| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B activation
1 Department of Microbiology and Immunology, Chang Gung University, Tao-Yuan, Taiwan 333, Republic of China
2 Graduate Institute of Biomedical Sciences and Department of Life Science, Chang Gung University, Tao-Yuan, Taiwan 333, Republic of China
3 Institute of Microbiology and Immunology, National Yang Ming University, Taipei, Taiwan 112, Republic of China
Correspondence
Szecheng J. Lo
losj{at}mail.cgu.edu.tw
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
B is linked to the ER stress that induces GFP–LD translocation. Combining this with results showing that tumour necrosis factor alpha (TNF-
) can also induce GFP–LD translocation, it was concluded that LDAg translocation correlates with ER stress and activation of NF-B. Nevertheless, TNF--induced GFP–LD translocation was independent of new protein synthesis, suggesting that a post-translational event occurs to GFP–LD to allow translocation. A supplementary table showing the distribution pattern of GFP–LD in HuH-7 cells is available in JGV Online.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Although the genome structures of HDV and plant viroids share a common evolutionary origin, unlike viroids, which do not encode any proteins, HDV encodes two antigen isoforms, the small and large forms (SDAg and LDAg, respectively) during its replication cycle. The SDAg and LDAg share 195 aa at their N terminus, while the LDAg contains 19 extra aa at its C terminus (Weiner et al., 1988). SDAg is required for HDV RNA replication, whereas LDAg suppresses this process and interacts with hepatitis B virus (HBV) envelope proteins (HBsAg) to assemble a mature virion for secretion and infection (Kuo et al., 1989; Chao et al., 1990; Chang et al., 1991; Ryu et al., 1992).
Replication of the HDV genome and antigenome is dependent on host cell RNA polymerases and occurs via a rolling-circle method, which produces multiple copies of HDV RNA in a linear form (Modahl et al., 2000; Macnaughton et al., 2002). Ribozymes in both the genome and antigenome then self-cleave the linear RNA into single units, which are then ligated into a circular form by cellular ligases (Lai, 1995; Reid & Lazinski, 2000; Taylor, 2003). During the HDV replication cycle, host enzymes called ADARs (adenosine deaminases that act on double-stranded RNA) edit a portion of the HDV RNA to convert the amber stop codon (UAG) of SDAg to a tryptophan codon (UGG), which results in the production of LDAg (Casey & Gerin, 1995; Sato et al., 2001; Jayan & Casey, 2002). Thereafter, LDAg inhibits HDV RNA replication and then LDAg together with SDAg and the HDV genome assemble into a ribonucleoprotein (RNP) complex (Ryu et al., 1993), which is then transported to the cytoplasm to form a mature virion with the HBsAgs.
In addition to the host RNA polymerases and ADARs, many other cellular enzymes involved in the post-translational modifications of HDAgs are important for the execution of HDAg's function (Mu et al., 2004; Li et al., 2004; Lai, 2005). For example, farenyl-transferase is required for LDAg isoprenylation, which is crucial to the interaction with HBsAg for secretion (Glenn et al., 1992; Hwang & Lai, 1993; Sheu et al., 1996). Different kinases are also required for SDAg and LDAg phosphorylation because the SDAg is phosphorylated at both serine and threonine, while the LDAg is phosphorylated only at serine (Chang et al., 1988; Mu et al., 1999), which may account for their distinct functions. Furthermore, the serine residues at positions 2 and 177 of SDAg have been demonstrated to modulate HDV RNA replication but have no significant role in subviral particle formation (Yeh et al., 1996; Yeh & Lee, 1998; Mu et al., 1999, 2001). Recently, methylation at arginine-13 and acetylation at lysine-72 of HDAg have been reported to play an important role in virus replication (Mu et al., 2004; Li et al., 2004). These lines of evidence fully support the hypothesis that post-translational modifications of HDAgs can drive HDAgs to specific cellular compartments and functions (for a review see Lai, 2005). However, signals and mechanisms involved in HDAg post-translational modification have not yet been well studied.
