera San Camillo-Forlanini, Roma Orlando Armignacco, Ospedale BELCOLLE, Viterbo Giustino Parruti, Ospedale Civile Spirito Santo, Pescara Maurizio Paoloni, Ospedale SS. Filippo e Nicola, Avezzano (AQ) Jacopo Vecchiet, Ospedale SS. Annunziata Chieti

Virology Group; University of Tor Vergata

Carlo Federico Perno Valentina Svicher Matteo Surdo Carmen Mirabelli Valeria Cento Ada Bertoli Claudia Alteri Francesca De Luca Michela Pollicita Velia Chiara Di Maio Francesca Ceccherini

PROJECT

Virology Group, Koln University

Maria Neumann-Fraune Jens Verheyen Rolf Kaiser