The expression of the PS fusion construct was drastically inhibited surface

The pre S1-specific domain of L contain ER retention signal that could override the secretary elements in the S domain. When L chains are overexpressed in the presence of M and S surface protein, the secretary ability of the M and S protein is inhibited in a dose-dependent manner. Although functional characterization of the pre S1 domain of L chain has mapped region essential for virion secretion that is localized in the C terminus of the pre S1 domain and extends into the adjacent amino acids 1 to 5 of the pre S2 domain, but their exact roles in surface particle secretion are unclear. As noted previously, since primary sequences were found to be greatly similar between both the L and the PS construct, first we performed to analyze the effect of the PS construct on Surface particle secretion among the several step of HBV life cycle. When the PS construct is overexpressed, the secretion of surface particle was largely inhibited in proportion to amounts of surface particle, that is, the increment of amount of surface antigen was more dramatically inhibited in PS construct than in PS-∆MS construct and the levels of surface particle secretion did not display the difference between PS construct and PS-∆MS construct. This result showed that the expression of the PS construct inhibits the secretion of surface particle and suggested that 29 amino acid adjacent to N-terminus of pre S1 region is dispensable on effect of surface particle.

Fig. 6. Effect of the PS construct for the surface protein secretions. Surface protein secretions from PS-∆MS (gray bar) (A) and PS (hatched bar) (B) constructs

transfected HuH7 cell. Amount of cotransfected construct are indicated at the bottom of the each figure. Supernatants derived from cotransfected cell were subjected to enzyme linked immunosorbent assay. Grapes are showed as the mean value of three experiments. (C) Endogycosidase H (EndoH) sensitivity analysis. The glycosylation of the PS fusion protein was tested by EndoH digestion. PS-∆MS transfected HuH7

cells at 2days posttransfection was lysed with RIPA and then immunoprecipitated with polyclonal anti-HBs antibody. Precipitated lysate was treated with EndoH (right lane) or without enzyme (left lane) and were subjected to 12% SDS-PAGE and then incubated with rabbit anti-TP antibody. HRP-conjugated secondary antibody and ECL were used to visualize proteins.

Fig. 6. Effect of the PS construct for surface protein secretion.

-P deficient-ΔLMS 2.3µg

µµµµ

gggg

-P deficient-ΔLMS 2.3µg

µµµµ

gggg

F. HBV DNA synthesis and core particle assembly were reduced and delayed by the expression of PS fusion construct

Previously many studies reported that various immune stimuli that alter transcription factor activities could negatively affect viral replication without affecting HBV transcription (Tang et al., 2001) and one of the akylated immuno Sugar was reduced nucleocapsid formation but not production of core protein. To determine whether the expression of PS construct influences both the capability of HBV DNA replication and the formation of core particle comprising the nucleocapsid and empty ones, we attempted to detect core particles by native agarose gel electrophoresis and Western blotting using the anti-HBc antibody. Core particles were detected from PS RNA-free plasmid transfected HuH7 cells but when we increased PS expression, the formation of core particle decreased gradually. To investigate effect of the PS on HBV DNA replication, cytoplasmic core particles were isolated from HuH7 cells transfected with construct of same set. Following extraction of HBV DNA from isolated core particles, Southern blot analysis and endogenous polymerase assay were performed to detect replicating HBV DNA.

When we increased PS construct expression, single-, double-stranded, and partially double-stranded relaxed circular forms of HBV DNA decreased gradually in dose dependent manner. A small amount of PS construct was inefficient but the increment of PS fusion RNA was negatively effective on HBV DNA replication and core particle formation. These results demonstrated that the PS construct could modulate HBV DNA replication and the formation of core particle.

Fig. 7. Effect of the PS construct on HBV replication. HBV polymerase is provided in trans (A) and in cis (B). Analysis of HBV core DNA mediated by the

PS-∆MS construct was performed by endogenous polymerase assay and southern blot

analysis. Amount of cotransfected construct are indicated at the top of the each figure.

