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Genes to Cells (2007) 12, 133-142. doi:10.1111/j.1365-2443.2007.01047.x
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Host factor Ebp1: Selective inhibitor of influenza virus transcriptase

Ayae Honda1,2,*, Takuto Okamoto2 and Akira Ishihama1,2

1 Department of Frontier Bioscience and Micro-Nano-Technology Research Center, Hosei University, Koganei, Tokyo 184–8584, Japan
2 Division of Molecular Biology, Nippon Institute for Biological Science, Ome, Tokyo 198–0024, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Influenza virus RNA polymerase is composed of three virus-coded proteins, and is involved in both transcription and replication of the negative-strand genome RNA. Subunit PB1 plays key roles in both the RNA polymerase assembly and the catalytic function of RNA polymerization. Using yeast two-hybrid screening, a HeLa cell protein with the molecular mass of 45 kDa was identified. After cloning and sequencing, this protein was identified to be Ebp1, ErbB3-binding protein. Epb1 specifically interacts with PB1 both in vitro and in vivo, and Epb1 contact site on PB1 was mapped at its binding site of transcription primers. Ebp1 was found to interfere with in vitro RNA synthesis by influenza virus RNA polymerase (3P complex), but no inhibition was observed for capped RNA endonuclease and RNA-cap binding, the intrinsic activities of RNA polymerase. Since inhibition was not observed against other nucleic acid polymerases tested, we propose that Ebp1 is a selective inhibitor of influenza viral RNA polymerase. Accordingly over-expression of Ebp1 interfered with virus production. The PB1-contact site on Ebp1 overlaps with the interaction site with ErbB3 (epidermal receptor tyrosine kinase), androgen receptor (AR) and retinoblastoma gene product (Rb), which are involved in controlling cell proliferation and differentiation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The genome of influenza virus consists of eight segments of negative strand RNA. Transcription and replication of the genome are carried out by the viral RNA-dependent RNA polymerase, which consists of three viral proteins, PB1, PB2 and PA (Krug et al. 1989; Honda & Ishihama 1997). Subunit PB1 plays major roles in subunit assembly (Toyoda et al. 1996), binding to vRNA (Li et al. 1998), binding of substrates for RNA synthesis (Asano et al. 1995), and catalytic function of RNA polymerization (Nakagawa et al. 1996). The RNA polymerase binds vRNA to form RNP complex and is packaged into mature virus particles. After uncoating of virus particles in influenza virus-infected cells, the RNP complex is transported into nuclei and the associated RNA polymerase transcribes vRNA into mRNA using host cell capped RNAs as primers (Krug et al. 1989). For generation of transcription primers, the RNA polymerase cleaves host cell capped RNAs by the RNA polymerase-associated endonuclease (Krug et al. 1989). Capped RNA is recognized and bound by subunit PB2 (Honda et al. 1999) even though the catalytic site for endonuclease was proposed to locate in PB1 (Li et al. 2001). Late after infection, the same RNA polymerase plays a catalytic role in replication of vRNA. The subunit PA is required for replication of the viral genome (Honda & Ishihama 1997). Several lines of evidence suggest the involvement of yet unidentified host protein(s) for the functional conversion of viral RNA polymerase from transcriptase to replicase (reviewed in Honda & Ishihama 1997, 2004). One approach for identification of the putative host factor(s) is to identify host proteins that interact with the vRNA polymerase. This approach may also be useful for identification of hitherto unidentified cellular proteins, which interact with influenza virus RNA polymerase and influence its function.

As an initial effort, we tried to identify a battery of cellular proteins, which specifically interact with one of the three subunits of influenza virus RNA polymerase. For this purpose we performed yeast two-hybrid screening using cDNA for each of the three P proteins and cDNA library from HeLa cells. In this report, we describe a HeLa cell protein, Ebp1 [ErbB3 (epidermal receptor tyrosine kinase) binding protein (Yoo et al. 2000)], which specifically interacts with the catalytic subunit PB1 of influenza virus RNA polymerase near the catalytic site for RNA polymerization, but not with other two subunits PB2 and PA. Ebp1 selectively inhibited RNA synthesis in vitro by the influenza virus RNA polymerase, but neither T7-, Escherichia coli-DNA-dependent RNA polymerases nor AMV reverse transcriptase was affected by Ebp1. Over-expression of Ebp1 reduced significantly the influenza virus production using the reverse genetics method. Taken together, we propose that Ebp1 is a selective inhibitor of influenza virus RNA polymerase. Mapping of the protein–protein contact sites between PB1 and Ebp1 indicates that the contact site on PB1 with Ebp1 overlaps with the catalytic region of PB1 while the PB1 contact site at the C-terminal proximal end of Ebp1 overlaps with its interacting sites with androgen receptor (AR) and retinoblastoma gene product (Rb).


