HBV Protein structure

HBV has been classified into at least 9 genotypes (A through H and J) and has been shown to have a distinct geographical distribution (H12).

MM 1

MM2

• picture

1. Dane particle (H1)
   Link: https://www.dropbox.com/s/01ictzaayyg7kkp/Dane%20particle.jpeg?dl=0
2. Real virus (H2)Link: https://www.dropbox.com/s/y1q07anlmkm1cnw/Real%20virus.jpeg?dl=0

Genome (H1)

General

schematic genome (H1)
Link: https://www.dropbox.com/s/q8f3olheczjwvqg/schematic%20gene.jpeg?dl=0

HBV has a small (3.2 kb), partially double-stranded, relaxed-circular DNA genome that encodes four overlapping ORFs.
The largest ORF encodes the viral polymerase, which also has reverse transcriptase (RT) activity that generates the first strand of the DNA genome from an RNA intermediate.
The second largest ORF encodes the three viral envelope proteins: large (L-), middle (M-), and small (S-) surface antigen (HBsAg).
Another ORF encodes precore, also referred to as HBV E antigen (HBeAg), and the core protein, which makes up the viral capsid.
Finally, the smallest ORF encodes the HBV X protein (HBx), a small regulatory protein that has been shown to be required for HBV replication both in vitro and in vivo.

a small fraction (5-20%) of virus contain Double stranded linear DNA

all ORF are in same direction. clockwise.

contains : 4 promotor, 2 enhancer, 1 polyadenilation signal

enhancer : EN1 and EN2 (to regulate the transcription)

activation of the Enhancer I/HBx promoter is a required first step in viral transcription, as this is believed to enhance transcription from downstream promoters. A number of the transcription factors that have been mapped to the EN1/HBx promoter are liver specific, including hepatocyte nuclear factor (HNF) 1, HNF3, and HNF4

Sifat minus strand:
Minus strand lebih panjang daripada positif strand.
Minus strand berlengketan dengan viral RT
mengandung terminal redundancy termed r (8-9 nucleotides) (penting untuk formasi RC DNA
mencode semua transcript
melekat dengan RT via phosphotyrosine bond

Positive strand : 2/3 panjang dari minus strand

 type

1.  genomic : 1H1

The genomic transcripts act as mRNAs for precore, core, and polymerase. The genomic transcript that encodes both core and polymerase is multifunctional and referred to as pregenomic RNA (pgRNA). The pgRNA is the template for HBV replication and is reverse transcribed to generate the HBV DNA genome

2.  subgenomic : 3H1

The subgenomic transcripts act only as templates for HBV proteins and consist of the 0.7 kb transcript, which encodes HBx, and the 2.1 kb and 2.4 kb HBsAg transcripts encoding M- and S-HBsAg, and L-HBsAg, respectively.

4 transcript utama
pre-C/C =3.5 Kb
pre-S = 2.4 Kb

S = 2.1 Kb

X = 0.7 Kb

Protein (H10)
none of the viral proteins are subject to post-translational proteolytic processing ;
in addition ,hepadnaviruses do not encode a proteinase.

2 types of Protein :

• HBV. Buoyant density1.24 - 1.26 g cm-3 in CsCl
• SVP. Non infectious SVP. Buoyant density1.18 g/cm-3 in CsCl

SVP :
form
Octahedral
Concentration in blood : until 10^12 per ml

Sphere:
M : S = 1:2. L little only


Rod
L:M:S = 1:1:4
diameter : 22 nm
In addition, an HBV-infected cell produces non-infectious subviral particles (SVP)
made primarily of S-HBsAg containing varying (but much lower) amounts of M-HBsAg and little to no L-HBsAg. These SVPs can reach a concentration 10,000-fold higher than infectious HBV particles in the serum of an infected individual.[44, 45]
SVPs are produced in two forms: 25 nm spheres, which are almost exclusively made up of S-HBsAg, and 22 nm filaments, which are made up primarily of S-HBsAg, with some M-HBsAg and potentially small amounts of L-HBsAg.
SVPs act to divert neutralizing antibodies away from infectious particles and that SVPs play a role in inducing the immune tolerance required to sustain a long-term chronic infection. A study of DHBV SVPs showed that the SVP-to-infectious-particle ratio plays a role in determining the efficiency of hepatocyte infection,

HBV :