Isolated core particles were incubated with EPA reaction buffer supplemented with 10 µCi of α-³²P-dATP (3000 Ci/mmol) and unlabeled dNTP mix at 37°C for overnight incubation, electrophoresed on a 1% native agarose gel, and subjected to autoradiography. Following EPA,³²P-labeled DNA was extracted, separated by 1%

agarose gel electrophoresis, and subjected to autoradiography. Isolated core DNA was resolved in agarose gel and transferred to nylon membrane and then hybridized with ³²P-labelled random-priming HBV specific probe. Single-, double-stranded, and partially double-stranded relaxed circular forms of HBV DNA are marked as SS, DS, and RC, respectively. Western blot analysis to detect core particles from a native agarose gel performed. Isolated core particles from cotransfected HuH7 cells were transferred to PVDF membranes and incubated with rabbit HBc antibody. HRP-conjugated secondary antibody and ECL were used to visualize proteins.

Fig. 7. Effect of the PS construct on HBV replication.

EPA

G. The expression of the PS construct modulate HBV life cycle by down-regulating the pgRNA but not the surface mRNA

Since HBV DNA DNA replication is dependent upon the formation of core particle, these process is subsequently dependent upon pg RNA levels, to investigate the effect of the PS construct on transcription of HBV RNA, the human hepatoma cell line Huh-7 was transiently co-transfected with the P deficient mutant, providing core protein and pgRNA but not all three surface proteins, Hpol-∆PS, proving polymerase in trans and do not occurring splicing event, and pcDNA3.1 was provided to tune final concentration of transfected DNA. We also constructed

pPB-∆PS produced polymerase, core and surface protein in cis. To detect and measure the levels of pgRNA, PS RNA and pre-S1 RNA expression upon the PS RNA gradually increased, we used an RNase protection assay. The design of the 470-nt probe was depicted in Fig. 6. Upon annealing of the probe to the three different HBV RNAs and digestion with RNase A and T1, protected fragments of 412, 235 and 148 nt, representing the pgRNA, preS1 RNA and PS spliced RNA, respectively, should be generated. Existence of the PS construct decreased amount of protected fragment of pgRNA. A 148 nt protected fragment of PS fusion RNA increased by adding the PS construct and at this time the level of preS1 RNA was not changed. This result also confirmed by northern blot analysis using the probe hybridized to a region common to all HBV transcripts. When the poly (A)⁺RNA isolated from the cell, this result was more evident than it presented in RPA procedure. These results demonstrated

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that the expression of the PS construct down-regulated the expression of pgRNA but not the surface mRNA regardless of providing of the HBV polymerase in trans or in cis. Furthermore these results suggest that the modulation of the life cycle of HBV mediated by the PS construct might be therapeutically beneficial.

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Fig. 8. Effect of the PS construct on HBV RNA transcription. (A) RNase protection analysis to detect HBV RNA. Total RNA from cotransfected HuH7 cells was hybridized to an antisense probe containing HBV sequence. Schematic diagram of antisense RNA and viral RNAs are indicated at bottom panel of figure 8 A. The heavy gray lines represent HBV sequence in the radiorabelled antisense probe or fragments that predicted to be protected by the different viral RNAs. The sizes of the protected fragments are indicated. Hybrids were digested with RNase A/T1. Yeast RNA was used as a reaction control. Amount of cotransfected construct are indicated at the top of the each figure. (B) Northern blot analysis to detect HBV RNA. Poly (A)⁺ mRNA was isolated using the oligo d (T) column and separated by 1%

formaldehyde gel electrophoresis, transferred to nylon membrane, hybridized with random-primed³²P labeled HBV specific probes, and subjected to autoradiography.

Each of HBV RNAs is marked. Spliced RNA is indicated as asterisk.

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Fig. 8. Effect of the PS construct on HBV RNA transcription.