Previously, we used a green fluorescent protein fused to LDAg (GFP–LD) to demonstrate that translocation of GFP–LD from the nucleolus to SC-35 speckles can be induced by treatment with the casein kinase II inhibitor, dichlororibofuranosyl benzimidazole (Shih & Lo, 2001), in which serine-123 of LDAg in the deposphorylated state is responsible for preferentially targeting to the SC-35 speckles (Tan et al., 2004). How cellular kinases and phosphotases are regulated in order to modify LDAg for translocation is still under question. We also demonstrated that the small form of HBsAg can facilitate the translocation of GFP–LD from the nucleus to the cytoplasm (Tan et al., 2004), but the mechanism remains unclear. In this study, we explore what signal molecules are generated by HBsAgs, which reside in the endoplasmic reticulum (ER) and can induce GFP–LD nuclear export.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and pN2LD encode authentic LDAg under the metallothionine (MT) and cytomegalovirus (CMV) promoter, respectively; (ii) pMTS, pMTMMS[S–] and pMTLS encode HBV small, middle and large (S, M and L) surface antigens, respectively (Sheu & Lo, 1994), and pN2LS encodes L HBsAg; (iii) those expressing different versions of HDAg fused to a GFP, pGFP–LD, pGFP–LDM, pGFP–LD(31–214) and pSD–GFP (Shih & Lo, 2001; Shih et al., 2004); and (iv) pIKK, which encodes the wild-type IB kinase subunit; pKA, which encodes a mutant form of IKK; and pNF-B-Luc, which contains the NF-B-response element (TGGGGACTTTCCGC)5 fused to a luciferase gene (kindly provided by Dr Y. S. Chang, Chang Gung University, Republic of China). The characteristics of the GFP fusion proteins encoded by the series (iii) plasmids are summarized as follows: (a) pGFP–LD encodes GFP fused to wild-type LDAg; (b) pGFP–LDM produces GFP fused to a non-isoprenylated mutant of LDAg; (c) pGFP–LD(31–214) produces GFP fused to an N-terminal deletion (aa 1–30) mutant of LDAg; and (d) pSD–GFP produces wild-type SDAg fused to GFP (Shih & Lo, 2001; Shih et al., 2004).
Cell culture and plasmid transfection.
Two human cell lines were used in this study; one is a well-differentiated hepatoma cell line, HuH-7, while the other is a cervical carcinoma cell line, HeLa. Most of the experiments were carried out using HuH-7 cells and a few were done with HeLa cells. Both cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum, penicillin (100 IU ml–1), streptomycin (100 µg ml–1), Fungizone (50 µg ml–1) and 2 mM L-glutamine, and grown at 37 °C under 5 % CO2. Plasmids in a supercoiled form were obtained using the Qiagen Plasmid Maxi kit and then used for transfection. Cells at 60–80 % confluence in a 10 cm Petri dish or six-well plate were transfected with 10–20 µg of the indicated plasmids by the calcium phosphate-DNA precipitation method (Graham & van der Eb, 1973) or by adding lipofectamine (Invitrogen). At 1 or 2 days post-transfection, cells were treated with various drugs for different time intervals: (i) tunicamycin (TM; 2–5 µg ml–1) or brefeldin A (BFA; 2–5 µg ml–1) for 2 h, and (ii) with or without pre-treatment with cycloheximide (10 µg ml–1) for 30 min and then followed by treatment with tumour necrosis factor alpha (TNF-; 10 ng ml–1) for 1 h.
Fluorescence microscopy.
To visualize the co-expression of HBsAgs and various forms of GFP fusion proteins, transfected cells were reseeded on 22x22 mm coverslips. After full attachment, cells were fixed and permeabilized using methanol for 30 min at –20 °C and then stained with anti-HBs antibody followed by the secondary antibody conjugated with rhodamine. In parallel, cells were stained with Hoechst 33258 for 10 min to show the nucleus. Finally, cells were mounted onto glass slides with mounting solution, and visualized using a fluorescence microscope (Olympus IX71) with a fluorescein isothiocyanate (FITC) or rhodamine filter. For quantification of the GFP fusion protein, the distribution of the nucleus only (N) versus the nucleus–cytoplasm (N+C) pattern was observed, and between 100 and 500 GFP-positive cells were randomly selected and their patterns classified as described previously (Tan et al., 2004). For simplicity in this study, type I, II and III distribution patterns were redesignated ‘N’ pattern, nucleus only, while the type IV was redesignated ‘N+C’, nucleus and cytoplasm.
Western blot analysis.
