Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit

ELSEVIER

Virus Research

Virus Research 42 (1996) 1-9

Recombinant-baculovirus-expressed PB2 subunit

of the influenza A virus R N A polymerase binds cap groups as an isolated subunit Licheng

S h i a'l, J o s e M . G a l a r z a a, D o n a l d

F. S u m m e r s a'b'*

aDepartment of Microbiology and Molecular Genetics, University of California, Irvine, CA 92717-4025, USA bDepartment of Medicine, University of California, Irvine, CA 92717-4025, USA

Received 7 October 1995; revised 8 January 1996; accepted 8 January 1996

Abstract

The influenza A viras RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the recognition of the 5' end cap structure of host cell hnRNA and the cleavage of the RNA substrate between 10 and 14 nucleotides from the 5' end to generate capped primers for initiation of transcription of virus-specific mRNAs. This report describes the use of an in vitro UV crosslinking and protein renaturation assay to identify the polymerase subunits which interact with the 5' end cap structure of an artificial RNA substrate. Our results showed, for the first time, that purified polymerase subunit PB 2 expressed by recombinant baculovirus in insect cells possessed cap-binding activity by itself after renaturation by Escherichia coli thioredoxin, whereas cleavage of the artificial capped substrate required the holoenzyme expressed in insect cells triply-infected with baculovirus containing all three polypeptide components, PB~, PB2, and PA. Purified polyclonal anti-PB2 I~gG inhibited the binding activity; anti-PBt and anti-PA IgGs did not. Keywords: Influenza A virus RNA polymerase; UV crosslinking; Protein renaturation; Cap binding activity

I. Introduction

Influenza A viruses are negative-stranded R N A viruses belonging to, the family Orthomyxoviridae. The virus genome consists of eight minus-sense, single-stranded R N A segments which are tightly * Corresponding author. Tel.: + 1 714 824 4534; fax: + 1 714 824 8598; e-mail: [email protected] ~Present address: Laboratory for Neurovirology, Department of Neurology, University of California, Irvine, CA 92717, USA.

associated with the viral nucleoprotein (NP) and the RNA-dependent R N A polymerase complex (PB1, PB2, and PA). These ribonucleoprotein complexes (RNPs) are responsible for the transcription and replication of the virus-specific R N A s in the nucleus of infected cells in a highly regulated process. During transcription a capped, polyadenylated, incomplete plus-strand is synthesized from each of the genomic segments, whereas during replication complete plus and minus-sense copies are synthesized without cap groups and polyadenylation (Hay et al., 1977; Robertson et

0168-1702/96/$15.00 © 11996Elsevier Science B.V. All rights reserved PH SO168-1702(96)01289-0

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L. S h i e t al./ Virus Research 42 (1996) 1 - 9

al., 1981). It has been assumed that replication is carried out by the same RNA-dependent RNA polymerase or a modified form of the enzyme that is responsible for transcription. The first step in influenza virus-specific m R N A synthesis is the recognition of the 5' end cap groups of host cell heterogeneous nuclear RNAs (hnRNAs) and the endonucleolytic cleavage of these RNA substrates to generate small capped RNA fragments which act as primers to initiate transcription (Plotch et al., 1981). It has been speculated that association of the capped RNA with the viral RNPs is caused by interactions other than base pairing with the viral template RNA. This means the cap structure is the only signal of recognition of the primer RNA. Therefore, it seems likely that some cap binding sites must be present in the polymerase complex. Striking homologies have been found between the amino acid sequence of the PB2 subunit and consensus sequences of cap-binding proteins (Rychlik et al., 1987). Previous studies have found that PB2 subunit in purified influenza A virus WSN strain viral cores binds to cap-labeled alfalfa mosaic virus (A1MV) R N A 4 fragments by using ultraviolet (UV) light-induced crosslinking and a two-dimensional gel electrophoresis assay (Ulmanen et al., 1981). Another research group, using a photoreactive derivative of mYGTP as an affinity label, showed that PB 2 protein of influenza virus PR8 binds cap structures and is most likely a cap-recognizing protein (Blaas et al., 1982). Genetic studies have also shown that the cap-dependent binding of a specific primer fragment (A~3 fragment of A1MV RNA 4) to influenza WSN strain temperature sensitive (ts) viral cores was temperature sensitive under conditions in which binding to wild type viral cores was not affected by increasing the temperature from 33 to 39.5°C (Ulmanen et al., 1983). A recent paper reported that PB2 was not required for influenza virus genome replication but was involved in capped m R N A synthesis (Nakagawa et al., 1995). These observations suggested that PB2 protein functions in the recognition of the 5' end cap structure that occurs as a required first step in the endonuclease cleavage and initiation of transcription.