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of Ebp1 as one of the PB1-interacting host proteins

As an initial effort for search of the host cell proteins interacting with influenza virus RNA polymerase, we performed yeast two-hybrid screening using pHybrex/Zeo for three P proteins insertion and pYES/Trp for cDNA library from HeLa cells. First screening was performed using each P protein fused to the DNA-binding domain, while HeLa cell proteins were fused to the activation domain. From this screening, we have isolated nine clones, which showed specific interaction with one of the three P proteins. Here we describe one of the PB1-interacting proteins, PB1c45 [PB1-contact protein with the molecular mass of 45 kDa]. After sequencing of intact PB1c45 cDNA clone, PB1c45 was found to be composed of 394 amino acid residues and identical with Ebp1.

In order to confirm the direct interaction between Ebp1 and PB1 and to identify the protein–protein contact sites, GST pull-down assay was performed using C-terminal fragments of Ebp1(C), and also yeast two-hybrid screening was carried out (see Fig. 2 and Table 1 for preparation of each fragments expression plasmid). When the C-terminal segment of Ebp1 (229–394) (Fig. 1A) was purified as GST-fusion (Fig. 1B), and subjected to GST pull-down assay with 35S-Met-labeled PB1, PB2 and PA proteins (Fig. 1C). PB1 protein formed complexes with Ebp1 (229–394) and intact Ebp1 (Table 1), but neither PB2 nor PA bound to GST-Ebp1(C) (Fig. 1D, left panel and Table 1). None of 3P subunits bound to purified GST (Fig. 1D, right panel).


Figure 2
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  		Figure 2  Binding of PB1 protein to Ebp1: Yeast two-hybrid screening assay. For confirmation of PB1-Ebp1 interaction and mapping of the contact site on each component, cDNA for each fragment of PB1, PB2 and PA, shown in (A), were inserted into pHybLex/Zeo "bait" plasmid so as to express as fusion proteins with the DNA-binding domain, while cDNA for each Ebp1 fragment (B) were inserted into pYESTrp2 "prey" plasmid for expression of fusion proteins with the activation domain. After co-transfection of bait and prey plasmids into yeast S. cerevisiae L40, the ß-gal assay was carried out. Results are summarized in Table 1. The gray bars on PB1 (A) and Ebp1 (B) indicate the regions involved in Ebp1 interaction.

 

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  		Table 1  Interaction between influenza vRNA polymerase subunits and Ebp1
 

Figure 1
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  		Figure 1  Binding of PB1 protein to Ebp1: GST pull-down assay. Panel (A) and (B): The C-terminal segment (229–394) of Ebp1 [Ebp1(C) in panel A] was expressed as GST-fused form in E. coli using pGEX-4T plasmid, and purified as described in Experimental procedures. In panel B, the purified GST-Ebp1(C) and GST were analyzed on SDS-10% PAGE and CBB staining. Lane 1, GST; lane 2, GST-Ebp1(C). (C) For preparation of [35S]Met-labeled P proteins, capped PB1-, PB2- and PA-RNA were synthesized in vitro under the control of SP6 promoter. In vitro translation was carried out in rabbit reticulocyte lysate for 1 h at 37 °C in the reaction mixture containing [35S]Met as a labeled substrate, and amino acid mixture without methionine. To check [35S]Met-labeled proteins, the reaction products were fractionated by SDS-8% PAGE and the gel was exposed to X-ray film. Lane 1, [35S]Met-labeled PB1; lane 2, [35S]Met-labeled PB2; lane 3, [35S]Met-labeled PA. (D) Mixtures of each one of [35S]Met-labeled P proteins and GST-Ebp1 (left panel) or GST (right panel) were incubated for 60 min at 30 °C, and then mixed with glutathione-bound resin. Resin-bound proteins were eluted by increasing concentrations of glutathione, and analyzed by SDS-8%PAGE. The gel was exposed on X-ray film. Lane 1, [35S]Met-labeled PB1; lane 2, [35S]Met-labeled PB2; lane 3, [35S]Met-labeled PA.