To detect the amount of GFP fusion proteins in the nucleus and cytoplasm, transfected cells were fractionated into nuclear and cytoplasmic fractions using a nuclear extraction kit (Active Motif), according to the manufacturer's instructions. Protein samples were separated by SDS-PAGE and then electro-transferred onto PVDF membranes. The membranes were incubated with 5 % non-fat milk for 1 h at room temperature for blocking and then reacted with anti-GFP, anti-HBsAg, anti-GRP78, anti-NF-B p65 or anti-tubulin antibodies (depending on the purpose of the experiment) in the presence of 5 % non-fat milk overnight at 4 °C. This was followed by incubation with the secondary antibody conjugated with horseradish peroxidase and the blots were then developed by enhanced chemiluminescence using a commercial kit (Amersham).
Luciferase activity assay.
Measurement of NF-B activity was carried out as described by Wu et al. (1998). Briefly, transfected cells were lysed using a buffer supplied in a commercial luciferase assay kit (Promega) according to the manufacturer's instructions. The luciferase activity of each cell lysate was determined by using an Autolumat LB953 luminometer (Berthold). All experiments were carried out in duplicate and repeated three times.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In this study, we first confirmed that HBsAg expression correlates with GFP–LD in the cytoplasm by co-transfection of pMTS and pGFP–LD into HuH-7 cells. A representative fluorescence microscopy image shows that three cells that have GFP appearing in an N+C pattern, while one cell has GFP located in the nucleus (N pattern) only [Fig. 1a(i) and a(ii)]. This particular N pattern cell had no HBsAg expression in contrast to the three N+C cells [Fig. 1a(iii)], indicating clearly that HBsAg can facilitate GFP–LD translocation to the cytoplasm. Consistently, the presence of authentic LDAg in the cytoplasm was highly correlated with the expression of HBsAg in HeLa cells [Fig. 1b(i), (ii) and (iii)], indicating that HBsAg can affect similarly on both GFP–LD and authentic LDAg. We then tested whether HBsAg can facilitate on other HDAg fusion proteins by performing single, double and triple plasmid transfection experiments, in which each of four plasmids [pGFP–LD, pGFP–LDM, pGFP–LD(31–214) and pSD–GFP] expressing a different version of HDAg fused to GFP was co-transfected with or without pMTS and pMTLD. As shown in Table 1, the percentage of N+C pattern cells for expressing GFP–LD (GFP fused to wild-type LDAg), GFP–LDM (GFP fused to a non-isoprenylated mutant of LDAg), GFP–LD(31–214) [GFP fused to an N-terminal deletion (aa 1–30) mutant of LDAg] or SD–GFP (wild-type SDAg fused to GFP) ranged from 1.1 to 5.1 % in single plasmid transfections. In the presence of HBsAg, the N+C pattern of GFP–LD cells increased to 26.5 %, while that of GFP–LDM, SD–GFP and GFP–LD(31–214) expressing cells increased ranging from 2.5 to 5.1 %, suggesting that HBsAg only facilitates the full-length of LDAg translocation to the cytoplasm but not the single point mutation, which cannot be isoprenylated (GFP–LDM), N-terminal deletion mutation [GFP–LD(31–214)] or SDAg. As compared with the result of double plasmid transfection, results of triple plasmid transfection showed 0.3 % or no increase of N+C pattern in cells expressing GFP–LD(31–214) and GFP–LD, while 13.5 and 12.9 % increase in cells expressing GFP–LDM and SD–GFP, respectively (see the last column of Table 1). A higher increasing percentage of N+C pattern in GFP–LDM and SD–GFP expressing cells as compared with that of GFP–LD(31–214) cells in the presence of HBsAg and LDAg suggested that LDAg can form a complex with GFP–LDM and SD–GFP via the coiled-coil domain and this allows them export to the cytoplasm, in contrast, the coiled-coil domain of GFP–LD(31–214) is truncated (Chang et al., 1992; Shih & Lo, 2001). No significant increase of N+C pattern in GFP–LD cells between double and triple plasmid transfections suggested that GFP–LD behaves similarly to LDAg under the effect of HBsAg.