In this study, we utilized an UV crosslinking and protein renaturation assay to investigate interactions between influenza virus RNA polymerase and cap group at the 5' end of artificial RNA substrate in vitro. We report here that purified polymerase subunit PB2 expressed by recombinant baculovirus in Sf9 cells possessed specific cap-binding activity by itself after renaturation by Escherichia coli thioredoxin. We also showed that A1MV R N A 4 and cap analog inhibited the cap-binding activity. Purified polyclonal anti-PB2 IgG blocked the binding, but antiPA and anti-PB~ IgGs did not. These results suggested that recombinant baculovirus expressed PB 2 subunit of influenza RNA polymerase was able to bind the cap groups of host cell hnRNAs as an isolated subunit, but cleavage only occurred with holoenzyme.

2. Materials and methods

2.1. Isolation of virion RNPs The procedure for purification of virion RNPs was described before (Shi et al., 1995). Briefly, influenza A WSN virus was grown in 10-day-old embryonated chicken eggs and concentrated from allantoic fluid by centrifugation through a 20% sucrose cushion, then the concentrated virus pellet was further purified by centrifugation in a 20-45% (w/v) potassium tartrate gradient at 10 000 g for 12 h at 4°C. The viral particles were disrupted by 1% Triton X-100 and 0.2% lysolecithin, loaded on a 25% glycerol cushion and centrifuged at 100 000 g for 4 h at 4°C. RNP complexes were collected from the bottom of the centrifuge tube from the glycerol cushion and stored at - 70°C.

2.2. Expression and purification of polymerase subunit PB 2 by recombinant baculovirus in Sf9 cells

Sf9 cells were infected with baculovirus-polymerase subunit PB2 recombinant at a multiplicity of 5 pfu/cell. After 4 days of infection, the cells (2 X 107) were harvested and disrupted in 1 ml NT

L. Shi et al. / Virus Research 42 (1996) 1-9

buffer (10 mM Tris-HC1, pH 7.4, 100 mM NaC1) containing 1% NP 40, and cytoplasm and nuclei were separated by centrifugation (1000 g, 10 rain). Expressed PB 2 protein was analyzed by electrophoresis in sodium dodecyl sulfate (SDS)8% polyacrylamide gel. The infected Sf9 whole cells were boiled for 3 rain in gel sample buffer and resolved by electrophoresis in a 8% polyacrylamide-SDS gel running at 200 V for 3 h. An Immobilon-P transfer membrane (Millipore) was prepared for protein transfer by rinsing with 100% methanol followed by transfer buffer (20 mM Tris-HC1, pH 8.0, 150 mM glycine). Proteins were transferred at 4°C for '.2 h at 50 V from the gel to the membrane in transfer buffer. Following transfer, the membrane was immersed in 0.1'7o amido black for 10 min to stain the proteins. The membrane was destained in water until the proteins could be visualized. The PB 2 band was excised and cut into small pieces to fit in an Eppendorf tube, and 300 121 of elution buffer (50 mM Tris-HC1, pH 9.0, 2% SDS, 1% Triton X-100, 150 mM N aC1) was added to elute the protein at 4°C overnight. The liquid was collected, and the protein was precipitated from the eluates with 4 V of cold acetone and incubated for 30 rain in a dry ice-ethanol bath. The precipitated proteins were pelleted by centrifugation in a microcentrifuge at 14000 rpm for 20 min. The pellet was washed with 80% cold acetone in buffer A (50 mM Tris-HC1, pH 8.0, 20% glycerol, 150 mM NaC1, 1 mM DTT, and 0.1 mM EDTA). The pellet was resuspended in 20 /21 of 6 M guanidine-HCl in buffer A and incubated for 15 rain at room temperature to completely dissolve the pellet, and then the guanidine concentration was lowered by the addition of 180 /ll of buffer A and dialyzed for 4 h at 4°C against buffer A to remove the guanidine. The purified protein was further analyzed by Western blotting using a polyclonal rabbit antiserum against virion PB2. Sf9 cells were also infected with baculovirus recombinants containing the PB~ gene. The expressed PB~ protein was then purified as described above.