 
The above observation indicates that Epb1 interacts specifically with PB1 and PB1-contact site on Ebp1 locates at its C-terminal region downstream from residue 229. For fine mapping of the protein–protein contact site on both PB1 and Ebp1, we performed yeast two-hybrid screening using cloned cDNAs for four segments of PB1 (Fig. 2A), inserted into pHybrex/Zeo, and cDNAs for both full-length Ebp1 and three segments of Ebp1 (Fig. 2B), inserted into pYes/Trp. As controls, we also cloned cDNAs for three segments and intact of PB2 (Fig. 2A) and four segments and intact of PA (Fig. 2A) into pHybrex/Zeo. Three segments of Ebp1 and intact Ebp1, and eleven segments and three intact P proteins were subjected to yeast two-hybrid screening in all possible combinations. Results are summarized in Table 1. Among the Ebp1 segments tested, the C-terminal segment Ebp1 (325–394) and the intact Ebp1 were found to interact with the PB1 subunit (see Fig. 2B for the PB1 contact site on Ebp1). Among four segments of PB1, an internal segment, PB1 (111–360), was found to interact with both intact Ebp1 and Ebp1 (325–394) (see Fig. 2A). Except for this PB1 segment (111–360), none of the other P segments (three PB1, three PB2 and four PA segments) showed positive signal of binary complex formation with Ebp1 (Table 1). The PB1 segment (111–360) carries the primer-binding site for transcription initiation (Kolpashchikov et al. 2004). Surprisingly, one of the PA fragments (residues 347–452) alone induced the ß-galactosidase activity in the absence of activation domain expression, indicating that this fragment itself contains the sequence, which directly interacts with transcription apparatus.

Molecular interaction of Ebp1 with viral RNA polymerase in virus-infected cells

To examine the molecular interaction between Ebp1 and PB1 in virus-infected cells, MDCK cells were infected with influenza virus A PR8 for 8 h at 34 oC and labeled with [35S]Met for 30 min. As a control, uninfected cells were also labeled with radioactive Met for 30 min. The radio-labeled nuclear extracts were subjected to immunoprecipitation using antibodies raised against each of purified viral P and NP proteins and host protein Ebp1. When the nuclear extract was incubated with anti-NP, NP proteins were recovered in antigen-antibody complexes (Fig. 3A, lane 2). When the nuclear extract was incubated with anti-PB1, viral proteins PB1, PB2, PA and NP were precipitated (Fig. 3A, lane 3) [note that PA and PB2 migrate apparently to the same position by PAGE (see Figs 1C and 3C)], indicating that most of the P proteins are assembled into 3P complex and associated with RNP core. When uninfected cell extracts were analyzed, however, no cross-reactive proteins were detected with anti-PB1 and anti-NP antibodies (Fig. 3A, lanes 5 and 6).


Figure 3
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  		Figure 3  PB1-Ebp1 interaction assay using co-immunoprecipitation method. MDCK cells were infected with influenza virus PR8 at moi of 5, and labeled in the presence of [35S]Met for 1 h at 8 hpi. The cells were harvested by centrifugation and cell lysates were prepared as described in Experimental procedures. (A) An aliquot of the whole cell lysate was mixed with pre-immune serum (lanes 1 and 4), anti-NP (lanes 2 and 5) or anti-PB1 antibody (lanes 3 and 6). The reaction mixtures were incubated for 1 h on the ice, and after addition of protein A, incubated for another 1 h. Protein A-bound proteins were separated by SDS-PAGE and the gel was exposed to an X-ray film. Uninfected cells were treated as above. (B) Anti-Ebp1 or pre-immune sera were added into the cell lysate and immunoprecipitated as in [A]. Lanes 1 and 2 show the result from immunoprecipitant using pre-immune serum while lanes 3 and 4 show the result from immunoprecipitant using anti-Ebp1. (Note that a small amount of NP in lane 3 in this experiment is due to overflow of lane 4 sample, as confirmed by separate experiments). (C) Immunoprecipitation of virus-infected cell lysate with anti-Ebp1. After SDS-8%PAGE of immunoprecipitates, the gel was subjected to Western blotting using anti-PB1, anti-PB2 and anti-PA antibodies. Migration positions of marker proteins, 103K and 77K, are shown on left.

 
When the same nuclear extract was incubated with anti-Ebp1 antibodies, not only PB1 but also PA, PB2 and NP proteins were recovered in immunoprecipitates (Fig. 3B, lane 4) (note that a small amount of NP in lane 3 was due to overflow from lane 4), even though the isolated individual viral proteins PA, PB2 and NP did not cross-react with anti-Ebp1 (data not shown). These results indicate that Ebp1 interacts with PB1 in the RNP complex in influenza virus-infected cells. This finding also agrees with the observation that in virus-infected cells, most of three P proteins exist as assembled, forming RNP cores (A. Honda, unpublished data). To confirm that all three P proteins exist in the immunoprecipitates with anti-Ebp1, we checked the gel by immunoblotting using each of anti-PB1, -PB2 and -PA antibodies. Result shown in Fig. 3C indicates the presence of all three P proteins in the anti-Ebp1 precipitates.