|
|
). We were thus interested to see whether or not the three different forms of HBsAg exert different capability on the translocation of GFP–LD. Co-transfection of pGFP–LD with pMTS, pMTMMS[S–] or pMTLS, which express S, M and L HBsAg, respectively, into HuH-7 cells was performed. The N and N+C pattern of the GFP–LD cells were analysed at 24, 48 and 72 h post-transfection. As shown in Table 2, GFP–LD cells had an increased percentage of the N+C pattern when they were co-expressed with S, M and L HBsAg (10.4, 26.4 and 34.1 %, respectively) at 24 h post-transfection. A similar trend was also observed in the 48 h post-transfected cells (25.2, 36.3 and 47.0 %, respectively) as well as in the 72 h post-transfected cells (34.7, 47.8 and 56.5 %, respectively). Since the three forms of HBsAgs reside in the ER, it was speculated that the presence of HBsAg may induce ER stress and then in turn cause GFP–LD translocation. Western blot analyses were performed to test this supposition. Results clearly show that the GRP78 protein/BiP, an ER stress-induced marker, which functions as a protein chaperone at the ER cisternae (Schröder & Kaufman, 2005; Lee, 2005), was increased in the total lysate of cells co-expressing GFP–LD with S, M and L HBsAg (Fig. 2, lanes 3–5, bottom line) compared with cells that were mock transfected or expressed GFP–LD alone (Fig. 2, lanes 1 and 2, bottom line). For an unknown reason, the increasing folds of GRP78/BiP induced by different HBsAgs varied and were not reproduced consistently. In contrast, the amount of p65 subunit of NF-B consistently increased in the nuclear fraction of cells co-expressing S, M and L HBsAg (Fig. 2, lanes 3–5, line 2). A decreased amount of GFP–LD in the nuclear fraction (Fig. 2, lanes 2–5, line 1) and, conversely, an increased amount of GFP–LD in the cytoplasmic fraction (Fig. 2, lanes 2–5, line 4) were found in cells expressing GFP–LD alone or co-expressing GFP–LD with S, M, or L HBsAg. These Western blot results are highly correlated with the results of the fluorescence microscopic observation (Table 2), indicating that all three forms of HBsAg have the capability to induce GFP–LD translocation, but to various degrees.
|
|
, to further correlate with ER stress and GFP–LD distribution in the cytoplasm, we treated GFP–LD expression cells with the ER stress inducers, TM and BFA, for 2 h and then determined the percentage of N and N+C pattern cells by fluorescence microscopy. Around 8.8 to 9.3 % N+C pattern increase was observed when they were treated with 5 µg TM and BFA ml–1 (see Supplementary Table S1 available in JGV Online). Consistently, Western blot results showed a decreased amount of GFP–LD present in the nuclear fraction, while an increased amount of GFP–LD present in the cytoplasm when cells were treated with TM (Fig. 3a, lanes 2–5, lines 1 and 3) and BFA (Fig. 3b, lanes 2–5, lines 1 and 3). In addition, a slightly increased amount of GRP78/BiP was also observed in a dose-dependent manner when cells were treated with lower doses of TM and BFA (2–4 µg ml–1) but not at higher doses (5 µg ml–1) (Fig. 3a and b, line 6). The highest dose was unable to induce the highest amount of GRP78/BiP and GFP–LD in the cytoplasm (Fig. 3a and b lane 6, line 6), which could be due to a desensitization effect. Nevertheless, the data showing a good correlation between increasing GFP–LD in the cytoplasm and expression of GRP78/BiP indicate that even in the absence of HBsAg, inducing ER stress by an exogenous drug also causes GFP–LD translocation to the cytoplasm. Therefore, induction of ER stress is suggested to be an early event when HBsAg is expressed in cells to induce GFP–LD translocation.