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2.3. Protein renaturation after elution from Immobilon membrane E. coli thioredoxin (1 raM) (Promega) was preincubated with 0.2 mM DTT for 30 min at 37°C, then 100 /2M reduced thioredoxin was mixed with 1 /~g eluted PB 2 protein in buffer A. The mixture was incubated overnight at 4°C for protein renaturation and the recovered protein activity was determined by testing the cap-binding activity. 2.4. Synthesis of' R N A substrate for the polymerase cap-binding activity

The construction of a recombinant plasmid containing a partial A1MV R N A 4 sequence (nt 7 18) downstream from a SP6 promoter in pSP6/ T7-19 plasmid was described previously (Shi et al., 1995). This plasmid was used as the template for RNA synthesis in vitro, and transcription was performed by using SP6 R N A polymerase. The R N A transcripts were purified by using a G-50 Sephadex column (Boehringer) and the R N A was then capped with vaccinia virus guanylyltransferase (GIBCO BRL) was further purified by electrophoresis on a 10% polyacrylamide gel as described (Shi et al., 1995). 2.5. Cap-binding reaction and U V crosslinking

The standard reaction for the R N A polymerase-associated cap-binding activity contained in a final volume of 20/21:20 mM HEPES, pH 7.5, 0.5 mM MgC12, 2 mM DTT, 25 mM potassium acetate, 50 ng capped RNA substrate or uncapped radiolabeled RNA substrate and 0.5 /2g purified virion RNPs or 1 /2g renatured, baculovirus-expressed PB2 subunit. The reaction was carried out for 20 rain at 30°C, then the reaction mixtures were irradiated at 254 nm for 45 min on ice for UV crosslinking at a distance of 8 cm with 40 watt UV lamps, producing a power of about 3000 /~watts/cm2 by using Stratalinker UV Crosslinker (Strategene). Samples were subsequently treated with 20 /2g/ml RNase A for 20 rain at 37°C. The crosslinked products were analyzed by 8% polyacrylamide-SDS gel running at

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L. Shi et al. / Virus Research 42 (1996) 1 - 9

200 V for 3 h. The gels were either transferred to the membrane for Western blotting assay or dried to reveal the radiolabeled proteins by autoradiography.

2.6. Blocking experiment with purified polyclonal IgGs against the three polymerase subunits Rabbit polyclonal IgGs against PA, PB 1 and PB2 were prepared by using recombinant baculovirus expressed polymerase subunits as antigens and purified by Protein-A-Sepharose column chromatography as described (Sambrook et al., 1989). Purified IgGs (0.5/tg) were mixed with 0.5 /~g viral RNPs or 1 /tg purified PB2 protein, preincubated for 20 rain at 30°C and cap-binding reactions were carried out after adding the RNA substrate.

2. 7. Inhibition of the cap-binding activity by ApG and cap analog Different concentrations of ApG (Sigma) and cap analog (mVGpppGm) (Pharmacia) were mixed with 0.5/~g RNPs and preincubated for 20 min at 30°C, then 50 ng cap-labeled RNA substrate was added to the reaction and incubated for an additional 20 min. After UV crosslinking and RNase A digestion, the reaction mixtures were analyzed by SDS-PAGE.