Influence of Ebp1 on in vitro RNA synthesis by influenza virus RNA polymerase

Possible influence of Ebp1 on PB1 functions was analyzed using in vitro assay systems. Using the baculovirus expression system, we expressed and purified the 3P complex (Honda et al. 2002), which can be converted into an active form after interaction with vRNA (Honda et al. 2001). Ebp1 was expressed in insect cells using recombinant baculovirus, and purified from nuclear extract (Fig. 4A). Using the purified Ebp1, the effect of Ebp1 on RNA synthesis activity of viral RNA polymerase was analyzed. In vitro RNA synthesis was performed in the standard reaction mixture with the purified 3P complex, v53 nucleotide-long model vRNA template (Parvin et al. 1989) and the purified Ebp1 from nuclear extract. Figure 4B shows the effect of Ebp1 on in vitro RNA synthesis by influenza virus polymerase. The primer-dependent RNA synthesis was significantly reduced by the addition of Ebp1 in dose-dependent manner. Ebp1, however, did not affect on DNA synthesis by AMV reverse transcriptase (Fig. 4C), and RNA synthesis by T7 RNA polymerase, E. coli RNA polymerase (data not shown).


Figure 4
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  		Figure 4  Effect of Ebp1 on viral RNA polymerase in vitro. (A) Sf9 cells were infected with recombinant baculovirus RBVHEbp1 at moi 5, and incubated for 4 days at 27 °C. Ebp1 was purified from infected cell extract as described in Experimental procedures. (B) In vitro RNA synthesis by influenza virus RNA polymerase was carried out at 30 °C for 60 min in the standard reaction mixture (Honda et al. 2002) using 200 ng purified 3P complex and 1 pmol model template (v-sence or c-sence) and the indicated amount of Ebp1. RNA was extracted with phenol–chloroform, ethanol precipitated, and then analyzed on 10% polyacrylamide gel in the presence of 7 M urea. A typical pattern of v-sense template-directed and primer-dependent RNA synthesis is shown. (C) AMV reverse transcriptase assay was carried out in the reaction mixture containing mRNA from influenza virus-infected cells, primers for NS1 mRNA and increasing amounts of Ebp1. (D) Endonuclease assay was performed at 30 °C for 30 min using [32P]capped poly(A) in the presence or absence of 200 ng 3P complex, 1 pmol model vRNA(53 nts) and increasing amounts of Ebp1. (E) Capped RNA binding assay was performed at 30 °C for 30 min using 200 ng 3P complex, 1 pmol model vRNA (53 nts), [32P]capped poly(A) and increasing amounts of Ebp1. The reaction mixtures were then exposed to UV irradiation followed by treatment with RNase A for 15 min at 37 °C. Reaction products were separated on PAGE and the gel was exposed on X-ray film.

 
Influence of Ebp1 on capped RNA binding and cleavage of capped RNA by influenza virus RNA polymerase

When the influenza virus RNA polymerase functions as transcriptase, capped RNA primers are generated after endonucleolytic cleavage of host cell capped RNA (Krug et al. 1989). PB2 is involved in recognition of RNA 5'-cap structure and binding (Honda et al. 1999), while PB1 is proposed to play a major role in the catalysis of endonucleolytic cleavage of capped RNA (Li et al. 2001). Possible influence of Ebp1 on both capped RNA binding and cleavage was then examined using the purified Ebp1 (Fig. 4D,E). Capped-poly(A) with 32P only at the cap structure was cleaved into capped fragment of 10–12 nucleotides in length by the 3P complex in the presence of v53 model vRNA (Fig. 4D, lane 2) in agreement with our previous observations (Kawakami et al. 1983; Honda et al. 1999). This reaction was, however, not affected by the addition of increasing amounts of Ebp1 (Fig. 4D, lanes 2–4). In the absence of 3P complex, capped RNA substrate was not degraded by the purified Ebp1 (Fig. 4D, lane 5).

Possible influence of Ebp1 on the binding of 3P complex to RNA5'-cap structure was also analyzed. The capped RNA binding assay was carried out in the standard reaction mixture with the purified 3P, the purified Ebp1 and 32P-labeled capped RNA for 30 min at 30 °C followed by UV irradiation and RNase A digestion. The UV-cross-linked proteins were separated on SDS-PAGE and exposed on X-ray film. Result shown in Fig. 4E indicates that the addition of Ebp1 did not inhibit RNA5'-cap binding but rather enhanced. These findings agree with the findings that RNA5'-cap is recognized by PB2 (Honda et al. 1999) and Ebp1 specifically binds to the PB1 subunit (see above).

