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Virology Group, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Road, New Delhi 110067, India
Correspondence
Sunil K. Lal
sunillal{at}icgeb.res.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Bβ and 
, were transcriptionally downregulated in ORF3-transfected cells. The constitutive expression of fibrinogen genes can be significantly upregulated by interleukin (IL)-6, an important mediator of liver-specific gene expression during an acute-phase response. Transcription of fibrinogen genes after IL-6 stimulation was less in ORF3-expressing cells compared with controls. This report adds one more biological function to, and advances our understanding of, the cellular role of the ORF3 protein of HEV. The possible implications of these findings in the virus life cycle are discussed. Two supplementary tables detailing the plasmids and primers used in this study are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Although recognized as an important pathogen, studies of HEV biology and pathogenesis have been severely restricted by the lack of a well-established cell culture system or small animal models of viral infection. Thus, to understand viral replication and pathogenesis, subgenomic expression strategies have been used to study the properties and functions of HEV gene products (Jameel et al., 1996; Zafrullah et al., 1997). HEV has three proteins, ORF1, ORF2 and ORF3 (Purcell & Emerson, 2000). ORF3 is a small 123 aa viral phosphoprotein, which associates with the cytoskeleton upon cell fractionation (Zafrullah et al., 1997). However, ORF3 has now been shown actually to be 9 aa shorter than previously believed (Wang et al., 2000; Graff et al., 2006; Huang et al., 2007). ORF3 has gained attention as a viral regulatory protein due to its presumed role in modulating cellular signalling (Korkaya et al., 2001; Kar-Roy et al., 2004; Moin et al., 2007).
The ORF3 protein is known to interact with other cellular proteins (Tyagi et al., 2004, 2005; Surjit et al., 2006) such as the 1-microglobulin bikunin precursor (Tyagi et al., 2004) and its two processed proteins, 1-microglobulin (Tyagi et al., 2004) and bikunin (Tyagi et al., 2005). In an earlier study, we postulated that the observed enhanced secretion of immunosuppressive 1-microglobulin in the presence of ORF3 was a mechanism used by the virus to maintain an immunosuppressed milieu surrounding the infected hepatocytes, thus helping the virus during the natural course of its infection (Surjit et al., 2006). Transfection studies indicate that ORF3 modulates the host-cell environment for efficient viral replication (Kar-Roy et al., 2004; Surjit et al., 2006; Moin et al., 2007) and promotes cell survival.
To elucidate further the role of ORF3, we used a yeast two-hybrid screen of a human cDNA liver library and identified the fibrinogen (FBG) Bβ chain as a cellular interaction partner for ORF3. FBG is an acute-phase protein synthesized constitutively by liver epithelium, the synthesis of which is upregulated two- to tenfold following infections, tissue injury and inflammation (Crabtree & Kant, 1982; Otto et al., 1987). In addition to its well-established role in haemostasis, FBG and its thrombin cleavage product, fibrin, have been proposed to play roles as the central regulators of the inflammatory/acute-phase response (Esmon et al., 1991; Levi et al., 2003).
FBG is a large, 340 kDa dimeric molecule, each unit of which is composed of three non-identical subunit proteins: A, Bβ and (Blomback, 1996). Each of the FBG chains is encoded by a separate gene (Redman & Xia, 2001) and each gene is separately transcribed and translated. During the acute phase of inflammation, their expression is coordinately regulated (Crabtree & Kant, 1982) and this effect can be mimicked by recombinant interleukin (IL)-6 in vitro (Fuller & Zhang, 2001). IL-6 is the main mediator of acute-phase-induced FBG synthesis (Otto et al., 1987; Lutticken et al., 1994; Zhong et al., 1994). IL-6 upregulation of FBG genes involves binding of activated STAT3 transcription factor to its cognate type II IL-6 response elements (Zhang et al., 1995, 1997).