|
B plays a role in GFP–LD transportation from the nucleus to the cytoplasmB in the nucleus has been demonstrated by Western blot in cells co-expressing GFP–LD and S, M or L HBsAg (Fig. 2), whether the nuclear form of NF-B can actively transcribe its target genes remains to be determined. Similar experiments described in Table 2 were therefore conducted, in which pNF-B-Luc containing the luciferase gene followed by the NF-B-response element was co-transfected with pGFP–LD and plasmids encoding S, M or L HBsAg into HuH-7 cells. After 24, 48 and 72 h post-transfection, cells were lysed and luciferase activities were determined. Results clearly show that the luciferase activity (2.7–3.2x105 luminescence intensity unit, liu) was higher in cells co-expressing GFP–LD with S, M or L HBsAg as compared with in those expressing GFP–LD without HBsAgs (1.3x105 liu) or in pNF-B-Luc transfection cells (8.2x104 liu) at 24 h post-transfection (Fig. 4). A similar trend was also observed in 48 h post-transfection cells (3.1–4.1x105 liu versus 1.4–1.7x105 liu) and in 72 h post-transfection cells (4.3–7.3x105 liu versus 2.1x105 liu). Among the cells co-expressing various HBsAgs at 72 h post-transfection, the L HBsAg expression showed the highest luciferase activity (7.3x105 liu), while the S HBsAg expression cells showed the lowest activity (4.3x105 liu). These results are consistent with the Western blot results and indicate that the nuclear form of NF-B is induced by three forms of HBsAgs (Fig. 2, line 2) and this varies to a range of different levels.
|
B in HBsAg-induced GFP–LD translocation, we co-transfected plasmid pKA, which expresses a dominant negative form of IKK that results in inactivation of NF-B, to examine whether the functioning of HBsAg will be blocked or not. Western blot results show that the GFP–LD remaining in the nucleus was increased proportionally to the amount of pKA transfected (Fig. 5a, line 1). Unlike the reciprocal change of GFP–LD between the nucleus and cytoplasm detected and shown in Fig. 2, a constant and similar amount of GFP–LD was present in the cytoplasm of cells with or without pKA transfection (Fig. 5a, line 3). However, the luciferase activity assay showed several fold decrease in cells expressing various amounts of mutant IKK (data not shown), suggesting that NF-B was indeed inhibited in those cells and this resulted in a higher amount of GFP–LD being retained in the nucleus. When cells co-expressed GFP–LD and various amounts of active form of IB kinase subunit, an increasing amount of GFP–LD, proportional to the amount of pIKK that was transfected was observed in the cytoplasmic fraction of cells (Fig. 5b, line 3). Conversely, a decreasing amount of GFP–LD was observed in the nuclear fraction of cells (Fig. 5b, line 1). Cells having the higher amount GFP–LD in the nucleus were highly correlated with the higher luciferase activity (data not shown). Taken together the evidence shown in Figs 4, 5(a) and (b) conclude that NF-B indeed plays a significant role in HBsAg-induced GFP–LD translocation.
|
treatment also induces GFP–LD translocation to the cytoplasm in the absence of protein synthesis induces cell responses largely through activation of NF-B, to correlate further with activation of NF-B and GFP–LD distribution, GFP–LD expressing cells were treated with TNF- for 1 h and the percentage of N+C cells was determined by fluorescence microscopy. The results revealed that 1 h after TNF- treatment induced more than 50 % of GFP–LD expressing HuH-7 or HeLa cells appeared to have the N+C pattern, while treatment with TM and BFA induced only 8.8 and 9.3 % of the cells to have the N+C pattern (Supplementary Table S1 available in JGV Online). The robust effect of TNF- allowed us to test whether the induction of GFP–LD translocation into the cytoplasm requires newly synthesized proteins or not. This kind of experiment could not be performed in cells expressing HBsAg because the protein translation inhibitor will also block HBsAg production. After pre-treatment with cycloheximide for 30 min, cells were treated with TNF- for 1 h and the N+C pattern of GFP–LD was examined. Around 53.3 % of cells appeared to have the N+C pattern (Supplementary Table S1), indicating that GFP–LD translocation into the cytoplasm does not require newly synthesized proteins. The inhibition effect by cycloheximide was also shown to occur by the luciferase activity assay, which showed that activity was greatly reduced in cells treated with both TNF- and cycloheximide (data not shown). However, the Western blot result shows that the amount of NF-B in the nucleus was similar after TNF- treatment with or without cycloheximide but it was higher than that in TNF- non-treated cells (Fig. 6, line 3). Western blot analysis also shows that the amount of GFP–LD in the nucleus and cytoplasm (Fig. 6, lines 1 and 4) correlated well with the fluorescence microscopic observations.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wang et al., 2005). However, signal molecules involved in HDV RNP export and the underlying mechanisms are largely unknown. In this study, we apply a previously established system, in which HBsAg can facilitate GFP–LD translocation into the cytoplasm, to investigate signals participating in the GFP–LD translocation. Several lines of evidence have demonstrated that ER stress and activation of NF-B, which are induced by HBsAg, play a significant role (Figs 2, 4 and 5a). It is known that ER stress induced by many different viral proteins leads to various cellular effects (Su et al., 2002; Tardif et al., 2002; Dimcheff et al., 2003), including full-length and truncated forms of the HBV L and M surface antigens (Wang et al., 2003; Hsieh et al., 2004; Hung et al., 2004). The current study is the first report to show that induction of ER stress results in a nuclear protein translocation into the cytoplasm.