Form 
outer :
spheris

42 nm
made up of 180 dimer

Ratio L:M: S = 1:1:4

inner (core)
- 22 nm
made up of 120 dimer
T=4 icosahedral





Hbcore

Core- 183-185 aa long polypeptide
- MW 21 KD
- contain arginine rich protamine, located at its C-terminus, harboring nuclear localization signal (play a role in packaging of pregenomic RNA, , in DNA replication and perhaps in the recruitment of SRPK1 and SRPK2)
-dimeric structure with Cys 61 forming the disulfide bridge
-alfa helical
- naturally : 120 dimer, T= 4structure
in lab : 90 dimer, T = 3 structure
- none of viral proteins exhibit kinase activity
but SR protein kinase 1 dan 2 (SRPK1, SRPK2) associate with cores and phosphorilate the 3 serine residues. which kinase play a role is still unknown.

1. structure (H1)
   The 21 kD HBV core protein, or HBcAg, is the organizing framework for the virion [Figure 1c]. When expressed in cells, core mainly exists as soluble dimers, or in T = 3 or T = 4 icosahedral capsids. About 95% of mature nucleocapsids isolated from Dane particles contain T = 4 capsids made up of 120 core dimers, with the remaining 5% being the smaller T = 3 with 90 dimers.[47] Core is translated from the pgRNA and the first 149aa of core form the assembly domain, which is sufficient for in vitro formation of capsids that are indistinguishable from capsids isolated from Dane particles.[48] The remaining 34–36aa makes up the arginine-rich C-terminal domain (CTD); phosphorylation of various aa in the CTD regulates multiple stages of the HBV life cycle

function

1. In fact, core protein binds to HBV covalently closed circular DNA (cccDNA), potentially to regulate spacing of nucleosomes on cccDNA;
2. In addition, the CTD is required for pgRNA packaging,[54]
3. and core protein also plays an active role in initiating reverse transcription[55–57] and in mature nucleocapsid envelopment

Hb polymerase

polMW : 90 KD, 844 aa

3 functional domain
N terminus encodes Terminal protein domain
C terminus encodes RNAseH (RT) domain + Reverse transcriptase
1 hinge region = spacer

1. structureH1
   The 90 kD, 838aa polymerase protein of HBV (reverse transcriptase/RT/Pol/P) is made up of 3 functional domains and a variable spacer region.

TP DomainH1

1. At the N-terminus is the terminal protein (TP) domain, which is important for multiple facets of the initiation of genome replication. This region, despite its important role in P binding to the pgRNA, RNA packaging, and protein-priming,[62–64] is a unique domain that is not shared by any non-hepadnavirus RTs.
2. In fact, only 3 cysteine residues within the Cterminal end of the spacer region, along with a fourth in the N-terminal side of the RT domain, are thought to be important for RT/pol function.[66]

RT DomainH1

The RT domain is responsible for genome replication by reverse transcribing the pgRNA to form the (-)-strand of the DNA genome and subsequent use of the (-)-strand as a template for synthesis of the (+)-strand of the DNA. This domain shares significant homology to the RT of other retroviruses.[67] The RT domain is the only current anti-HBV therapeutic target,

RNase H domain (H1)

The final domain, P, is the RNase H domain. This domain is responsible for degrading the pgRNA template during synthesis of the (-)-strand of the DNA genome. Coordination of metal ion binding, which is important for RNase H activity, is achieved through 4 conserved carboxylates.[70] Studies of the RNase H domain have also shown that purified recombinant  RNase H domain is functional in vitro and that the RNase H domain of P is important for pgRNA packaging

Reverse TranscriptaseMW = 90 KD

terdiri dari 3 functional domain:
- Terminal protein : priming of minus strand DNA synthesis
- Reverse transcriptase : DNA syntesis
- RNAseH : degradation of pgRNA

Hepadnaviral polimerase are strictly template spesific. It requires binding of the polypeptide to the packaging signal, termed epsilon, located at the 5' end of pgRNA.
Priming activity of Pol also requires cellular factor
it is dependent on chaperones including heat shock protein 90 (Hsp90), Hsp70, and p23. HBV replication is sensitive geldamicin (bind to N terminal of Hsp90) and novabiocin (bind to C terminal of Hsp90)

Structural information about the polymerases is lacking.

single polymerase polypeptide catalyzes one complete round of DNA replication.
1 virion of DNA = -0,7 polymerase molecules