The biological significance of ORF3–Bβ interaction and the possible consequences of decreased FBG synthesis on HEV pathogenesis are discussed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, pRSV-Neo-Bβ and pRSV-Neo- plasmids (Roy et al., 1990, 1991), containing the full-length cDNAs encoding the three FBG chains, were kindly provided by Dr C. M. Redman (Lindsley F. Kimball Research Institute, New York Blood Centre, USA), and pcEF-Myc STAT3 wild-type (WT) and dominant-negative (DN) constructs were provided by Dr L. M. Pfeffer (Health Science Centre, University of Tennessee, USA). All DNA manipulations were carried out as described by Sambrook et al. (1989).
Yeast two-hybrid techniques.
All two-hybrid media and protocols for library screening, transformations, yeast plasmid isolation and filter lift, and liquid β-galactosidase assays were as described in the Clontech manual for the Matchmaker GAL4 two-hybrid system and in the Clontech Yeast Protocols Handbook. The yeast strain AH109 was from Clontech.
Cell culture and transfection.
HuH-7 and COS-1 cells were maintained and transfected as described previously (Ratra et al., 2008).
Metabolic labelling.
At 44 h post-transfection, cells were starved for 1 h in cysteine/methionine-deficient Dulbecco's modified Eagle's medium supplemented with 1 % glutamine and 0.1 mg heparin ml–1 when secreted FBG was to be isolated. Cells were then labelled with 100 µCi (3.7 MBq) 35S-labelled cysteine/methionine promix ml–1 for 3 h, washed with PBS and lysed in immunoprecipitation buffer. The lysates were collected and clarified. Monensin (Sigma) was added at a final concentration of 5 µM in starvation and labelling media, wherever used.
Immunoprecipitation and immunoblotting.
Equal amounts of protein were immunoprecipitated and analysed by SDS-PAGE, followed by immunoblotting or fluorography, as described previously (Ratra et al., 2008). For isolation of secreted FBG, the labelling medium was directly immunoprecipitated. All images were acquired by scanning the autoradiographs with a Scan Jet 3400C (Hewlett-Packard).
Immunofluorescence and fluorescence resonance energy transfer (FRET) analysis.
The procedure for co-localization and FRET analysis has been described previously (Kar-Roy et al., 2004). COS-1 cells plated on coverslips were transfected with enhanced cyan fluorescent protein (ECFP)–ORF3 and enhanced yellow fluorescent protein (EYFP)–Bβ expression constructs. FRET was detected using the acceptor photobleaching approach. In dual-labelling immunofluorescence assays in HuH-7 cells, immunofluorescence staining was carried out as described previously (Tyagi et al., 2004). The primary antibodies used were monoclonal anti-ORF3 or polyclonal anti-FBG (Dako), both used at a 1 : 500 dilution. Polyclonal anti-FBG antibody recognizes A, Bβ and FBG chains individually and also the assembled complex. Secondary antibodies were used at a 1 : 1000 dilution and were goat anti-rabbit or anti-mouse IgG coupled to either Alexa Fluor 488 or Alexa Fluor 594 dye (Molecular Probes). All images were acquired using a Bio-Rad confocal microscope and prepared using Laser Sharp 2000 software.
Northern blot and RT-PCR analyses.
FBG mRNA levels were analysed by both Northern blot hybridization and RT-PCR. Total RNA was extracted from ORF3-transfected or vector-transfected cells with Trizol (Invitrogen), according to the manufacturer's instructions. Northern hybridization was carried out as described by Sambrook et al. (1989). RNA was hybridized separately with nick-translated [-32P]dCTP-labelled A, Bβ or cDNA. RT-PCR analysis was also carried out using a standard protocol (Sambrook et al., 1989). First-strand cDNA synthesis was primed by oligo(dT) using 2 µg total RNA isolated from transfected HuH-7 cells and aliquots of the reverse-transcribed samples were used for semi-quantitative PCR using specific primer pairs as described in Supplementary Table S2. Where specified, IL-6 (PeproTech) was used at a concentration of 50 ng ml–1 for 90 min to stimulate the cells before harvesting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was used as bait. A human liver cDNA AD fusion library constructed in AD vector pACT2, such that the proteins encoded by the inserts were fused to the GAL4 AD, was obtained as Escherichia coli transformants from Clontech. Several cellular proteins were found to interact with HEV ORF3 protein, such as BAC clone RP11-701P16 (GenBank accession no. AC084871
[GenBank]
); haemopexin mRNA (NM_000613
[GenBank]
) (Ratra et al., 2008); STAG-3-like mRNA (NM_001025202); START domain containing 5 (STARD5), transcript variant 1, mRNA (NM_181900.2
[GenBank]
); protein phosphatase 6, catalytic subunit (PPP6C) mRNA (NM_002721
[GenBank]
); mRNA for FLJ00221 protein (AK074148
[GenBank]
); sulfatase modifying factor 2 (SUMF2), transcript variant 3, mRNA (NM_001042469); chromosome 11, clone RP11-793I11 (AC023232
[GenBank]
); homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1, mRNA (BC000086
[GenBank]
); and cytochrome P450, family 8, subfamily B, polypeptide 1 (CYP8B1), mRNA (NM_004391
[GenBank]
) among others, including the previously reported mRNA for 1-microglobulin and bikunin precursor (NM_001633
[GenBank]
) (Tyagi et al., 2004).