3. Results

3.1. Specific cap-binding activity of influenza RNA polymerase PB2 We previously established an in vitro assay to investigate the endonuclease activity mediated by the influenza virus RNA polymerase. We found that all three polymerase subunits from recombinant baculovirus triply-infected Sf9 cells were needed to cleave the artificial RNA substrate containing [32P]-labeled cap structure and partial A1MV RNA 4 sequence (Shi et al., 1995). Also, Hagen et al. (1994) showed that all three polymerase subunits from HeLa cells that had been triply-infected with recombinant vaccinia virus

vectors containing the three influenza polymerase subunits were required for the endonuclease activity (Hagen et al., 1994). In this report, we initially used the same substrate and purified viral RNPs to study the protein-cap interaction by UVcrosslinking and SDS-PAGE. The cap-labeled RNA substrate or uncapped UTP-labeled RNA substrate were incubated with purified virion RNPs, then the mixture was exposed to UV light to crosslink the cap to the proteins recognizing it. These samples were treated with RNase A and analyzed by electrophoresis on a 8% polyacrylamide SDS gel to identify the [32p]-radiolabeled proteins in viral polymerase. In the presence of the cap-labeled RNA, one major band was observed at the expected position of PB 2 subunit (Fig. I(A), lane 1). The subsequent Western blotting showed that this labeled band was PB 2 by using specific anti-PB2 IgG (data not shown). No band was shown in the presence of uncapped UTP-labeled RNA substrate (Fig. I(A), lane 2). To further investigate the specificity of cap-binding activity of PB2 in viral RNPs, the effect of A1MV RNA 4 and tRNA on the cap binding activity were studied. The results showed that the cap-binding activity was inhibited by A1MV RNA 4 but not inhibited by tRNA (data not shown). Further immunoprecipitation studies showed that anti-PB2 IgG precipitated the PB2-cap complex specifically, but anti PB1 and anti-PA IgGs did not precipitate any labeled protein (data not shown). These experiments confirmed that the PB2 subunit in polymerase complexes was the cap recognizing protein (Ulmanen et al., 1981; Blaas et al., 1982; Braam et al., 1983).

3.2. Effect of antipolymerase subunit IgGs on crosslinking of subunit protein to cap structure In an attempt to determine whether anti-subunit IgGs would inhibit cap-binding, virion RNPs were crosslinked in the absence or presence of the anti-polymerase subunit IgGs. As shown in Fig. I(B), only purified IgG against PB2 subunit blocked the crosslinking of the protein to the cap structure (Fig. I(B), lane 3); whereas, crosslinking of protein to the labeled cap was not reduced in the absence of any IgG (Fig. I(B), lane 1) or in

L. Shi et al. / Virus Research 42 (1996) 1-9 the presence o f antii-PB 1 and anti-PA I g G s (Fig. I(B), lanes 2 and 4).

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3.3. Expression and purification o f polymerase subunit PBe from baculovirus-infected Sf9 cells Sf9 cells infected with PB2 recombinant baculovirus were analyzed by 8% polyacrylamideSDS gel. The results showed that PB 2 protein was expressed at high levels in S19 cells and almost all o f the expressed PB2 was localized in the cell nuclei (data not shown), as had been observed by others (Angelo et al., 1987; K o b a y a s h i et al., 1992). To study the cap-binding activity o f baculovirus expressed PBz, the PB z protein was purified by elution o f the protein from an I m m o bilon m e m b r a n e after S D S - P A G E separation and electroblotting. PBz protein was eluted from the membrane, acetone precipitated and dialyzed after being dissolved in 6 M guanidine. Purified PB 2 was analyzed by 8% polyacrylamide-SDS gel and visualized by silver staining (Fig. 2(A), lane 2). Baculovirus expressed PB2 with virion PB2 in Western blotting using rabbit polyclonal serum against PB2 showed that the expressed PB2 was immunologically similar to virion PB2 (Fig. 2(B)).