The current study also provides direct evidence that three different forms of HBsAgs induce various levels of NF-B activities (Fig. 4) as well as 34.7–56.5 % cells having GFP–LD in the cytoplasm (Table 2). The various capabilities exhibited by different forms of HBsAgs could be due to their intrinsic properties, i.e. capability of ER retention (Bruss & Ganem, 1991; Sheu & Lo, 1994; Chau et al., 2005). The effects of HBsAg on NF-B activity and GFP–LD translocation could be explained by either that they are two parallel events or that the GFP–LD translocation is the downstream event following the NF-B activation. We prefer the second explanation because in the absence of HBsAg, GFP–LD cells treated with TNF-, which induces a higher level of NF-B, also appear to have a higher percentage of N+C pattern (Supplementary Table S1). At the present time, no direct evidence that HBsAg can trap the newly synthesized LDAg in the cytoplasm has been demonstrated. However, we favour the hypothesis that LDAg must enter into the nucleus and then translocate into the cytoplasm after post-translational modification, in which HBsAgs exert signals to facilitate the modification. This hypothesis is supported by results that cells co-expressing GFP–LD(31–214) and HBsAg with or without LDAg show a low N+C pattern from 1 to 3 days post-transfection (Table 1), while in 6 days post-transfected cells expressing GFP–LD(31–214) can be co-secreted with HBsAg (Shih & Lo, 2001). Therefore, the current study suggests that the sequence located between aa 1 and 30 may be important in helping LDAg translocation into the cytoplasm and post-translational modification of serine-2 and/or arginine-13 (Mu et al., 2004; Li et al., 2004) is likely to be involved in facilitating LDAg translocation into the cytoplasm.
In the absence of HBsAgs, GFP–LD translocation can also be facilitated by adding ER stress inducing drugs, TM and BFA (Fig. 3), but the effect is fivefold lower than that of L HBsAg on 72 h post-transfected cells (Table 2), suggesting that many signal pathways and molecules may participate in the HBsAg-induced GFP–LD translocation. This may explain why inactivation of NF-B by co-transfection of dominant negative IKK does not significantly abolish the cytoplasmic distribution of GFP–LD (Fig. 5a) and the expression of GRP78/BiP and activity of NF-B is not perfectly matched in some cases. Interestingly, no new proteins are required for TNF- to induce GFP–LD translocation, indicating that a post-translational modification occurs to GFP–LD to allow this translocation (Fig. 6 and Supplementary Table S1). Results of low translocation of GFP–LD(31–214) and GFP–LDM in the presence of HBsAgs (Table 2) suggest that multimerization and farnesylation of GFP–LD are two important modifications for facilitating GFP–LD translocation. The present study cannot distinguish which step, farnesylation or multimerization, comes first when facilitating LDAg nuclear export but one report has shown that multimerization between SDAg and LDAg increases farnesylation of LDAg (O'Malley & Lazinski, 2005). Therefore, the farnesyl-transferase and other unidentified molecules that are directly or indirectly modified by the activated NF-B are of interest for future exploration.
In conclusion, the current study presents the first report showing the presence of signals in the cross-talk between HDV and HBV using a system of GFP fusion proteins. However, to identify the target enzymes downstream to NF-B and to understand how they are activated to modify LDAg are a great challenge for future studies. Moreover, whether the HDV RNP translocation into the cytoplasm can be facilitated by HBV and TNF- remains to be tested.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Casey, J. L. & Gerin, J. L. (1995). Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA. J Virol 69, 7593–7600.[Abstract]
Chang, M.-F., Baker, S. C., Soe, L. H., Kamahora, T., Keck, J. G., Makino, S., Govindarajan, S. & Lai, M. M. C. (1988). Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity. J Virol 62, 2403–2410.
