Herpatitis B virus Flashcards
How is the immune system reacting to HBV?
Firstly the immune reaction is severely delayed (months after infection) due to the virus being non cythopathogenic and the liver being an immuno privileged organ.
Immune pathology:
– Cytotoxic T cell response
– Natural killer cells
– Elimination of HBV-producing hepatocytes
– Cytokines (IFN-γ, TNF-α)
– Extrahepatic disease manifestation by immune complexes
• Arteritis, Vasculitis
What are the problems with HBsAg testing?
-genotypes of HBV react differently
-suboptimal sensitivity: best ELISA detects 10pg HBsAg/ml which is not reached in early infection stage
-HBsAg is highly variable when under immune pressure
→this could lead to a wrong negative result - infection over blood transfer etc possible
What are the options of prophylaxis and therapy are there agains HBV?
- vaccine: first development in 1981 -HBsAg purified from chronic infected patients - risk of infection over vaccine! Since 1986 recombinant vaccine from yeast
- Prophylactic passive immunisation: human anti-HBsAg Immunoglobulin
- Therapy: Pegylated interferon alpha, RT-inhibitors: Lamivudine (3 TC), Adefovir (ADV) and Entecavir - Problem: virus can become drug resistant
How was HBV discovered?
Blumberg tied to find a „aborigini-gen“ in Australia and isolated the viral genome - german Assistent was used as negative control, after a while the „aborigini-gen“ was detected in her samples as well
What are the structural components of HBV?
- Partial dsDNA with bound terminal protein and RT
- capsid
- 3 different surface proteins: SHB, MHB and LHB
HBV surface proteins also form filaments and spheres that are not infectious and can be used for vaccine development as they are better recognised by Immunsystems due to their size compared to sigle protein.
What are the genomic characteristics of HBV?
The HBV genome is enormously condensed. Has overlapping readingframes
partially ds DNA:
complete (-)-strand (longer than 1 copy)
incomplete (+)-strand (gap)
circular, but not covalently closed: Relaxed Circular (RC)-DNA (vs. cccDNA)
5 ́end of (+)-strand DNA covalently linked to viral RNA with Cap
5 ́end of (-)-strand DNA with covalently linked P-protein
incomplete (+)-strand, structure maintained by hybridization to complementary (+)-strand
each nucleotide has coding function
overlapping ORFs: > 50 % of bases encode in 2 different ORFs
all regulatory elements (promoters, enhancers, poly-A-signal, replicational-cis- elements) overlap with ORFs
ORFs are modular:
PreC/C, PreS1/PreS2/S > C and S modules are used in different proteins
Explain the replicative cycle of HBV?
- cell entry over endocytosis (NTCP receptor)
- genome amplification in nucleocapsid and genome release at nuclear pore
- conversion to cccDNA and mRNA synthesis
- sg(subgenomic)RNAs and pg(pregenomic)RNAs are ttranslocated to cytoplasms
- translation
- assembly of immature nucleocapsid with pgRNA, P-protein and core proteins
- RT-step → mature nucleocapsid
- another replicational cycle or virion assembly ER and Golgi and release
What is the specialty of the translation of the P protein?
All other transcripts have their own mRNA (only first cistron is translated), the p protein is encoded in the second cistron of the pgRNA
Exact mechanism of translation is unclear (Theories: leaky scanning, reinitiation, shunting)
P protein: TP(terminal protein) - Spacer - Pol/RT - RNaseH
How is the packaging of pgRNA into immature nucleocapsid regulated?
RNA stem-loop (ε) at 5 ́end of pgRNA - serves as packaging signal recognized by the P-protein
- no pgRNA packaging without P-protein
- no pgRNA packaging without intact 5 ́end
What are the steps of initiation and (-) strand DNA synthesis of HBV?
- Initiation: RT binds to epsilon RNA stem loop of pgRNA and synthesises 3 bp recombinant to UUC sequence in 3’ end DR1. - priming of (-)DNA synthesis. First template switch: RT transfer to 3‘ end DR1 and primer annealing
- (-)DNA synthesis: elongation of (-)DNA to 5‘ end and degradation of RNA (Template) by RNaseH domain of P protein with the exception of the 5‘ end including DR1 witch serves as primer for (+)DNA synthesis.
3a. (10%) (non productive way): (+)DNA synthesis: Remaining 5 ́- end fragment of pgRNA serves as primer for (+)-DNA synthesis - ds linear DNA only side product
3b. (90%) (productive way): 2. Template switch: RNA primer transfer from DR1 to DR2 and elongation towards 5‘ end of (-)DNA. 3. Template switch: (+)-DNA 3 ́-end of (-)-DNA 5 ́R to 3 ́R
- relaxed circularisation - elongation of (+)strand DNA
What is the driving force for template switching?
Thermodynamics of strand interactions = genome gymnastics
- Template switch: Formation of stemloop in DR1 in (-) DNA „truncates“ sequence complementary to primer - longer complementary sequence now in DR2 more stable base pairing with DR2.