3.4. Baculovirus recombinant expressed PB2 subunit binds cap groups as an isolated subunit

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Fig. 1. (A) Identification of a cap-binding protein in influenza viral RNPs. The cap-labeled RNA substrate or uncapped UTP-labeled RNA substrate were incubated with purified influenza A viral RNPs. After incubation for 20 min at 30°C, the samples were UV crosslinked and treated with 20 /tg/ml RNase A for 20 rain a L37°C. The proteins were analyzed by SDS-PAGE. Lane 1, RNPs (0.5 /~g) plus 50 ng cap-labeled RNA substrate; lane 2, RNPs (0.5 /tg) plus 50 ng uncapped radiolabeled transcript. (B) Effect of anti-polymerase subunit IgGs on crosslinking of polymerase subunit to labeled cap. RNPs (0.5 #g) were preincubated with 0.5 ~g purified lgGs against PB1, PB2 and PA for 20 min at 30°C, then 50 ng cap-labeled RNA substrate was added to each reaction and incubated for an additional 20 rain before UV crosslinking. Lane 1, RNA (50 ng) plus 0.5 ,ug RNPs; lane 2, same as lane 1, but 0.5/tg anti-PB I IgG was added; lane 3, same as lane l, but 0.5/tg anti-PB2 IgG was added; lane 4, same as lane 1, but 0.5 /tg anti-PA IgG was added.

O u r previous results had shown that all three polymerase subunits were required for the endonuclease activity (Shi et al., 1995). We also wanted to see whether PB 2 protein alone was able to bind the cap g r o u p o f artificial R N A substrate, or whether all three subunits were required. Initially, purified PB 2 protein expressed by recombinant baculovirus in insect cells was used to study the cap-binding activity, but no activity was observed (data not shown). It was possible that either PB 2 protein expressed in Sf9 cells was denatured and, therefore, was unable to bind the cap group, or that all three polymerase subunits were required for cap-binding activity as was the case for cleavage. To study these possibilities, PB 2 subunit expressed in Sf9 cells and purified was renatured with D T T and E. coli thioredoxin (Szewczyk et al., 1988), and cap-binding activity was tested following this renaturation. The results showed that PB2 alone could bind the cap structure o f the R N A substrate (Fig. 3(A), Lane 1),

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3.5. Effect of cap analog and ApG on the cap-binding activity

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but not bind to the uncapped UTP-labeled same RNA substrate (Fig. 3(A), lane 2). This binding activity was inhibited by increasing amount of A1MV RNA 4 (Fig. 3(B), lanes 2 and 3) but not inhibited by tRNA (data not shown). In a control experiment, polymerase subunit PB~ was purified from baculovirus infected Sf9 cells renatured and assayed for cap-binding activity. PB~ protein did not show any cap-binding activity (Fig. 3(C), lane 2). The effect of anti-polymerase IgGs on the cap-binding was also studied, as expected, only anti PB2 IgG blocked the cap-binding activity of PB2 subunit expressed in Sf9 cells (data not shown).

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|"-'~[ Fig. 3. (A) Specific cap-binding activity of polymerase subunit PB 2 expressed by recombinant baculovirus. The cap-labeled RNA substrate or uncapped radiolabeled RNA substrate were incubated with purified renatured polymerase subunit PB 2 expressed in Sf9 cells. After incubation for 20 min at 30°C, the samples were UV crosslinked and treated with 20 /~g/ml RNase A for 20 min at 37°C. The proteins were analyzed by SDS PAGE. Lane l, PB 2 (1 pg) plus 50 ng cap-labeled RNA substrate; lane 2, PB 2 (1 /~g) plus 50 ng uncapped RNA substrate. (B) Dose response effect of the competitor RNA on cap-binding activity. Purified PB2 was preincubated with different amount of A1MV RNA 4, the cap-binding reaction was carried out by adding the cap-labeled RNA substrate. Lane 1, PB 2 (1 l~g) plus 50 ng RNA substrate; lane 2, same as lane 1, but 100 ng AIMV RNA 4 was added; lane 3, same as lane 1, but 500 ng AIMV RNA 4 was added. (C) Purified polymerase subunits PB~ and PB 2 were incubated with cap-labeled artificial RNA substrate and then crosslinked. The results were analyzed by S D S - P A G E . Lane l, PB 2 (1 ~lg) plus 50 ng RNA substrate; lane 2, PB~ (1 ,ug) plus 50 ng RNA substrate.