HEV ORF3 protein interacts with FBG Bβ
From this yeast two-hybrid screen, FBG Bβ mRNA (GenBank accession no. NM_005141
[GenBank]
) was also identified and the interaction was studied further. Results of the yeast two-hybrid studies are shown in Fig. 1(a). The AD–Bβ (library clone) was reintroduced into AH109 along with BD–ORF3. As a positive control for the assay, self-association of ORF3 (Tyagi et al., 2001) was observed. ORF3 and Bβ, when present together inside the yeast host, allowed growth on synthetic dropout Leu–Trp–His–Ade– medium and gave a positive filter β-galactosidase assay result. The liquid β-galactosidase level (shown graphically) of ORF3-Bβ co-transformants was almost 16-fold higher compared with the negative controls but about 1.5-fold lower compared with the positive control. The histidine reporter construct has residual histidine expression that is overcome by growing cells in the presence of 3-amino-1,2,4-triazole (3-AT). The test ORF3 and Bβ co-transformants and the positive control were able to grow in the presence of 20 mM 3-AT.
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, Bβ and full-length cDNAs was confirmed in COS-1 cells (Fig. 1b
, upper panel, lanes 1–3, respectively). The COS-1 cell line does not express FBG endogenously and represents a widely used in vitro system for transient or stable expression of FBG (Roy et al., 1991). FBG processing in COS-1 cells mimics that in hepatocytes. This cell line has also been shown to be valuable for ORF3 expression (Jameel et al., 1996). The Bβ protein co-immunoprecipitated with ORF3 (upper panel, lane 5).
The human hepatoma cell line HuH-7 constitutively expresses and secretes FBG. ORF3 protein was shown to interact specifically with endogenous FBG Bβ chains in these cells (Fig. 1c, lane 2). Immunodepletion (lane 3) was carried out to deplete ORF3 protein from the sample, but not to completion. The resulting supernatant was again immunoprecipitated using anti-ORF3 antibody. As expected, both ORF3 and FBG band intensities were significantly reduced (lane 3). Co-immunoprecipitation assays in COS-1 and HuH-7 cells showed that ORF3 interacted specifically with the Bβ chain and not with A or .
Deletion mapping of the interacting domains of the ORF3 and fibrinogen Bβ proteins using the yeast two-hybrid system
To characterize the ORF3 domain involved in the ORF3–Bβ interaction, an array of deletion mutations of ORF3 cloned into the yeast two-hybrid BD vector were used as described previously (Ratra et al., 2008). Pair-wise combinations of ORF3 deletion mutants and full-length AD–Bβ were tested for protein–protein interactions by the yeast two-hybrid assay. The strength of these interactions was investigated by measuring the relative β-galactosidase activity and by the ability to grow in medium lacking histidine and adenine in the presence of 0, 5, 10 and 15 mM 3-AT. The latter results indicated the strength of the protein–protein interaction as a function of histidine prototrophy.