Hb precore (H12)
HBeAg is a small secretory antigen that can cross the placenta from the mother to the fetus

preC/pre core = 15 KD
HbeAg = 161aa
- not have role in viral replication
- regulation in immune response against core protein

how to produce (H1)
. The HBeAg ORF encodes an endoplasmic reticulum (ER)
targeting sequence that co-translationally traffics the peptide to the ER, where the protein is processed to the final 15 kD HBeAg that is secreted from HBV-infected cells. Post translantional processing

structure  (H1)

clinical aspectH1
HBeAg is an important marker of HBV replication, and the levels of serum HBeAg are generally considered to correlate with viral titer. In fact, HBeAg seroconversion is considered an important aspect of the transition to the inactive carrier state of infection

Precore- second product
- a signal sequence directs the translation of HbeAg to ER membrane
- mengalami pemotongan 34 aa dari C terminus sebelum disekresi dari sel sebagai 15 Kd protein
- peranan yang mungkin : suppress immune response to the virus

Avihepadnaviruses:
-80 aa longer
Precore- second product
- a signal sequence directs the translation of HbeAg to ER membrane
- mengalami pemotongan 34 aa dari C terminus sebelum disekresi dari sel sebagai 15 Kd protein
- peranan yang mungkin : suppress immune response to the virus

Surface 

L, M, S, HbsAgL, M, S, HbsAg
Li and colleagues (Yanetal.,2012) used the myristylated preS1-derived peptide to isolate receptor
candidates expressed in primary hepatocytes cultures derived from tree shrews ,which can beinfected with HBV and HDV. They identified the bile acid transporter, sodium taurocholate cotransporting polypeptide (NTCP) and demonstrated that it is the bona fide
HBV/HDV receptor . NTCP is expressed on the basolateral membrane of hepatocytes, which is exposed to the space of Disse and separated from the blood stream by a fenestrate dendothelium

preS/SS = 24-27 KD
M = 31 KD (10-15%)
L = 42 KD (1-2%)

structureH1

HBV encodes three envelope proteins, or surface antigens, that make up the viral envelope: large (L), middle (M), and small (S) surface antigen [Figure 1a and c]. The smallest envelope protein, or S (24 kD), is 226 amino acids (aa) in length and makes up a shared C-terminal region of the two longer envelope proteins. The M protein (31 kD) contains the S sequence with a 55aa N-terminal extension known as preS2. Expression of the M- and S-encoding mRNA is driven by the S promoter, with translation initiating from an upstream (M) or downstream (S) AUG. The L protein (39 kD), the largest of the envelope proteins, contains S, preS2, and an additional 108aa or 119aa (depending on the genotype) N-terminal extension known as preS1. L-HBsAg is encoded by its own mRNA transcript that is controlled by the preS1 promoter.

EnvelopeFungsi dari protein L, M, S:
1. komponen dari virus envelope
2. membentuk subviral particles

contain 2 topogenic signal I and II that determine their orientation in lipid bilayers.

potongan L dan M ditemukan dalam liver pasien infeksi kronis
L myristate group st gly 2

has 2 conformations:
1. N terminus is located in the cytosol, required for binding capsid and assembly of virus.
2. N terminus is present in the ER lumen, exposed on the surface of viral particles, where it play a role in the infection of hepatocytes)

Perubahan konformasi difasilitasi oleh molekul chaperon : Hsc70/Hsp40 and Bip

paling penting : N terminal 55 aa of L

L tertahan di dalam membran kalau disintesis tanpa kehadiran S dan M
dapat trans aktivasi beberapa promoter
akumulasi L dapat menyebabka e stress sehingga meningkatkan ekspresi M dan S.
L export process controlled by its S domain. S dan M contai the necessary signals

M is N terminally acetilated
the role of M in replication is not yet understood
it is not required for the production of Dane particles

S2 topogenic signal
signal I : is located in N terminus
signal II : acting as a stop transfer sequence, and signal sequence

G28
Pre-S1 : cccDNA regulator
Pre-S2 : fusion sequence of the virus

H2
The distribution of the three envelope glycoproteins varies among the types of viral particles, with little or no L and M protein in the 20-nm particles but relatively more L protein in the Dane particles.


