1 Department of Medical Molecular Biology, University of Lübeck, Ratzeburger Allee 160, D-23562 Lübeck, Germany
2 Department of Medical Microbiology, Department of Virology, Basel, Switzerland
Correspondence
Yuri Y. Kusov
koussov{at}molbio.uni-luebeck.de
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ABSTRACT |
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MAIN TEXT |
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There is increasing evidence that the biological properties of the hepatitis A virus (HAV) are unique among those of the picornaviruses. HAV replication in cell culture is slow, persistent and highly asynchronous, and little is known about the rate-limiting steps in the virus life cycle. To gain further insight into the regulatory steps of HAV replication, we analysed the role of the cell cycle and the length of the viral 3' poly(A) tail.
To assess the role of the 3' poly(A) tail of the HAV genome, we examined the replication efficiency of HAV RNA with and without a poly(A) tail in quiescent cells. In vitro transcripts were prepared from linearized plasmids (Kusov & Gauss-Müller, 1999; Kusov et al., 2001
) that encoded the genome of HAV strain 18f with a tail of 14 (18f-A14) or no (18f-A0) adenosine residues, followed by the genomic ribozyme of hepatitis
virus (HDV). The 3' product of the HDV ribozyme was shown to serve as substrate for polyadenylation (Düvel et al., 2003
). After RNA transfection into human hepatoma cells (Huh-7) that were rendered quiescent by serum starvation (Feuer et al., 2002
), virus replication was assessed by RT-PCR amplification of a 0·3 kb fragment within the 5' NTR (Kusov et al., 2001
) and by viral-particle identification using a particle-specific ELISA (Kusov & Gauss-Müller, 1999
). RNA 18f-A0 was non-infectious, as neither viral particles (Fig. 1a
, open circles) nor infectious virus (data not shown) was recovered [see also Kusov et al. (2001)
]. In contrast, HAV RNA with a poly(A) tail of 14 residues was infectious, with viral antigen detectable approximately 14 days post-transfection (p.t.) (Fig. 1a
, filled circles). Input RNA 18f-A0 was demonstrable by RT-PCR (day 0) and became undetectable at 6 days p.t. (Fig. 1b
, upper panel). In contrast, RNA 18f-A14 was detectable for 3 days, proving its enhanced stability in vivo (Fig. 1b
, lower panel). No or very small amounts of RT-PCR products, detectable on days 6 and 9 p.t., indicated loss of input RNA below the detection limit and a delay of de novo RNA synthesis. Newly synthesized viral genomes of RNA 18f-A14 were found 15 days p.t. The poly(A)-tail length of the rescued virus was determined by a method described previously and consisted of about 60 residues, a tail length similar to that of HAV isolated from infected patients or cell culture (Siegl et al., 1981
; Kusov et al., 2001
, 2002
). The results implicate that, possibly owing to its reduced stability, the tailless HAV RNA was unable to initiate genome replication in quiescent cells.
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For cytolytic picornaviruses (e.g. poliovirus and coxsackievirus), it was shown that the cell-cycle status affects virus replication, persistence and reactivation (Eremenko et al., 1972; Feuer et al., 2002
). Moreover, stationary and dividing somatic cells differ in their content of factors that regulate the poly(A)-tail length (Kazazoglou et al., 1987
; Colgan et al., 1996
; Mendez & Richter, 2001
). These reports and our observations (Fig. 1c
, open circles) that the tailless vector-encoded viral genome was infectious prompted us to study the role of cell division in the infectivity of HAV transcripts lacking a poly(A) tail. In contrast to the experiment shown in Fig. 1(a)
, RNAs 18f-A0 and 18f-A14 were transfected into Huh-7 cells that were actively dividing by passaging them at 1 day p.t. and then every 3 days. The data show clearly that RNA 18f-A0 (Fig. 2a
, open circles) was able to produce increasing amounts of viral particles, albeit with delayed kinetics compared to those of RNA 18f-A14 (filled circles). Compared to quiescent cells (Fig. 1a
), replication of RNA18f-A14 was faster in dividing cells. The poly(A)-tail length of viral isolates originating from RNA 18f-A0 transfection and rescued 30 days p.t. was found to be 3660 residues, as determined by the G-tailing method described previously (Kusov et al., 2001
). The replication-defective mutant RNA 18f-A0-mut was unable to initiate an infectious replication cycle (Fig. 2a
, open triangles).
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Final evidence for HAV genome replication after RNA transfection was achieved by FISH detection of minus-strand RNA with a strand-specific riboprobe. Minus-strand RNA yielded a less strong signal than plus-strand RNA and was detectable only late after transfection in 26 % of cells (Fig. 2c, right panels; Fig. 2b
, grey and black columns, dimensions on the right-hand axis). Cells transfected with RNA 18f-A0-mut or mock-transfected cells were negative in all tests (Fig. 2b, c
; data not shown). Thus, HAV RNA 18f-A0 is replication-competent when transfected into actively dividing cells, suggesting that the cell cycle might affect HAV replication.
Based on the observation that HAV replication is affected by host-cell replication, we next studied virus replication in cells arrested at various steps of the cell cycle. We employed widely used cell-cycle inhibitors that were applied, for example, in a study on coxsackievirus B3 replication (Feuer et al., 2002). To determine HAV genome replication independently of its uncoating and packaging, RNAs of the HAV replicon (pT7-18f-Luc) and its replication-deficient mutant 18f-Luc-mut, both containing a poly(A) tail of 20 residues, were transfected into arrested Huh-7 cells (Gauss-Müller & Kusov, 2002
). Expression of the reporter gene 18 h p.t. was suppressed in cells arrested at the G0 phase and G2/M transition of the cell cycle, but was hardly affected in cells in the G1 and G1/S phases (Fig. 3a
). Similar effects were observed when the reporter activity of a replication-defective, yet translation-competent replicon carrying a mutation in the polymerase gene was analysed (data not shown). When the transfected and arrested cells were permitted to divide after the removal of inhibitors, reporter-gene expression was restored to normal levels, indicating that the limited protein expression is not due to irrevocable damage of the cells by the inhibitors (data not shown). Similar results were obtained when HAV infection was analysed (Kusov et al., 1996
). HAV replication, as detected by the accumulation of viral antigen, was reduced when the cells were arrested at the G0 phase and G2/M boundary, but not at the G1 or G1/S phases (Fig. 3b
). In conclusion, our data show clearly that cell division favours efficient HAV RNA translation and replication.
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ACKNOWLEDGEMENTS |
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Received 28 September 2004;
accepted 14 January 2005.
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