ORF3 deletion mutants showed differences in their ability to interact with full-length Bβ in the two-hybrid assay (Fig. 1d). The ORF3 (1–91) and ORF3 (1–77) C-terminally truncated proteins interacted with full-length Bβ in the yeast two-hybrid assay, but with a strength of 1.7-fold compared with that obtained with full-length ORF3 (1–123). C-terminal truncations beyond aa 77 [ORF3 (1–63)], however, completely abolished the interaction. ORF3 (83–123) also did not interact in the two-hybrid assay. These initial experiments suggested that the region of ORF3 from aa 63 to 77 is critical for the interaction. The C-terminal amino acids beyond aa 77 may contribute to the strength of the interaction; however, the presence of this region is not sufficient to give a positive two-hybrid result, as was evident from the fact that there was no interaction obtained with the deletion construct ORF3 (83–123). This observation was further substantiated by the fact that ORF3 (63–123) interacted with Bβ with strength comparable to full-length ORF3–Bβ interaction. ORF3 (33–123) also interacted with a strength comparable to full-length ORF3. All of the above results indicated that the C-terminal half of ORF3 from aa 63 to 123 contains the interaction domain, and possibly that the presence of aa 63–77 of ORF3 is critical for the association.
Co-localization of ORF3 with endogenous FBG
The subcellular localization of ORF3 and FBG was determined by indirect antibody labelling. Distribution of ORF3 (Fig. 2a, panels ii and v), as observed previously (Korkaya et al., 2001; Tyagi et al., 2002), was cytoplasmic and displayed punctate staining. Endogenous FBG (Fig. 2a, panels i and iv) was cytoplasmic in distribution with distinct perinuclear localization, possibly in the endoplasmic reticulum and Golgi complex characteristic of secretory proteins. In the merged images (Fig. 2a, panels iii and vi), distinct golden yellow stained regions were observed, indicating co-localization of ORF3 with FBG in these areas.
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, panel iii). A mean FRET efficiency of 30±12.7 % was found in the regions of co-localization as opposed to 5.4±3.38 % in control areas. The difference in pixel intensity of ECFP, before and after photobleaching of EYFP, in the areas of co-localization and the regions where the two proteins did not co-localize when compared for ten independent observations over two separate experiments showed high levels of significance with a P value of 1.3x10–6. The presence of FRET indicated an actual protein–protein interaction in vivo.
Hepatocytes expressing ORF3 protein show a shift in distribution of FBG
In ORF3-expressing HuH-7 cells, either endogenous FBG could not be detected at all or reduced levels were detected in a few cells (characterized as cells with less-intense staining). ORF3 non-expressing cells, on the other hand, stained well for FBG, showing a characteristic perinuclear staining. Fig. 2(c) shows a representative field. No co-localization of ORF2 and FBG, nor a shift in distribution, was observed, indicating that the observed phenomenon was specifically due to ORF3 expression. This observation prompted us to ask questions about the fate of FBG in ORF3-expressing liver cells. The disappearance of endogenous FBG could either mean that we were witnessing the same effect that we observed previously for 1-microglobulin (Tyagi et al., 2004) where 1-microglobulin secretion was being expedited by ORF3, or it could be that ORF3 is mediating FBG degradation or downregulating its synthesis. Further experiments were designed to address this issue.
ORF3-expressing hepatocytes secrete less FBG
The level of secreted FBG was studied in cells expressing ORF3. The results (Fig. 3a) clearly showed that the radioactivity of the three component chains of secreted FBG in the vector control was greater than in ORF3-transfected cells, implying that ORF3 was not expediting FBG secretion. This phenomenon was specific to ORF3, as secreted FBG in ORF2-transfected cells was comparable to that in the control.
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). This method was used to take measurements independent of any difference in secretion processes in the ORF3- and vector-transfected cells. As expected, the intracellular levels of the component FBG chains in monensin-treated vector-transfected cells (Fig. 3b, lane 2) were higher than in the non-treated cells (lane 1). Under the same conditions, when intracellular levels of FBG chains were compared in monensin-treated vector-transfected (lane 2) and ORF3-transfected (lane 3) cells, the latter clearly showed lower levels of each of the FBG chains. No secreted FBG could be immunoprecipitated from the medium of monensin-treated cells, indicating that FBG secretion was effectively blocked (data not shown). These results indicated that either ORF3 downregulates endogenous FBG expression or FBG is degraded in the presence of ORF3.
