Hepadnaviruses - Hepadnaviridae

1. Hepadnaviruses - Hepadnaviridae Virion Genome Genes and proteins Viruses and hosts Diseases Distinctive characteristics

2. Hepadnaviruses - Hepadnaviridae • Virion • Spherical enveloped particle. • Diameter 42 nm. • Icosahedral capsid, T=4. • Abundant smaller spherical and filamentous forms lack nucleocapsid and are not infectious.

3. Hepadnaviruses - Hepadnaviridae • Genome • Circular dsDNA with a ss gap on one strand, 3.2 Kb.

4. Hepadnaviruses - Hepadnaviridae • Genes and proteins • mRNAs transcribed by cellular RNA polymerase II from five promoters. • Four overlapping reading frames, seven viral proteins. • Three surface proteins: LS, MS, SS. • Core proteins: C, E. • Polymerase (reverse transcriptase) protein: P. • Regulatory protein: X.

5. Hepadnaviruses - Hepadnaviridae • Viruses and hosts • Hepatitis B viruses of human, chimpanzee, duck, gibbon, gorilla, ground squirrel, heron, orangutan,snow goose, woodchuck, woolly monkey.

6. Hepadnaviruses - Hepadnaviridae • Diseases • Hepatitis: incubation period 30–180 days. • Transmission by blood and sexual contact. • Acute disease can be mild or severe. • Chronic or associated diseases: cirrhosis, liver cancer, serum sickness. • Treatment: interferon, lamivudine. • Prevention: vaccination with recombinant hepatitis B surface antigens; injection with anti-hepatitis B immunoglobulin after exposure to virus.

7. Hepadnaviruses - Hepadnaviridae • Distinctive characteristics • Unusual partly ss, partly ds circular genome results from incomplete replication in cell. • Pregenome RNA is both an mRNA and a template for synthesis of genome DNA. • Reverse transcriptase (polymerase protein) generates genome DNA from pregenome RNA within capsid during virus assembly. • Makes large amounts of noninfectious spherical and filamentous particles. • Has a satellite virus: hepatitis delta virus (see Chapter 29, viroids).

8. Virion • At least seven distinct viruses cause human hepatitis • The discovery of hepatitis B virus • 1963, unknown protein related to type B hepatitis • 1973, Virus-like particles in the serum of type B hepatitis patients (Dane particle) • 1979, DNA genome cloned and sequenced

9. Virion • Dane particles are infectious virions; abundant noninfectious particles lack nucleocapsids Fig. 28.1 Structure of virions of hepatitis B virus.

10. Virion • The viral genome is a circular, partly single-stranded DNA with overlapping reading frames • Partly double-stranded with a single-stranded region of variable length • Neither DNA strands forms a covalently closed circle • (-) strand joined at 5’ end to P protein • (+) strrand joined at 5’ end to short capped RNA • 5’ ends of (-) strand and (+) strand genomic DNA are joined by short complementary regions named DR2 • Four partly overlapped ORFs : C, P, S, X

11. Genome • The 5 ends of minus-strand (inside circle) and plus-strand (outside circle) genomic DNA are joined by short complementary regions named DR2 (gray boxes). The 5 end of the minus strand is attached to the viral polymerase protein (orange sphere) and the 5 end of the plus strand is attached to a short, capped RNA (black line). • DNA polymerase extends the plus strand, filling in the ss gap. • Nucleases remove the 5 ends of both strands. • DNA ligase joins the 3 and 5 ends of each strand, forming a fully covalently closed double-stranded circular DNA. Fig. 28.2 Structure of hepatitis B virus genome, and its conversion to a covalently closed, circular DNA.

12. Genes and proteins Fig. 28.3 Coding and signaling regions on the hepatitis B genome.

13. Genes and proteins • Nucleocapsids enter the cytoplasm via fusion and are transported to the nucleus • No currently available in vitro culture cell lines • Initial steps of virus entry are poorly understood • Cleavage of large hepatitis B surface Ag exposes fusion peptide, resulting in viral and host cell membrane fusion • Released nucleocapsid into cytoplasm is transported to nuclear membrane • Viral genome is converted within nucleus into covalently closed circular form by host enzymes

14. Genes and proteins • Transcription of viral DNA gives rise to several mRNAs and a pregenome RNA a. Circular DNA is transcribed by host RNA polymerase II, which recognizes five promoters (preC, pg, preS1, S, and X) to generate five classes of capped mRNAs that share a common 3 polyadenylated end. b. Open reading frames and translation products are shown above each RNA. pg RNA also serves as a template for virus replication. Fig. 28.4 Transcription of hepatitis B virus DNA.

15. Genes and proteins • The roles of hepatitis B virus proteins • X protein • Implicated in HCC • Up-regulate c-src, Ras/raf/MAPk, SAPK, PKC etc • Interact with and sequester p53 • Inhibit apoptosis • Surface proteins. • Involved in envelope formation • Core and E proteins. • Core : major component of necleocapsid • E protein : may suppress host immune system • Polymerase protein. • RNA/DNA-dependent DNA polymerase (reverse transcriptase) • Ribonuclease H

16. Genes and proteins • The pregenome RNA is packaged by interaction with polymerase and core protein • pgRNA serves as mRNA for C and P, and also used as template to produce DNA genome • 5’ e stem-loop is functional in packaging, and only in the pg RNA • P protein recognizes and interacts with the e stem-loop, initiating both encapsidation and reverse transcription of pg RNA

17. Genes and proteins • Genome replication occurs via reverse transcription of pregenome RNA • Step 1. Initiation of the reverse transcription process begins when P binds to the 5 stem-loop. • Steps 2 and 3. Reverse transcription progresses for only a few base-pairs beyond the 5 stem-loop, after which the enzyme and the newly synthesized DNA are translocated onto an identical sequence in the direct repeat region 1 (DR1) near the 3 end of the pregenome RNA.

18. Genes and proteins • Genome replication occurs via reverse transcription of pregenome RNA • Steps 4 and 5. The P protein then makes a complete (minus strand) copy of the remainder of the pregenome RNA by extending the DNA chain to the 5 end of the RNA template. • Steps 6 and 7. The translocated RNA fragment serves as a primer for subsequent synthesis of plus-strand DNA.

19. Genes and proteins Fig. 28.5 Reverse transcription of pregenome RNA to form hepatitis B virus genome DNA.

20. Genes and proteins

21. Genes and proteins Fig. 28.6 Schematic comparison of replication cycles of retroviruses and hepadnaviruses.

22. Genes and proteins • Virions are formed by budding in the endoplasmic reticulum Fig. 28.7 Assembly of nucleocapsids and virions of hepatitis B virus.

23. Genes and proteins • Hepatitis B virus can cause chronic or acute hepatitis, cirrhosis, and liver cancer • Hepatitis B virus is transmitted by blood transfusions, contaminated needles, and unprotected sex • A recombinant vaccine is available • Antiviral drug treatment has real but limited success • Interferon-a • Lamivudine (3T3) • Interfere with DNA polymerase activity of P protein

25. Apoptosis Ascites Cirrhosis Complement Enteric Epitope Exocytosis Fulminant hepatitis Hepatitis Hepatocellular carcinoma Immunoprophylaxis Interferons Jak-Stat pathway Jaundice Myristate Myristoylation NF-B (nuclear factor-B) Ras Ribonuclease H Serum sickness Src kinases Tumor necrosis factor (TNF) Tumor suppressor protein Key Terms