A

-P deficient-∆LMS 2.3µg

PS-∆^MS

-P deficient-ΔLMS 2.3µg

PS-ΔMS

-P deficient-∆LMS 2.3µg

PS-∆^MS

-P deficient-ΔLMS 2.3µg

PS-ΔMS

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IV. DISCUSSION

In this study a spliced RNA species derived from transfected cells with HBV polymerase were detected regardless of the presences or absence of surface and X proteins. The combined evidence from RT-PCR, immunocytochemistry and immunoprecipitation analysis clearly demonstrates that a polymerase/surface (PS) RNA or protein is derived from RNA of the HBV polymerase construct as well as the pg RNA by deletion of the 450 bp fragment between 2447 and 2903. Amount of the PS RNA derived from HBV wt and core deficient mutant, which are provided as full length pg RNA, was remarkably decreased relative to amount of the PS RNA derived from various HBV polymerase mutant. First this result suggests an idea that several splice sites in the core gene may compete with the PS RNA because their site used to cellular splicing machinery. There is no specific transcript for polymerase protein and also previous studies of HBV showed that polymerase is not specific transcript for polymerase protein and polymerase is not synthesized as a capsid-polymerase fusion protein or by internal entry of ribosome. Second, this result suggest the possibility that since translation of core and polymerase protein is linked (Chen et al., 2005), the increment of the PS construct may decrease steady state levels of pg RNA. The potentiality of this hypothesis is guaranteed by later experiment in our study. Finally if polymerase of HBV carried mRNA of itself, the possibility exposure to splicing machinery may be likely to increase. Although the HBV resembles the retrovirus, evolutionally HBV may be carrying the bicistronic mRNA to achieve multiplication

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of viral genome. Another spliced species also have occurred splicing event using the same splice donor and acceptor site, which are already known to be occurring on the pgRNA. For HBV, 11 smaller than full-length genomes with single, double and triple splice RNAs were isolated and characterized so far from viral particles of sera from chronically infected patients (Terre et al., 1991; Rosmorduc et al., 1995; Gunther et al., 1997). However little is known about determinants governing the intracellular stability and translocation of HBV transcripts. Despite numerous well-conserved regions in the HBV genome that represent consensus sequences for splice donor and acceptor sites, neither a function for splicing in viral replication nor a role for splice transcripts has been firmly established (Chen et al., 1989; Su et al., 1989; Rosmorduc et al., 1995). Spliced L RNA described in DHBV showed to be functional in early steps of the viral life cycle and In the HBV, some reports demonstrated that a short region in the genome of HBV was critical for maintenance of 2.1 Kbp surface RNA levels (zu Putlitz et al., 1999) and 2.2 Kbp spliced RNA could enhance the replication of full-length HBV genome (Lin et al., 2002). Therefore biological function of each spliced RNA remains to be resolved. In this study one of them verified but the inter-correlation among the spliced RNAs need to be further studied.

Additionally, important point concerning for spliced RNAs were derived from their ability to encode for new viral proteins. Such viral proteins might play an important role in the viral life cycle, as demonstrated for other viruses. Until the now only two of these predicted proteins were reported and the distribution of them in transfected hepatoma cells was not directly demonstrated. Our results showed that

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the PS proteins were retained in ER like L protein. The PS proteins were distributed inside ER, close to nuclear lamina. The distribution of PS protein implied that myristoylation and 29 amino acids at N-terminus of preS1 domain in the L protein were not requisite for ER retention. This result was consistent with reports that the retention of L protein in the endoplasmic reticulum was mediated by a sequence containing a region of 35 amino acids of the preS1 C-terminus (Nemeckova et al., 1994). Although previous results have demonstrated that the PS fusion proteins could substitute for the LS protein for HBV maturation (Huang et al., 2000), exclusive retention of the PS protein implied that the possibility might be play another role in the viral life as well as a structural protein. In this study PS protein was co-localized with nuclear pore complex (NPC), intermediated filament vimentin and microtubule.

This result suggests that alternative spliced product; PS protein might be directly related to modulate gene regulation throughout nuclear/cytoplasmic transport machinery as Rev protein of HIV. The PS fusion protein exclusive binding to vimentin resembles to human immunodeficiency virus type-1 (HIV-1) virion infectivity factor (Vif) in various contexts (Lake et al., 2003). First, the Vif and the PS fusion proteins were connected between the cytoskeletal components based on co-localization of both proteins with vimentin. Second, the Vif and the PS fusion proteins were associated with Gag (Huang et al., 2000; Lake et al., 2003). Third, the Vif and the PS fusion proteins seemed to be modulating the viral infectivity. Even though the significance of the PS fusion-vimentin interaction remains unclear, this study extends these data to human hepatitis B virus.