Influence of Ebp1 on influenza virus replication in vivo

Inhibition of transcription at the initial step of virus growth may lead to reduction in viral proteins for replication and virion formation, ultimately resulting in inhibition of overall virus growth. Effect of Ebp1 on influenza virus replication was then examined using the reverse genetics method (Hoffmann et al. 2000). Eight vRNA-expressing plasmids and four recombinant plasmids for PB1, PB2, PA and NP expression were co-transfected, with or without an expression plasmid of Ebp1 (pCAGGSEbp1), into 293T cells by lipofectin method. Two days after transfection the culture medium was harvested and subjected to plaque assay and HA titration. Results indicated that under the experimental conditions employed, the level of virus production was at least 5 times lower than the control in the absence of Ebp1 expression plasmid (Fig. 5A). PB1 expression in each transformed 293T cells were analyzed using immunoblotting method. When the intact Ebp1 was over-expressed, the levels of PB1 (Fig. 5B) and PB2 (data not shown) were lower than the control culture without Ebp1 expression plasmid. After the over-expression of Ebp1, the cell viability was virtually unaffected as same as control cells by detection using trypan blue (data not shown). The expression level of {alpha}-tubulin was also unaffected (Fig. 5C). Taken together we concluded that Ebp1 interacts with the PB1 subunit of influenza virus RNA polymerase and inhibits the synthesis of virus mRNA and viral proteins, leading to the inhibition of virus replication.


Figure 5
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  		Figure 5  Effect of Ebp1 on virus production in vivo. Influence of Ebp1 on influenza virus growth was tested by using reverse genetics method (Hoffman et al. 2000). In brief, both a mixture of expression plasmids of PB1, PB2, PA and NP proteins and a whole set of virus RNA expression plasmids were transfected into 293T cells in the presence or absence of Ebp1 expression plasmid pCAGGSEbp1. (A) The production of progeny viruses was examined by plaque formation assay on MDCK cells. (B) The expression level of PB1 and Ebp1 was examined by immunoblotting assay using anti-PB1 and anti-Ebp1 antibodies. –, without pCAGGSEbp1; +, with pCAGGSEbp1. (C) The expression of Ebp1 and {alpha}-tublin in the plasmids transfected cells was assayed by immunostaining with anti-Ebp1 and anti-{alpha}-tublin after Western blotting. –, without pCAGGSEbp1; +, with pCAGGSEbp1.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Molecular nature of Ebp1

Using yeast two-hybrid screening system, we identified a set of HeLa cell proteins that interact with influenza vRNA polymerase. Ebp1 is one of the PB1-interacting proteins. Ebp1 specifically binds to PB1 (but not to PB2 and PA) near the primer-binding site within the catalytic domain for RNA polymerization (Fig. 6; also see Fig. 2) and thereby inhibits RNA synthesis in vitro by influenza vRNA polymerase (see Fig. 4B) [but by neither AMV reverse transcriptase (see Fig. 4C) nor T7 and E. coli RNA polymerases (data not shown)]. However, Ebp1 did not inhibit capped RNA binding nor cleavage of capped RNA (see Fig. 4D,E). Instead the capped RNA binding is rather enhanced by Ebp1 (see Fig. 4E). The interpretation that Ebp1 is a selective inhibitor of influenza vRNA polymerase, derived from the in vitro experiments, is further supported by the finding that Ebp1 inhibited the multiplication in vivo of influenza virus (see Fig. 5).


Figure 6
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  		Figure 6  Mapping of PB1-Ebp1 interaction sites. Functional map of influenza virus PB1 protein is described in previous reports (Honda & Ishihama 1997, 2004), while mapping of Ebp1 has been performed by Hamburger and colleagues (Zhang et al. 2002, 2003). The contact sites for PB1-Ebp1 interaction are described in this report. The Ebp1-contact site on PB1 overlaps with the binding site of transcription primers and the catalytic site for RNA synthesis by influenza vRNA polymerase. This agrees well with the inhibitory activity of Ebp1 on the RNA polymerase function. ErbB3, epidermal receptor tyrosine kinase-binding protein; HDAC, histone deacetylase; Rb, retinoblastoma gene product; AR, androgen receptor.