Degradation of FBG chains in ORF3-expressing hepatocytes
The effect of ORF3 on the rate of degradation of newly synthesized A, Bβ and chains in ORF3-transfected HuH-7 cells was examined. The densitometry data were plotted as percentage stability, assuming a relative densitometry unit at 0 h to be 100 %. Densitometry analyses were carried out using ImageJ software version 1.36b. Fig. 4 shows the rate of degradation of radiolabelled A, Bβ and chains in vector- and ORF3-transfected cells, respectively. About 50 % of A chains were degraded in about 2 h, and Bβ and in 3 h. In ORF3-transfected cells, the radioactivity of the FBG chains also decreased during the chase period, with an estimated half-life of 2 h for A and 3 h for Bβ and , respectively. These times, however, were not comparable to those reported previously in COS cells: 1 h, 80 min and 4 h for A, Bβ and , respectively (Roy et al., 1992; Xia & Redman, 1999). In addition, in HepG2 cells, the chain has been reported to have a half-life of 4 h (Roy et al., 1992; Xia & Redman, 1999). Danishefsky et al. (1990) reported that non-secreted Bβ chain was degraded intracellularly with a half-life of 5 h in COS-1 cells. It is evident that, in different cell lines and under different culture conditions, differences in the rate of degradation of FBG are observed. In our system, the data showed that the stability of A, Bβ and chains was not affected by ORF3.
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). As was expected, the mRNA levels of the three chains were found to be downregulated by two- to threefold in ORF3-transfected cells compared with mock-transfected cells, as seen clearly by densitometry analysis conducted to quantify the amount of RNA. The decreases in mRNA levels of the FBG genes were verified independently by RT-PCR analysis. Fig. 5(b) shows the RT-PCR results showing lower mRNA levels of FBG chains A, Bβ and in ORF3-transfected HuH-7 cells (lane 2) compared with vector-transfected cells (lane 1).
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). Although constitutive FBG expression was found to be downregulated, it was more interesting to observe whether the same effect was brought about by ORF3 under IL-6 stimulation, a condition that mimics the acute-phase response and is hence physiologically relevant to HEV infection. As expected, IL-6 induced FBG expression in HuH-7 cells (Fig. 6a) in a dose- and time-dependent manner. Data were verified at the protein level by Western blotting with anti-FBG antibody (data not shown). IL-6-activated STAT3 transcription factor is the main transactivator of FBG gene expression. That the observed increase in FBG expression by IL-6 was indeed due to STAT3 was demonstrated in two ways. Firstly, STAT3 WT overexpression (Fig. 6b) induced FBG expression in a dose-dependent manner. Secondly, a STAT3 DN mutant blocked IL-6-mediated stimulation of FBG gene expression (Fig. 6c). However, when ORF3-transfected cells were stimulated with IL-6, FBG gene expression was not induced. Fig. 6(d) shows that ORF3 inhibited induced FBG expression in a dose-dependent manner. The same effect was observed at the protein level by immunoblot analysis (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Zafrullah et al., 1997). Data from such experiments clearly indicate that the ORF3 protein modulates the host-cell environment in ways that support efficient viral replication and propagation. For example, by activating ERK (Kar-Roy et al., 2004), the ORF3 protein creates a pro-survival environment for the virus. Further credence was lent to this hypothesis in a study where it was shown that the ORF3 protein protects cells from mitochondrial depolarization and death following apoptotic insult by overexpression of the mitochondrial VDAC protein and hexokinase (Moin et al., 2007). On the other hand, by binding to a strong immunosuppressive molecule, 1-microglobulin, and promoting its secretion into the extracellular medium, the ORF3 protein is likely to have an impact on the host immune response (Surjit et al., 2006). Although these in vitro data should be interpreted with much caution, they nonetheless have provided unifying concepts and expanded our knowledge of HEV biology when extrapolated to conditions of natural infection. Thus, ORF3 has emerged as a viral regulatory protein that influences multiple cellular pathways that promote the establishment of viral infection contributing to viral pathogenesis. With recent advances in the development of an efficient cell culture system for HEV (Tanaka et al., 2007; Lorenzo et al., 2008), the role of ORF3 in HEV biology should become established. In the present study, two important results have been reported: firstly, the ORF3 protein of HEV associates with the Bβ chain of FBG, and secondly, the levels of FBG secreted from ORF3-transfected hepatoma cells decrease. We explored the mechanism of the ORF3-mediated decrease in the levels of secreted and intracellular FBG and attributed it to downregulation of FBG expression in the presence of ORF3.