Chang, F.-L., Chen, P.-J., Tu, S.-J., Wang, C.-J. & Chen, D.-S. (1991). The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc Natl Acad Sci U S A 88, 8490–8494.
Chang, M.-F., Chang, S. C., Chang, C.-I., Wu, K. & Kang, H.-Y. (1992). Nuclear localization signals, but not putative leucine zipper motif, are essential for nuclear transport of hepatitis delta antigen. J Virol 66, 6019–6027.
Chao, M., Hsieh, S.-Y. & Taylor, J. (1990). Role of two forms of hepatitis delta antigen: evidence for a mechanism of self-limiting genome replication. J Virol 64, 5066–5069.
Chau, P. K., Wang, R. Y.-L., Lin, M. H., Masuda, T., Suk, F.-M. & Shih, C. (2005). Reduced secretion of virions and hepatitis B virus (HBV) surface antigen of a naturally occurring HBV variant correlates with the accumulation of the small S envelope protein in the endoplasmic reticulum and Golgi apparatus. J Virol 79, 13483–13496.
Dimcheff, D. E., Askovic, S., Baker, A. H., Johnson-Fowler, C. & Portis, J. L. (2003). Endoplasmic reticulum stress is a determinant of retrovirus-induced spongiform neurodegeneration. J Virol 77, 12617–12629.
Glenn, J. S., Watson, J. A., Havel, C. M. & White, J. M. (1992). Identification of a prenylation site in delta virus large antigen. Science 256, 1331–1333.
Graham, F. L. & van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456–467.[CrossRef][Medline]
Hsieh, Y.-H., Su, I.-J., Wang, H.-C., Chang, W.-W., Lei, H.-Y., Lai, M.-D., Chang, W.-T. & Huang, W. (2004). Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis 25, 2023–2032.
Hu, H.-M., Shih, K.-N. & Lo, S. J. (1996). Disulfide bond formation of the in vitro-translated large antigen of hepatitis D virus. J Virol Methods 60, 39–46.[CrossRef][Medline]
Hung, J.-H., Su, I.-J., Lei, H.-Y. & 8 other authors (2004). Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-B and pp38 mitogen-activated protein kinase. J Biol Chem 279, 46384–46392.
Hwang, S. B. & Lai, M. M. C. (1993). Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B surface antigen. J Virol 67, 7659–7662.
Jayan, G. C. & Casey, J. L. (2002). Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2. J Virol 76, 3819–3827.
Kuo, M. Y.-P., Chao, M. & Taylor, J. (1989). Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol 63, 1945–1950.
Lai, M. M. C. (1995). The molecular biology of hepatitis delta virus. Annu Rev Biochem 64, 259–286.[CrossRef][Medline]
Lai, M. M. C. (2005). RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus. J Virol 79, 7951–7958.
Lee, A. S. (2005). The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35, 373–381.[CrossRef][Medline]
Lee, C.-H., Chang, S. C., Wu, C. H. & Chang, M.-F. (2001). A novel chromosome region maintenance 1-independent nuclear export signal of the large form hepatitis delta antigen that is required for the viral assembly. J Biol Chem 276, 8142–8148.
Li, Y. J., Stallcup, M. R. & Lai, M. M. C. (2004). Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication. J Virol 78, 13325–13334.
Macnaughton, T. B., Shi, S. T., Modahl, L. E. & Lai, M. M. C. (2002). Rolling circle replication of hepatitis delta virus RNA is carried out by two different cellular RNA polymerases. J Virol 76, 3920–3927.
Modahl, L. E., Macnaughton, T. B., Zhu, N., Johnson, D. L. & Lai, M. M. C. (2000). RNA-dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerase. Mol Cel Biol 20, 6030–6039.
Mu, J.-J., Wu, H.-L., Chiang, B.-L., Chang, R.-P., Chen, D.-S. & Chen, P.-J. (1999). Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens. J Virol 73, 10540–10545.
Mu, J.-J., Chen, D.-S. & Chen, P.-J. (2001). The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication. J Virol 75, 9087–9095.
Mu, J.-J., Tsay, Y. G., Juan, L. J., Fu, T. F., Huang, W. H., Chen, D.-S. & Chen, P.-J. (2004). The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis. Virology 319, 60–70.[CrossRef][Medline]
O'Malley, B. & Lazinski, D. W. (2005). Roles of carboxyl-terminal and farnesylated residues in the functions of the large hepatitis delta antigen. J Virol 79, 1142–1153.