L. Shi et al. / Virus Research 42 (1996) 1--9

specific competitors, either capped R N A such as fl-globin m R N A (Ulmanen et al., 1981) or cap analog (Blaas et al., 1982; Penn et al., 1982). Our previous data also showed that the endonuclease activity of purified influenza R N P s was specifically inhibited by the addition of A p G and cap analog (Shi et al., 1995). To evaluate the specificity of the cap-binding activity of PB2 with capped R N A substrate we tested the effects of cap analog (mVGpppGm) and A p G on the cap-binding function. The results showed that cap-binding activity was inhibited partially by the addition of 0.1 m M and 0.5 m M cap ar~alog and was inhibited completely by the addition of 1 m M cap analog (Fig. 4(A), lanes 2-4), but 0.5 m M A p G did not inhibit the cap-binding activity and 1 m M A p G only had partial inhibition in the same reaction conditions and same exposure time (Fig. 4(B), lanes 2 and 3).

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4. Discussion The roles of each subunit of influenza virus R N A polymerase have been studied by using viral R N P s and reconstituted R N A polymerase (Huang et al., 1990; Seong and Brownlee, 1992; Fodor et al., 1993). Prior genetic and in vitro UV crosslinking studies had shown that influenza virus polymerase PB2 possessed the cap-recognizing activity which was required as the first step for viral m R N A synthesis (Blaas et al., 1982; Braam et al., 1983) In this report, we utilized an in vitro UV crosslinking and protein renaturation assay to further investigate whether PBz alone, expressed by recombinant bac,ulovirus in Sf9 cells, was able to bind the 5' end cap structure of an artificial R N A substrate. Results showed that purified PB2 protein possessed thee ability to bind the cap structure of the R N A substrate after renaturation with E . coli thioredoxin but not bind to the same R N A substrate without cap. Purified polyclonal antiPB2 I g G specifically blocked the binding activity, but purified IgGs against PB], and PA did not. In addition, A1MV R N A 4 inhibited the binding, but uncapped R N A , such as t R N A , did not inhibit the binding. Furthermore, the purified PB1 expressed by recombinant baculovirus in Sf9 cells did not show any cap-binding activity by itself. These

Fig. 4. Effects of cap analog and ApG on the cap-binding reaction. RNPs (0.5 /~g) were preincubated with different concentrations of cap analog or ApG for 20 min at 30°C, then 50 ng RNA substrate was added to the reaction and incubated for 20 rain at 30°C. (A) Lane 1, RNPs (0.5 fig) plus 50 ng RNA substrate; lane 2, same as lane 1, but 0.1 mM cap analog was added; lane 3, same as lane 1, but 0.5 mM cap analog was added; lane 4, same as lane 1, but 1 mM cap analog was added. (B) Lane 1, RNPs (0.5 /tg) plus 50 ng RNA substrate; Lane 2, same as lane 1, but 0.5 mM ApG was added; lane 3, same as lane 1, but 1 mM ApG was added. The results were evaluated after same exposure time of the gel to X-ray film. results clearly demonstrated that PB 2 protein was capable of binding the 5' end cap group specifically as an isolated subunit. Our previous data had shown that anti-PB2 I g G specifically blocked the endonuclease activity (Shi et al., 1995) and transcription primed by A1MV R N A 4 (unpublished data). We speculated that anti-PB2 IgG blocked the cap-binding domain of PB2 protein, which lost the ability to recognize the cap groups of host cell hnRNAs. Therefore, subsequent endonuclease cleavage and transcription were inhibited due to lack of the primer providing substrates.