 
Ebp1, a member of the PA2G4 family, was initially isolated as an ErbB3 (an epidermal receptor tyrosine kinase)-binding protein (Yoo et al. 2000). Ebp1 with curved DNA-binding activity inhibits cell growth and represses transcription of E2F-regulated cell cycle genes. After sequence analysis, we found that: Ebp1 carries a nuclear localization signal at the carboxy terminus, in good agreement with its nuclear localization, and a sequence similarity to Met aminopeptidase on the amino-terminal region; and Ebp1 contains a number of potential phosphorylation sites by casein kinase II and protein kinase C.

Here we demonstrated that PB1, the catalytic subunit of influenza vRNA polymerase, directly interacts with the C-terminal domain of Ebp1 (Fig. 6). ErbB3 also interacts at the C-terminal region of Ebp1. Both AR (Zhang et al. 2002) and Rb (Xia et al. 2001) interact with the same region of Ebp1, thereby controlling cellular proliferation and differentiation. Interestingly histone deacetylase 2 (HDAC2) also interacts the same C-terminal region of Ebp1 (Zhang et al. 2003). Ebp1 suppresses AR-mediated transcription, resulting in tumorigenesis of prostate cancer (Zhang et al. 2005). We then propose a prediction that Ebp1 binds influenza vRNA polymerase via PB1 subunit, thereby leading to suppression of virus growth, as it controls cell growth and differentiation by recruiting key regulatory factors.

Mouse homologue p38-2G4 of human Ebp1 is expressed from G1 to S phase in cell cycle-dependent manner (Radomski & Jost 1995), implying possible involvement of Ebp1 in cell cycle control. Schizosaccharomyces pombe contains a homologue of Ebp1 with curved DNA-binding activity (Yamada et al. 1994). We then tried to construct knockout mutant of S. pombe Ebp1 homologue, but the mutant appeared to be lethal (A. Honda, unpublished data), suggesting that the S. pombe homologue of Ebp1 is essential for normal cell growth. These observations altogether indicate that Ebp1 plays an essential role in cell growth control.

Pilipenko et al. (2000) searched for host factors needed for positive-strand virus growth, and found that Ebp1 is required for cap-independent translation initiation at IRES on positive-strand virus genome RNA. In the case of positive-strand viruses, Ebp1 interacts possibly at specific ordered structure within viral RNA, thereby controlling its translation. By contrast, in the case of influenza virus, the same protein binds to vRNA polymerase to modulate its functions. It is noteworthy that the same host protein is involved in growth control of different viruses but in different manner.

Host factors as targets for development of anti-influenza virus agents

Influenza virus has long been a major target of systematic search for effective anti-viral inhibitors, yet the drug target has been focused on viral surface proteins, HA or NA. Recent development in technology of the drug design based on the three dimensional structures of NA molecules has led to create some new effective compounds, which are currently used for clinical treatment of influenza virus infection. However, drug-resistant viruses appear, sooner or later, due to frequent variations of these surface proteins (Kiso et al. 2004).

Mx proteins that are induced by interferons are also known as selective inhibitors of influenza virus growth. Mouse Mx1 protein with GTPase activity is a dynamin-like protein in cell nuclei and represses, directly or indirectly, viral transcription (Mayer & Horisberger 1984; Krug et al. 1985). In the case of human MxA protein, it associates with nucleoprotein (RNP) of Thogoto virus (Orthomyxoviridae) (Kochs & Haller 1999a) and inhibits its transport into infected cell nuclei (Kochs & Haller 1999b). The contact target of MxA protein has been suggested to locate on the NP protein of influenza virus (Kochs & Haller 1999a; Turan et al. 2004). On the other hand, influenza virus NS1 protein functions as an interferon antagonist by preventing the synthesis of IFNs during influenza virus infection (Wang et al. 2000). Influenza PB1-F2 protein, a short polypeptide encoded by the RNA segment containing the PB1-coding sequence, is transported into mitochondria and induces cell death, thereby resulting in down-regulation of the host cell response to influenza infection (Zamarin et al. 2005). Manipulation of PB1-F2 could be a potential target of a new strategy of influenza control. Hsp90 was also identified as a host protein, which interacted with influenza virus RNA polymerase subunit PB2 and stimulated vRNA synthesis (Momose et al. 2002).