Reid, C. E. & Lazinski, D. W. (2000). A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs. Proc Natl Acad Sci U S A 97, 424–429.
Ryu, W. S., Bayer, M. & Taylor, J. (1992). Assembly of hepatitis delta virus particles. J Virol 66, 2310–2315.
Ryu, W. S., Netter, H. J., Bayer, M. & Taylor, J. (1993). Ribonucleoprotein complexes of hepatitis delta virus. J Virol 67, 3281–3287.
Sato, S., Wong, S. K. & Lazinski, D. W. (2001). Hepatitis delta virus minimal substrates competent for editing by ADAR1 and ADAR2. J Virol 75, 8547–8555.
Schröder, M. & Kaufman, R. J. (2005). ER stress and the unfolded protein response. Mutat Res 569, 29–63.[Medline]
Sheu, S. Y. & Lo, S. J. (1992). Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts. Virology 188, 353–357.[CrossRef][Medline]
Sheu, S. Y. & Lo, S. J. (1994). Biogenesis of the hepatitis B viral middle (M) surface protein in a human hepatoma cell line: demonstration of an alternative secretion pathway. J Gen Virol 75, 3031–3039.
Sheu, S. Y., Chen, K.-L., Lee, Y.-H. W. & Lo, S. J. (1996). No intermolecular interaction between the large hepatitis delta antigens is required for the secretion with hepatitis B surface antigen: a model of empty HDV particle. Virology 218, 275–278.[CrossRef][Medline]
Shih, K.-N. & Lo, S. J. (2001). The HDV large-delta antigen fused with GFP remains functional and provides for studying its dynamic distribution. Virology 285, 138–152.[CrossRef][Medline]
Shih, K.-N., Chuang, Y.-T., Liu, H. & Lo, S. J. (2004). Hepatitis D virus RNA editing is inhibited by a GFP fusion protein containing a C-terminally deleted delta antigen. J Gen Virol 85, 947–957.
Su, H.-L., Liao, C.-L. & Lin, Y.-L. (2002). Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response. J Virol 76, 4162–4171.
Tan, K.-P., Shih, K.-N. & Lo, S. J. (2004). Ser-123 of the large antigen of hepatitis delta virus modulates its cellular localization to the nucleolus, SC-35 speckles or the cytoplasm. J Gen Virol 85, 1685–1694.
Tardif, K. D., Mori, K. & Siddiqui, A. (2002). Hepatitis C virus subgenomic replicons induce endoplasmic reticulum stress activating an intracellular signaling pathway. J Virol 76, 7453–7459.
Taylor, J. M. (2003). Replication of human hepatitis delta virus: recent developments. Trends Microbiol 11, 185–190.[CrossRef][Medline]
Wang, H.-C., Wu, H.-C., Chen, C.-F., Fausto, N., Lei, H.-Y. & Su, I.-J. (2003). Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 163, 2441–2449.
Wang, Y.-H., Chang, S. C., Huang, C., Li, Y.-P., Lee, C.-H. & Chang, M.-F. (2005). Novel nuclear export signal-interacting protein, NESI, critical for the assembly of hepatitis delta virus. J Virol 79, 8113–8120.
Weiner, A. J., Choo, Q.-L., Wang, K.-S., Govindarajan, S., Redeker, A. G., Gerin, J. L. & Houghton, M. (1988). A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta. J Virol 62, 594–599.
Wu, C. J., Leu, C. Y., Liu, S. T., Chow, K. P., Meng, C. L. & Chang, Y. S. (1998). Transcriptional activation of NF-B activity by Epstein–Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases. Gene Ther 5, 905–912.[CrossRef][Medline]
Yeh, T.-S. & Lee, Y.-H. W. (1998). Assembly of hepatitis delta virus particles: package of multimeric hepatitis delta virus genomic RNA and the role of phosphorylation. Virology 249, 12–20.[CrossRef][Medline]
Yeh, T.-S., Lo, S. J., Chen, P.-J. & Lee, Y.-H. W. (1996). Casein kinase II and protein kinase C modulate hepatitis delta virus RNA replication but not empty viral particle assembly. J Virol 70, 6190–6198.[Abstract]
Received 28 November 2005;
accepted 30 January 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL |
