HBV wt RH D750V
RT YMHA
TP Y65F
C def
IV. DISCUSSION
Cytoplsmic MT has been evolved for intracellular trafficking of vesicles, organelles, and proteins. Also it has been known that MT are involved in the transport of many viruses (Fuchs and Yang, 1999; Ploubidou and Way, 2001; Sodeik, 2000). Intermediate filaments are needed for mechanical integration of the cytoplasm in higher eukaryotes, and impart intracellular mechanical strength. Several studies reported that vimentin, one of the components of intermediate filament, was shown to interact with viruses or viral proteins and be reorganized in virus replication process (Birkenbach et al., 1989; Chen et al., 1986; Lake et al., 2003; Mora et al., 1987; Nedellec et al., 1998). Although early steps of hepadnavirus infection and viral trafficking are poorly characterized, recent study reported that cytoplasmic trafficking of DHBV viral particles and emergence of nuclear cccDNA require MT for specific time periods during entry (Funk et al., 2004). From these backgrounds, it is presumed that HBV also use host cytoskeleton for movement of core particles through host cytoplasm after core particle assembly as well as during entry for infection.
The HBV virion release from hepatocyte must be tightly regulated events. The current dogma is that the only core particles with mature hepadnavirus genome are preferentially exported from the intracellular compartment (Gerelsaikhan et al., 1996;
Perlman and Hu, 2003; Wei et al., 1996). This means that core particles with mature
genome are ready for envelopment. This readiness for envelopment may be applied to nuclear targeting for core particle recycling. And previous report showed the differences in nuclear import and genome release between the immature and the mature core particles (Rabe et al., 2003).
In this study, four different replicative stages of core particles were established.
Each replicative stage is the encapsidation stage of core particles which contain pgRNA only, the initiation stage of core particle which contain pgRNA and short oligomer of minus strand DNA, the elongation stage of core particles which contain minus-strand DNA and pgRNA hybrid, and the mature stage of core particles which contain relaxed circular DNA genome. With series of mutants arrested at different DNA maturation stages of core particles, intracellular trafficking of core particles was investigated focusing on retrograde movement for core particle recycling.
Core particles were observed predominantly in cytoplasm, regardless of replication stages in this study. Predominant cytoplasmic expressions of HBV core particles are observed in actively proliferating (Ozer et al., 1996; Serinoz et al., 2003).
The experimental system in this study is actively proliferating HuH7 hepatoma cell line in which HBV was expressed and replicated. Thus this may explain predominant cytoplasmic distribution of core particle.
From series of replicative stages of core particles, only core particle in late stages of HBV DNA replication with completed minus-strand DNA or relaxed circular DNA genome, can co-localize with MT, vimentin and NPC of host cells.
transport through cytoplasm and may bind to NPC for nuclear import. From these results, the model for retrograde transport of core particles can be proposed. In this model, HBV core particles with mature genome or completed minus-strand DNA interact with MT and move toward nucleus by MT mediated active transport mechanism. Since MT network is originated from MT organizing center (MTOC) in peri-nuclear region, HBV core particle in peri-nuclear region may use another way, intermediate filament, to move to NPC. Then, core particles associate with NPC to enter nucleus.
In addition, core particles with completed minus-strand DNA can be speculated to be ready for nuclear targeting like HBV wt mature core particles with relaxed circular DNA genome, since the core particles with pgRNA-minus-strand DNA hybrid showed similar intracellular localization like wt. This study shows the intracellular transport of RNase H mutant core particles. The result is consistent with previous report that RNase H mutant virus was secreted as virions (Gerelsaikhan et al., 1996; Perlman and Hu, 2003). Taken together, it is believed that core particles undergo some distinctive changes from immature core particle to mature core particle after minus-strand DNA synthesis is completed to move back into nucleus or to get envelope from intracellular membrane.
The factors for different phenotype between immature and mature core particles are still ambiguous. However, from duck hepatitis B virus (DHBV) and HBV model, core particle trafficking to release genome into nucleus is regulated by phosphorylation of core particle (Kann et al., 1999; Kock et al., 2003). Since genome
maturation is correlated with core particle phosphorylation (Rabe et al., 2003), core particle phosphorylation status is probably one of the factors for different trafficking while DNA replication proceeds.
This study showed different intracellular trafficking of HBV core particles and the involvement of cytoskeleton in core particle trafficking or the targeted transfer to nucleus according to DNA genome maturation stages of core particle, with series of replicative stages of core particles.
V. CONCLUSION
In this study, the differences in intracellular trafficking of HBV core particle between DNA genome maturation stages were investigated. To accomplish this goal, immature and mature core particles are subdivided into four stages: the encapsidation stage in which core particles contain pgRNA only, the initiation stage of minus-strand DNA synthesis with short oligomer of minus-minus-strand DNA, the elongation stage of minus-strand DNA synthesis in which core particles contains minus-strand DNA and pgRNA hybrid, and the final stage of HBV DNA replication with mature core particles with relaxed circular DNA genome. With this core particles, which cytoskeleton involved in core particle trafficking or which stage of core particle can target nucleus was examined.
As a result, the core particles in late stages of HBV DNA replication with completed minus-strand DNA or relaxed circular DNA genome are co-localized with MT, vimentin and NPC. These results suggest that HBV core particles use MT and intermediate filament to move through host cytoplasm, and can interact with NPC for nuclear transport. From these results, it is reasonable to speculate that HBV core particles with mature genome or completed minus-strand DNA can interact with MT and move toward nucleus by MT-mediated active transport mechanism. To move toward nucleus from peri-nuclear region, MTOC, core particles may use intermediate filament and then associate with NPC to enter nucleus. In addition, core particles
with completed minus-strand DNA can be speculated to be ready for nuclear targeting like HBV wt mature core particles with relaxed circular DNA genome, since the core particles with pgRNA-minus-strand DNA hybrid showed similar intracellular localization like wt.
REFERENCES
1. Bailey CJ, RG Crystal, PL Leopold: Association of adenovirus with the microtubule organizing center. J Virol 77:13275-13287, 2003
2. Bartenschlager R, M Junker-Niepmann, H Schaller: The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol 64:5324-5332, 1990
3. Bartenschlager R, H Schaller: Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. Embo J 11:3413-3420, 1992
4. Beck J, M Nassal: Formation of a functional hepatitis B virus replication initiation complex involves a major structural alteration in the RNA template. Mol Cell Biol 18:6265-6272, 1998
5. Birkenbach M, D Liebowitz, F Wang, J Sample, E Kieff: Epstein-Barr virus latent infection membrane protein increases vimentin epression in human B-cell lines. J Virol 63:4079-4084, 1989
6. Blum HE, E Galun, TJ Liang, F von Weizsacker, JR Wands: Naturally occurring
missense mutation in the polymerase gene terminating hepatitis B virus replication. J Virol 65:1836-1842, 1991
7. Chang LJ, RC Hirsch, D Ganem, HE Varmus: Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase. J Virol 64:5553-5558, 1990
8. Chen M, R Goorha, KG Murti: Interaction of frog virus 3 with the cytomatrix. IV.
Phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites. J Gen Virol 67:915-922, 1986
9. Fuchs E, Y Yang: Crossroads on cytoskeletal highways. Cell 98:547-550, 1999
10. Funk A, M Mhamdi, L Lin, H Will, H Sirma: Itinerary of hepatitis B virus:
Delineation of restriction points critical for infectious entry. J Virol 78:8289-8300, 2004
11. Ganem D, RJ Schneider: Hepadnaviridae: The viruses and their replication. In Fields Virology 4th ed (ed. P. M. Howley) Philadelphia, Lippincott-Raven Publishers, pp. 2923-2969, 2001
does not occur without genomic DNA synthesis. J Virol 70:4269-4274, 1996
13. Gilbert JM, IG Goldberg, TL Benjamin: Cell penetration and trafficking of polyomavirus. J Virol 77:2615-2622, 2003
14. Hirsch RC, JE Lavine, LJ Chang, HE Varmus, D Ganem: Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as wel as for reverse transcription. Nature 344:552-555, 1990
15. Jeong JK, GS Yoon, WS Ryu: Evidence that the 5'-end cap structure is essential for encapsidation of hepatitis B virus pregenomic RNA. J Virol 74:5502-5508, 2000
16. Kann M, B Sodeik, A Vlachou, WH Gerlich, A Helenius: Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex.
J Cell Biol 145:45-55, 1999
17. Kelkar SA, KK Pfister, RG Crystal, PL Leopold: Cytoplasmic dynein mediates adenovirus binding to microtubules. J Virol 78:10122-10132, 2004
18. Kim H-Y, G-S Park, E-G Kim, S-H Kang, H-H Shin, S Park, KH Kim: Oligomer synthesis by priming deficient polymerase in hepatitis B virus core particle.