Systematic screening has, however, never been performed for search of the drugs against the viral RNA polymerase, the key enzyme for virus replication, because of lack of high-level production system of the viral RNA polymerase. Here we demonstrated, for the first time, that Ebp1 is a selective inhibitor of transcriptase activity of influenza virus. Controlled expression of Ebp1 could be a potential anti-viral therapy against influenza virus infection. In progress analysis of other influenza virus RNA polymerase-interacting host proteins will provide novel strategies for influenza virus growth control.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Yeast two-hybrid screening

For initial screening of P protein-interacting host proteins, a full-length cDNA of each P protein was inserted into a bait vector (pHybLex/Zeo) to express fusion proteins with the DNA-binding domain, while a HeLa cell cDNA library (Invitrogen) was inserted into a prey vector (pYes/Trp2) to construct an expression plasmid library of fusion proteins with the activation domain. After co-transfection of two species of plasmid into yeast S. cerevisiae L40, transformants were subjected to first screening on the selection medium containing 300 mg/mL Zeocin without Trp, His and Lys. Viable colonies were picked up and subjected to second screening for detection of ß-galactosidase activity. Zeocin-resistant and ß-galactosidase-positive cDNA clones were isolated. Each cDNA sequence was determined using plasmid amplified in E. coli. For confirmation, two-hybrid-screening was performed in opposite combinations. Finally cDNA of positive clone was cloned from mRNA of HeLa cell.

Yeast two-hybrid screening was also used for confirmation of interaction between the identified HeLa cell proteins and influenza virus P proteins, and for mapping of the contact sites between the two proteins. Each P protein cDNA or its segments were inserted into pHybLex/Zeo vector so as to express the respective fusion proteins with the DNA-binding domain while cDNA of Ebp1 and its segments were expressed as fusion proteins with the activation domain.

Purification of the viral 3P complex and Ebp1

Construction of the recombinant baculoviruses for expression of each P protein of influenza vRNA polymerase was described in elsewhere (Honda et al. 2002). The recombinant baculovirus for each P protein was co-infected into Tn5, and cultured for 72 h at 27 °C. Cells were disrupted in lysis buffer, and cell lysates were centrifuged at 800 g for 5 min at 4 °C. The 3P complex were purified from nuclear extracts using Cobalt-chelating resin as described in Honda et al. (2001).

The full-sized cDNA of Ebp1 was cloned into pGEX 4T-1 plasmid for expression of GST-fused in E. coli. For construction of recombinant baculovirus of Ebp1, the full-sized cDNA was inserted into pFastBac (GibcoBRL), and the resulting pFastBacHEbp1 (his-tagged Ebp1) was transfected into E. coli DH10Bac competent cells. Transformants were screened on Bluo-gal agar plate (Gibco-BRL) containing Kanamycin and Tetracyclin. After checking the DNA inserts in bacmids isolated from white colonies, the recombinant bacmid DNA was purified by centrifugation in cesium chloride and transfected into Sf9 insect cells by electroporation to generate recombinant baculovirus RBVHEbp1.

Sf9 cells were infected with RBVHEbp1 at moi 5 and after incubation for 3 days at 27 °C, the cells were harvested by centrifugation at 800 g for 5 min. Cell pellet was homogenized with Dounce homogenizer in a hypotonic buffer containing 20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 0.1% Triton X-100 and 10 mM KCl. Cell extract was centrifuged at 800 g for 5 min, and the nucleus was homogenized with Dounce homogenizer in an extraction buffer containing 20 mM HEPES (pH 7.5), 0.3 M NaCl, 1.5 mM MgCl2, 0.1% Triton X-100, 25% glycerol and 5 mM DTT. After centrifugation at 8000 g for 1.5 h, the supernatant was subjected to Ni-NTA column chromatography. The column was washed extensively with a washing buffer containing 20 mM HEPES (pH 7.5), 0.5 M NaCl, 40 mM imidazole, 1% Triton X-100 and 20% glycerol, and then Ni-NTA-bound proteins were eluted with an elution buffer containing 20 mM HEPES (pH 7.5), 0.3 M NaCl, 0.1 M imidazole and 20% glycerol.

Preparation of [35S]methionine-labeled P proteins

35S-labeled P proteins were translated in vitro in the rabbit reticulocyte lysate, containing amino acid mixture with [35S]methionine instead of methionine and mRNA for each P protein, for 60 min at 37 °C. mRNA of each P protein was transcribed in the reaction mixture with cap analogue, ATP, GTP, CTP, UTP and SP6 RNA polymerase.

GST pull-down assay

[35S]Met-labeled P protein was mixed with GST-fused C-terminal region of Ebp1 (229–394) in 20 mM HEPES (pH 7.5), 1.5 mM Mg acetate and 1 mM DTT, incubated for 60 min at 30 °C, and then mixed with glutathione-bound resin. Resin-bound proteins were eluted by increasing concentrations of glutathione, and analyzed by SDS-PAGE.