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L. Shi et al. / Virus Research 42 (1996) I - 9

A recent report from Cianci et al. (1995) showed that the recombinant polymerase expressed in HeLa cells, using recombinant vaccinia virus vectors containing the three influenza polymerase subunits, possessed the cap-binding activity after binding to 5' end viral R N A (Cianci et al., 1995). We also found that partially purified polymerase expressed by recombinant baculovirus in triply-infected Sf9 cells was able to bind the cap structure of the artificial R N A template (data not shown). On the basis of this, we further studied the cap-binding activity of PB 2 alone using a protein purification and renaturation assay which had been successfully used previously (Szewczyk et al., 1988; Szewczyk and Summers, 1988). This method will enable us to study the functions of each polymerase subunit in viral transcription and replication. Using this procedure, we found that purified PB 2 expressed in Sf9 cells was initially inactive in cap-binding reaction, but did possess cap-binding activity by itself after renaturation with E. coli thioredoxin. We previously showed that only the recombinant polymerase from triplyinfected insect cells possessed the endonuclease activity and complexes containing only two or single subunits did not have any cleavage function. These diversities of function were probably due to different configurations. It had been previously shown that cap analog and the dinucleotide, ApG, were able to inhibit the endonuclease activity of influenza viral polymerase (Kawakami et al., 1983). Therefore, we investigated their effect on the cap-binding step. We found that cap analog was able to inhibit binding activity of PB2 completely, whereas only 1 mM ApG showed minor inhibition in the same reaction condition and exposure time. It has been shown that ApG is able to substitute in some manner for capped oligonucleotide but is a 1000 fold less efficient than priming by globin m R N A (Plotch and Krug, 1977; Bouloy et al., 1978). Also, previous reports had shown that the transcription reaction primed by ApG was stimulated by 7-methylated cap analogs indicating that the two binding sites most likely were different (Bouloy et al., 1980; Kawakami et al., 1985). Therefore, the inability of ApG to inhibit cap-binding might suggest that ApG and cap bind to different sites in the polymerase complexes.

Elution of proteins from SDS PAGE with subsequent recovery of activity by renaturation is potentially a very useful technique. The activity of some enzymes has been recovered by using different renaturation procedures (Hager and Burgess, 1980; Weissman et al., 1994) and it was previously suggested that E. coli thioredoxin might catalyze the formation of correct disulfide bonds during protein renaturation because of its ability to act as an efficient oxidoreductant (Holmgren, 1979; Pigiet and Schuster, 1986). Previously we had been successful using S D S - P A G E and subsequent renaturation with thioredoxin and DTT of influenza A virus R N A polymerase. After renaturation of the individual purified subunits, they were recombined and enzyme activity was recovered (Szewczyk et al., 1988). In the present study, we used a similar method to purify PB 2 protein expressed in Sf9 cells and renature it by using DTT and E. coli thioredoxin. Following renaturation we showed that purified PB2 possessed capbinding activity, but PB 2 which had not been reacted with thioredoxin showed no activity. We had no means of establishing what level of activity had been recovered and, therefore, the efficiency of renaturation. However, since this procedure has been successful in recovering PB2 function we are now studying the use of this procedure in recovering holoenzyme activity from triply-infected Sf9 cells and recombining subunits from single infections.

Acknowledgements This work was supported by National Institute of Health grant AI 12316. We would like to thank Drs. Shirley A. Harmon, Oliver Richards and Ellie Ehrenfeld for stimulating discussions and helpful advice.

References Angelo, C.ST., Smith, G.E., Summers, M.D. and Krug, R.M. (1987) Two of the three influenzaviral polymeraseproteins expressed by using baculovirus vectors form a complex in insect cells. J. Virol. 61, 361 365.

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