The interaction between ORF3 and FBG Bβ was confirmed by yeast two-hybrid and in vitro co-immunoprecipitations from COS-1 and HuH-7 cells. The results were substantiated with co-localization and FRET studies. The critical regions for protein–protein interaction were mapped by a yeast two-hybrid assay. The results indicated that the region between aa 63 and 77 of ORF3 is critical for the interaction, but that the minimal region that supports full interaction is the entire C-terminal half from aa 63 to 123. Interestingly the interaction domain found in our studies involving the C-terminal half of ORF3 overlaps with the ORF3 dimerization domain (aa 81–123; Tyagi et al., 2001), the bikunin interaction domain (aa 83–123; Tyagi et al., 2005) and the SH3-binding domain (aa 75–113; Korkaya et al., 2001). Our data, along with these previous observations, clearly suggest that the C-terminal region of ORF3 is a multifunctional domain. The 41 aa binding domain contains a small stretch of hydrophobic amino acids from residues 84 to 95 and also contains two polyproline regions. Strategic overlapping of these interaction domains may be a possible mechanism to ensure regulation of these interactions. It should be noted that all of these studies were carried out using genotype 1 ORF3 protein; whether these interactions also occur in other HEV genotypes remains to be established.
By pulse–chase analysis, it was determined that the stability of A, Bβ and chains was not affected by ORF3. Hepatocytes of all species studied have unequal amounts of the three FBG chains. Due to variations in the origin of cell lines and culture conditions in vitro, differences in the proportions of the A, Bβ and chains are seen. Synthesis of the Bβ chain is the rate-limiting factor in the production of human FBG (Yu et al., 1984; Roy et al., 1990); thus, FBG synthesis is more sensitive to small changes in Bβ. As ORF3 interacts specifically with Bβ, we speculated that the observed decrease could be due to the interaction precluding Bβ from participating in the assembly process. Only fully assembled FBG, and not individual unassembled A, Bβ and or intermediates of the assembly process, is secreted by hepatocytes (Yu et al., 1984; Roy et al., 1991). This implies that, if Bβ is not available for assembly, the surplus A and chains are unable to form the complex and be secreted from ORF3-transfected cells. Consequently, the levels of secreted FBG would decrease in ORF3-expressing cells. Furthermore, our experiments also suggested that, in ORF3-transfected cells, FBG assembly or its secretion was not abolished altogether because the assembled FBG complex and its subunit chains were detectable in the culture medium, albeit at lower levels compared with vector-transfected cells, implying that the synthesized chains were assembled intracellularly and then secreted.
mRNA levels were next determined for FBG in the presence and absence of ORF3. The results of Northern blot hybridization, verified by RT-PCR analysis, demonstrated a decrease in the levels of all three FBG chain mRNA species in the presence of ORF3. Consequently, the intracellular level as well as secretion of FBG was decreased. One obvious question arising from this result is how ORF3 expression and its interaction with FBG Bβ modulates FBG gene transcription. It is unlikely that ORF3 by itself acts as a transcription factor, as all previous studies have shown that ORF3 is confined to the cytoplasm and is not expected to enter the nucleus. The mechanisms by which FBG genes are coordinately expressed and regulated are complex. Earlier experiments suggested that there are probably feedback mechanisms that maintain a steady intracellular proportion of FBG chains. Other studies have indicated that the extracellular concentration of FBG itself regulates its gene expression. Our studies suggest that the ORF3–Bβ interaction may trigger such an event, which translates into negative feedback leading to transcriptional downregulation of all three genes.