Co-immunoprecipitation assay

Anti-Ebp1, anti-NP and anti-PB1 were raised in rabbits after immunization with the respective purified proteins. For co-immunoprecipitation test, the cell lysate labeled with [35S]Met was mixed with anti-Ebp1, anti-PB1 or anti-NP, and incubated for 60 min on ice. After addition of protein A, incubation was continued for another 60 min. Protein A-bound immunoprecipitants were eluted with SDS-sample buffer and separated by SDS-PAGE. Gels were exposed to imaging plate, which were analyzed with BAS (Fuji Film Co., Japan).

In vitro RNA polymerase assays

RNA synthesis in vitro by influenza virus 3P complex was carried out at 30 °C for 60 min in the standard reaction mixture, which contained 20 mM HEPES (pH 7.8), 0.1 M NaCl, 1.5 mM Mg acetate, 1 mM DTT, 0.1% Triton X-100, 0.25 mM each of ATP, CTP and GTP, 0.1 mM UTP, 5 µCi [{alpha}-32P]UTP, globin mRNA or 0.25 mM ApG as primer, 200 ng purified 3P complexes, 1 pmol model template (v-sense or c-sense) (Parvin et al. 1989) and in the presence or absence of various concentrations of Ebp1. After in vitro RNA synthesis, RNA was extracted with phenol–chloroform, precipitated with ethanol, and then analyzed by electrophoresis on 10% polyacrylamide gel containing 7 M urea.

Escherichia coli RNA polymerase was purified in this laboratory and assayed under the standard reaction conditions in the presence or absence of various concentrations of Ebp1.

Reverse transcriptase assay

The reverse transcriptase assay by AMV reverse transcriptase was carried out in the presence or absence of various amount of Ebp1 at 37 °C for 60 min in the reaction mixture, which contained 3 µg RNA from influenza virus-infected cells, 1 fmol primer for NS1 gene, 0.2 mM each of dATP, dGTP and dTTP, 10 µM dCTP and 0.5 mCi [{alpha}-32P]dCTP. Reaction products were analyzed by PAGE on 6% denatured gel.

Capped RNA cleavage and RNA-cap binding assays

To check possible influence of Ebp1 on the endonuclease activity of influenza vRNA polymerase, capped RNA with 32P only at cap position was prepared using 100 pmol of poly(A), [{alpha}-32P]GTP, 10 mM S-adenosyl methionine (SAM) and 5 U vaccinia virus guanylytransferase (Ambion) at 37 °C for 60 min. The endonuclease reaction was carried out in the reaction mixture (Honda et al. 1999), which contained 20 mM HEPES (pH 7.8), 0.1 M NaCl, 1 pmol v53 template, 1 mM DTT, 0.1% Triton X-100, 1.5 mM Mg acetate and purified 3P complex and in the presence or absence of various amounts of Ebp1, at 30 °C for 30 min. Products were analyzed by 12% PAGE in the presence of 7 M urea followed by autoradiography.

RNA-cap binding assay was carried out as described previously (Honda et al. 1999). The reaction mixture contained poly A with radioactive cap structure, 20 mM HEPES (pH 7.8), 0.1 M NaCl, 1.5 mM Mg acetate, 0.1% Triton X-100, 1 pmol v53 template (Parvin et al. 1989), purified 3P complex, and in the presence or absence of various amounts of Ebp1. After incubation for 30 min at 30 °C, the mixtures were subjected to UV cross-linking for 30 min on ice and digested with RNase A for 15 min at 37 °C. Proteins cross-linked with radioactive cap were analyzed by SDS-PAGE followed by autoradiography.

Influenza virus production using reverse genetics method

Influenza virus production using reverse genetics method was performed essentially according to Hoffmann et al. (2000). For expression of Ebp1, the cDNA of Ebp1 was inserted into an expression plasmid pCAGGS. The expression levels of viral proteins and Ebp1 were checked by immunostaining. The virus replication was assayed by plaque formation and HA titration. The cell viability was determined using Trypan Blue (Gibco).

Western blotting assay for Ebp1 and {alpha}-tubulin

The Ebp1 over-expressed cells and control cells were disrupted in the extraction buffer and subjected to SDS-10%PAGE followed by blotting onto the PVDF membrane. The membrane was incubated with anti-Ebp1 or anti-{alpha}-tubulin and then anti-rabbit/mouse IgG conjugated with horseradish peroxydase followed by detection with DAB.


   Acknowledgements
 
We thank Y. Kawaoka (University of Tokyo) for supply of plasmids for the reverse genetics system. This work was supported by Grant-in-Aid to AH (17076017) and AI (17076016) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Hiroshi Handa

* Correspondence: E-mail: ayhonda{at}k.hosei.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 16 June 2006
Accepted: 22 October 2006




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