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To date, several studies have provided controversial evidence that the domain(s) of viral surface proteins and the N-linked glycosylation implicated in the surface particle secretion of HBV (Mehta et al., 1997). There are known that the pre S1-specific domain of L protein contains signals that can override the secretory elements in the S protein and when L proteins are overexpressed in the presence of M and S chains, the ability of the M and S chains to be secreted is inhibited in a dose-dependent fashion (Ganem and Schneider, 2001). Previously short domains of pre S1 that were able to positively or negatively influence the release of surface proteins were identified by internal deletion (Le Seyec et al., 1999). Our data was shown that the PS fusion protein was N-linked glycosylated and the PS fusion RNA was drastically inhibited surface particle secretion. These results suggest that primary sequences of PS but not itself and glycosylation of the PS proteins were important for surface particle inhibitory activity.

An alkylated imino sugar with a galactose-based head group has anti-HBV and anti-bovine viral diarrhea virus activity (Block et al., 1998; Mehta et al., 2001;

Jordan et al., 2002). Various immune stimuli that alter transcription factor activities involved in regulating viral RNA synthesis can negatively affect viral replication without affecting HBV transcription (Tang et al., 2001). IL-4 suppressed that the production of the 2.4, 2.1 and 3.5-Kbp pg RNA, which not only encoded the core and polymerase proteins but also served as a template for reverse transcription, and then resulted in the inhibition of HBV replication (Lin et al., 2003). In this study, the step in the virus life cycle affected by the PS fusion RNA was explored. Since the

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alkylated imino sugar, n,n-DGJ, reduced the amount of replicative forms of HBV DNA detected in HepG2.2.15 cells mediated by inhibition of nucleocapsid formation or stability without apparently inhibiting either the viral polymerase or synthesis of viral envelope proteins (Lu et al., 2003), whether the reduction in HBV replication mediated by the HBV RNA is due to depletion of HBV nucleocapsid and reduction of 3.5 Kbp pg RNA was determined. Unlike presented above, these results suggest that the PS fusion RNA influence pg RNA level but not surface mRNA. Since the level of 3.5 Kbp pg RNA synthesis is approximately proportional to the level of viral replication in cell culture, the reduction synthesis of HBV DNA and formation of core particle was explained by reflecting the reduction of pg RNA. The down-regulation mechanism of pg RNA mediated by the PS fusion RNA further will be analyzed. In this study demonstrated that the PS fusion RNA is functionally essential for virus replication of HBV and suggest the possibility that the PS fusion RNA might be available as therapeutic target.

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V. CONCLUSION

Even though all protein of hepadnavirus constituted as viral component are translated from unspliced transcripts, spliced transcripts of HBV recently been identified in HBV DNA-transfected cell lines, in transgenic mice, in HBV-infected liver and in liver tissues of hepatocellular carcinoma patients. Little is known about determinants governing the functional role of HBV transcripts in their life cycle. In this study, the functional importance of the spliced RNA or protein in the HBV transfection system was investigated. Not only the spliced RNAs and spliced product of polymerase-surface (PS) fusion protein but also another spliced RNAs in HBV polymerase expressing cells were detected. In contrast to middle and small surface protein, the PS fusion protein partially distributed in part of endoplasmic reticulum and nuclear pore complex and also was associated with microtubule and vimentin. The secretion of surface particle, the replication of HBV DNA and the formation of core particle were drastically inhibited by the expression of the spliced RNA. These phenomenons were attributed to the primary sequence of the PS construct and the down-regulation to the expression of pgRNA. It suggested that the expression of the PS construct might modulate the HBV life cycles and suggested that the PS construct is functionally essential for virus replication of HBV and might be available as therapeutic target.

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