how to works (H1)
N-terminal signal sequence initiates insertion of S into the ER membrane,
. This orientation forms two loops; one loop, between aa 23–79, remains on the cytosolic side, while the other loop, between aa 99–169, remains in the ER lumen.
A major characteristic of L is that it exists in two conformations that vary in the localization of the N-terminal domain. In the first conformation of L, the preS1 and preS2 domains are present in the cytosol. This conformation of L is essential for binding of capsids and for the assembly of HBV virions. In the second conformation of L, the N-terminus is located in the ER lumen and, as a result, exposed on the surface of viral particles.
The conformational change is facilitated by interactions of molecular chaperones Hsc70/Hsp40 and BiP with L

functionH1
1. The main function of the surface antigen proteins is to form the HBV envelope.
2. In addition, an HBV-infected cell produces non-infectious subviral particles (SVP)
made primarily of S-HBsAg containing varying (but much lower) amounts of M-HBsAg and little to no L-HBsAg. These SVPs can reach a concentration 10,000-fold higher than infectious HBV particles in the serum of an infected individual.[44, 45]
SVPs are produced in two forms: 25 nm spheres, which are almost exclusively made up of S-HBsAg, and 22 nm filaments, which are made up primarily of S-HBsAg, with some M-HBsAg and potentially small amounts of L-HBsAg.
SVPs act to divert neutralizing antibodies away from infectious particles and that SVPs play a role in inducing the immune tolerance required to sustain a long-term chronic infection. A study of DHBV SVPs showed that the SVP-to-infectious-particle ratio plays a role in determining the efficiency of hepatocyte infection,

Hbx H10

XDHBV express X like protein
soluble cytoplasmic protein
short half life : 15-20 minutes
X is required for efficient infection in vivo
exact role is not known
154 aa

soluble cytoplasmic protein

also found in cytoskeleton and nucleus

HBx is also required for transcription from cccDNA in HepaRG cells and most likely in HepG2cells infected with HBV (Lucifora et al.,2011;SeegerandSohn,2014). However, the exact mechanism of HBx action is still obscure .Evidence for direct binding of X to the mini -chromosome has been obtained with chromatin immuno-precipitation experiments ( Bellonietal.,2009). However, the best-
documented interaction of X is with DNA damage-binding protein1 (DDB1) and the cullin4A-RINGDDB1-ubiquitin ligase

G28
Hbx : pleiotropic effect
encoding the polymerase promoter
structureH1
It is a 154aa, 17 kD protein that is encoded by the smallest HBV ORF.

functionH1
HBx plays an essential role during HBV replication. Specifically, studies have shown that HBx is bound to cccDNA,[73] that HBx is required for transcription from
cccDNA
This indicates that while HBx may not be absolutely required for HBV replication
in these systems, it undoubtedly enhances the levels of replication. Moreover, studies of direct HBV infection of mice with humanized livers demonstrated that only infection with wild type HBV, and not HBx-deficient virus, could result in HBV replication
While HBx is generally considered to have oncogenic potential, it is yet to be determined if it is directly oncogenic or simply acts as a co-factor in HCC development, as both effects have been demonstrated in different HBx-tg mouse models.
Avian hepadnaviruses, which do develop a chronic infection but do not cause HCC, either do not express an X protein or express a highly divergent form
HBx can modulate calcium (PyK2/FAK --> Src family kinase),[84, 85] apoptosis,[83, 86] and proliferation signals,
among other pathways, and can activate numerous transcription factors, including activator proteins 1[94] and 2[95] (AP-1 and AP-2), nuclear factor of activated T cells (NFAT),[96] and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).[97–99] HBx can also regulate cellular signaling factors, such as Wnt/β-catenin,[100] p53,[101] and Akt,[86, 102] that have been implicated in HCC
The presence of HBx in these cells could mean that HBx might be active in these HCC cells, even in the absence of replicating HBV, and potentially contribute to HCC development or maintenance.


HBxis required for efficient and replication in vitro
difficult to elucidate because interact with many cellular factor

functions :
- regulation of gene expression, relatively weak transactivator : NFkb, ap-1, ap-2, c/EBP, ATF/CREB, NFAT
- inhibition of DNA repair :inhibit NER, through binding to DDB1
- activation of several signal transduction

HbX bind to p53, RBP5, ERCC2

no mouse model to investigate the HBx in a viral replication during natural infection.
with primary cultures, there is evidence for a role of Hbx in promoting progression of the cell cycle from G0 to G1.