Virology 322:22-30, 2004
19. Kock J, M Kann, G Putz, HE Blum, Fv Weizsacker: Central role of a serine phosphorylation site within duck hepatitis B virus core protein for capsid trafficking and genome release. J Biol Chem 278:28123-28129, 2003
20. Koschel M, D Oed, T Gerelsaikhan, R Thomssen, V Bruss: Hepatitis B virus core gene mutations which block nucleocapsid envelopment. J Virol 74:1-7, 2000
21. Lake JA, J Carr, F Feng, L Mundy, C Burrell, P Li: The role of Vif during HIV-1 infection: interaction with novel host cellular factors. J Clin Virol 26:143-152, 2003
22. Lanford RE, L Notvall, H Lee, B Beames: Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase. J Virol 71:2996-3004, 1997
23. Mora M, K Partin, M Bhatia, J Partin, C Carter: Association of reovirus proteins with the structural matrix of infected cells. Virology 159:265-277, 1987
24. Nedellec P, P Vicart, C Laurent-Winter, C Martinat, MC Prevost, M Brahic:
72:9553-9560, 1998
25. Ogawa-Goto K, K Tanaka, W Gibson, E Moriishi, Y Miura, T Kurata, S Irie, T sata: Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid. J Virol 77:8541-8547, 2003
26. Ozer A, VI Khaoustov, M Mearns: Effect of hepatocyte proliferation and cellular DNA synthesis on hepatitis B virus replication. Gastroenterology 110:1519-1528, 1996
27. Perlman D, J Hu: Duck hepatitis B virus virion secretion requires a double-stranded DNA genome. J Virol 77:2287-2294, 2003
28. Ploubidou A, M Way: Viral transport and the cytoskeleton. Curr Opin Cell Biol 13:97-105, 2001
29. Rabe B, A Vlachou, N Pante, A Helenius, M Kann: Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci USA 100:9849-9854, 2003
30. Radziwill G, W Tucker, H Schaller: Mutational analysis of the hepatitis B virus P gene product: domain structure and RNase H activity. J Virol 64:613-620, 1990
31. Roychoudhury S, AF Faruqi, C Shih: Pregenomic RNA encapsidation analysis of eleven missense and nonsense polymerase mutants of human hepatitis B virus. J Virol 65:3617-3624, 1991
32. Ruby-Phelps K: Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol 192:189-221, 2000
33. Serinoz E, M Varli, E Erden, K Cinar, A Kansu, O Uzunalimoglu, C Yurdaydin, H Bozkaya: Nuclear localization of hepatitis B core antigen and its relation to liver injury, hepatocyte proliferation, and viral load. J Clin Gastroenterol 36:269-272, 2003
34. Sodeik B: Mechanisms of viral transport in the cytoplasm. Trends in Microbiol 8:465-472, 2000
35. Tavis JE, D Ganem: RNA sequences controlling the initiation and transfer of duck hepatitis B virus minus-strand DNA. J Virol 69:4283-4291, 1995
36. Tavis JE, D Ganem: Evidence for activation of the hepatitis B virus polymerase by binding of its RNA template. J Virol 70:5741-5750, 1996
37. Tavis JE, B Massey, Y Gong: The duck hepatitis B virus polymerase is activated by its RNA packaging signal, epsilon. J Virol 72:5789-5796, 1998
38. Tavis JE, S Perri, D Ganem: Hepadnavirus reverse transcription initiates within the stem-loop of the RNA packaging signal and employs a novel strand transfer. J Virol 68:3536-3543, 1994
39. Wands J, H Blum: Primary hepatocellular carcinoma. N Engl J Med 325:729-731, 1991
40. Weber M, V Bronsema, H Bartos, A Bosserhoff, R Bartenschlager, H Schaller:
Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription. J Virol 68:2994-2999, 1994
41. Wei Y, JE Tavis, D Ganem: Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses. J Virol 68:6455-6458, 1996
42. Whittaker GR, M Kann, A Helenius: Viral entry into the nucleus. Annu Rev Cell Dev Biol 16:627-651, 2000
43. Zoulim F, C Seeger: Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase. J Virol 68:6-13, 1994
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