During an acute-phase response to infection or inflammation, the production of FBG by the liver increases in response to pro-inflammatory agents, particularly IL-6 and glucocorticoids (Otto et al., 1987; Huber et al., 1990). Increased levels of plasma FBG augment the immune response of the host to restore homeostasis. Fibrin(ogen) deposition locally within the inflammatory foci also regulates the immune response. There is increasing evidence showing that FBG plays a multifaceted role in the immune and inflammatory response (Ugarova & Yakubenko, 2001). FBG interacts with leukocyte cell-surface integrins CD11b/CD18 and CD11c/CD18 expressed on neutrophils, monocytes, macrophages and several subsets of lymphocytes. By binding to these integrins, FBG regulates cytokine (Walzog et al., 1999) and chemokine (Smiley et al., 2001) gene expression of polymorphonuclear neutrophils, promotes leukocyte adhesion and migration (Altieri et al., 1993), and modulates neutrophil functionality (Rubel et al., 2002) and degranulation. FBG has been reported to activate the NF-
B pathway in mononuclear phagocytes (Sitrin et al., 1998) and to delay apoptosis of activated neutrophils (Rubel et al., 2003).
Our data showed that ORF3 downregulated FBG expression, even under IL-6 stimulation. This result is significant, as ORF3 downregulated not only the basal transcription but also FBG expression under conditions mimicking an acute-phase response. Promoter elements for constitutive and IL-6-regulated expression of the human FBG genes have been described (Mizuguchi et al., 1995). Inspection of the regulatory regions of FBG genes show that common transcription binding motifs for all three genes and unique motifs for each gene are both present (Fuller & Zhang, 2001). Studies are under way to elucidate the molecular mechanisms of how the ORF3 protein downregulates FBG transcription. This must involve regulation at different levels, as the basal transcriptional machinery for A and Bβ expression differs from that for . Our data strongly indicate that the ORF3 protein downregulates FBG transcription through an indirect mechanism that may involve STAT3. Our results also substantiate a recently published study suggesting that ORF3 downregulates the expression of important acute-phase proteins (APP) in liver cells, such as C-reactive protein, haptoglobin, haemopexin and 1-antitrypsin (Chandra et al., 2008). It is proposed that, by downregulating the transcription of APP genes, ORF3 can potentially attenuate inflammatory responses and create an environment for increased viral replication and survival (Chandra et al., 2008). It was also reported that ORF3 delays the degradation of epidermal growth factor receptor, which blocks activated STAT3 nuclear transport and results in reduced transcriptional activity of the activated APP genes. This phenomenon could explain the decreased transcription of fibrinogen genes observed in our study. Current experiments are aimed at understanding further the molecular mechanisms of fibrinogen downregulation.
In an in vivo context, when an inflammatory stimulus is present, FBG gene expression and protein production are upregulated in liver cells, but this process would probably be counteracted in the presence of ORF3, which in our studies was shown to downregulate FBG gene expression and protein production. This would be advantageous for the virus, as the localized immune response around the virus-infected cells would consequentially be inhibited/delayed. Our results substantiate our previous observations where ORF3 was proposed to create an immunosuppressive environment around the infected cell by virtue of enhanced secretion of immunosuppressive 1-microglobulin. In the present study, ORF3 is also proposed to achieve a similar goal to create an immunosuppressive environment around the infected cell, but this time by virtue of decreased secretion of immunomodulatory FBG.
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ACKNOWLEDGEMENTS |
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, pRSV-Neo-Bβ and pRSV-Neo- plasmids and Dr L. M. Pfeffer (Department of Pathology, Health Science Centre, University of Tennessee, USA) for the pcEF-Myc STAT3 WT and DN constructs. Technical help from Ravinder Kumar and Vaibhao Janbandhu is gratefully acknowledged. R. R. was a senior research fellow of the Council of Scientific and Industrial Research, India. This work was supported by internal funds from the International Centre for Genetic Engineering & Biotechnology, New Delhi, and the Swedish International Development Cooperation Agency (SIDA). |
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Received 1 December 2008;
accepted 19 February 2009.
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