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• W2078796009 abstract "Article1 July 1999free access Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates Susana González Susana González Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Juan Ortín Corresponding Author Juan Ortín Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Susana González Susana González Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Juan Ortín Corresponding Author Juan Ortín Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain Search for more papers by this author Author Information Susana González1 and Juan Ortín 1 1Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:3767-3775https://doi.org/10.1093/emboj/18.13.3767 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of ∼7×10−8 M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5′ and 3′ arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5′ and 3′ arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates. The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates. Introduction The influenza A viruses are members of the family Orthomyxoviridae that produce epidemics of respiratory infections every year and occasionally severe pandemics. Their genome consists of eight single-stranded RNA segments of negative polarity with a coding capacity for 10 proteins (for reviews, see Krug et al., 1989; Lamb, 1989). The transcription and replication of these RNA segments take place in the nucleus of the infected cells (Herz et al., 1981; Jackson et al., 1982) and are mediated by ribonucleoprotein (RNP) complexes in which the vRNA template is associated with the nucleoprotein (NP) and the three P proteins (PB2, PB1 and PA) (reviewed in Krug et al., 1989; Lamb, 1989). Replication of vRNA molecules takes place by generation of full-length RNA copies of positive polarity (cRNA) that form RNP complexes analogous to those present in the virions (Hay, 1982). Transcription is initiated by cap stealing of cellular hnRNAs (Krug et al., 1979). The capped primers are elongated by the polymerase present in the vRNPs to generate viral mRNAs that are polyadenylated at an oligo(U) signal located next to the RNA panhandle structure at the 5′ terminus of the vRNA templates (Robertson et al., 1981; Poon et al., 1999). Polyadenylation appears to require the proximity of the panhandle structure (Luo et al., 1991) and the interaction of the polymerase with the conserved 5′-terminal sequences of the template (Poon et al., 1998; Pritlove et al., 1998, 1999). The viral polymerase consists of a heterotrimer formed by the PB1, PB2 and PA proteins (Kato et al., 1985; Detjen et al., 1987; Digard et al., 1989; Honda et al., 1990). All three subunits are required for viral RNA replication (Perales and Ortín, 1997). A map of intersubunit contact regions has been established. Thus, the N-terminal sequences of PB1 interact with the C-terminal region of the PA subunit, while sequences next to the C-terminus of PB1 interact with the N-terminal region of the PB2 subunit (Pérez and Donis, 1995; González et al., 1996; Perales et al., 1996; Toyoda et al., 1996; Zürcher et al., 1996). The interaction of the polymerase with vRNA template has been studied using virion cores or the enzyme complex reconstituted by co-expression of the subunits from vaccinia recombinant viruses. Both PB1 and PB2 subunits of the virion core could be cross-linked to the 3′-terminal sequence of vRNA (Fodor et al., 1993) and all three subunits were cross-linked to the vRNA 5′-terminal sequence (Fodor et al., 1994). The enzyme complex bound to the 5′-terminal sequence with higher affinity than to the 3′-terminal one (Tiley et al., 1994). In recent years, our knowledge about the roles of the different subunits in the transcription and replication processes has improved considerably. The PB2 subunit has cap-binding and cap-dependent endonuclease activities (Ulmanen et al., 1981; Blaas et al., 1982; Licheng et al., 1995; Shi et al., 1996). Consistent with these findings, cap primer-dependent in vitro RNA synthesis is affected by mutations in the PB2 gene (Perales et al., 1996; P.Gastaminza, B.Perales and J.Ortín, unpublished results). Nevertheless, both transcription and cap-dependent endonuclease activity require the presence of the three subunits of the polymerase and the RNA template (Hagen et al., 1994; Cianci et al., 1995). Genetic evidence (reviewed in Mahy, 1983) suggests a role for the PA subunit in vRNA synthesis. The PA subunit is a phosphoprotein (Sanz-Ezquerro et al., 1998) whose expression by transfection leads to the degradation of co-expressed proteins (Sanz-Ezquerro et al., 1995). The regions of the PA subunit responsible for this activity map to the N-terminal third of the protein (Sanz-Ezquerro et al., 1996), close to the nuclear localization signal (NLS) (Nieto et al., 1994). The PB1 protein is responsible for the polymerase activity. It contains amino acid motifs typical of RNA-dependent RNA polymerases (Poch et al., 1990), and mutations of the conserved residues abolish the transcriptional activity (Biswas and Nayak, 1994). Furthermore, extracts from baculovirus-infected cells expressing PB1 show some polymerase activity in vitro (Kobayashi et al., 1996). Several functional domains have been identified in the PB1 subunit. Thus, in addition to the regions responsible for the polymerase activity (Biswas and Nayak, 1994), the locations of the NLS and the putative nucleotide-binding domains have been described (Nath and Nayak, 1990; Asano and Ishihama, 1997). Likewise, the protein regions involved in binding to the vRNA template have been identified (Li et al., 1998; González and Ortín, 1999). Two different regions of the protein, located at the N- and C-terminus, appear to interact with both arms of the panhandle structure of the template, the interaction with the 5′ arm being of higher affinity than that with the 3′ arm (González and Ortín, 1999). In this report, we have extended these studies by analyzing the interaction of the isolated PB1 subunit with the cRNA template. The individual PB1 protein bound cRNA specifically, with an apparent Kd of ∼7×10−8 M. PB1 bound both the 5′ and the 3′ arm of the panhandle with similar affinities. Although the N-terminal region of the protein was also responsible for the binding to the cRNA template, the central region of the protein, rather than the C-terminal portion, was involved in the recognition of the cRNA template. Results The PB1 subunit of the polymerase specifically binds virus complementary RNA We have shown previously that the PB1 subunit of influenza virus RNA polymerase is able to bind specifically a vRNA analog probe (González and Ortín, 1999). To study whether isolated PB1 is also able to bind cRNA, we followed a similar experimental strategy. The protein was expressed by transfection of PB1 cDNA, cloned under control of the T7 promoter, into vaccinia–T7 virus-infected COS-1 cells. The infected–transfected cells were labeled continuously with [35S]methionine and [35S]cysteine, and soluble extracts were prepared. The recombinant PB1 protein present was immunoprecipitated with specific antibodies and the immunoprecipitates were used for the in vitro binding of a cRNA labeled probe (cNSZ, containing a deleted CAT gene between the termini of the NS segment; Perales and Ortín, 1997). As a control, an unrelated probe (G3N) of similar length was used. The binding was carried out in the presence of excess yeast RNA and, after extensive washing, the complexes were split into two identical fractions. The protein present in one of the fractions was analyzed by SDS–PAGE and the bound RNA was isolated from the other fraction and analyzed by polyacrylamide–urea gel electrophoresis. The results are presented in Figure 1. It is clear that PB1-containing immunoprecipitates were capable of retaining the specific cNSZ probe (Figure 1A, left panel), but not the unrelated G3N probe (Figure 1A, right panel). As shown in the bottom panels of Figure 1A, the PB1-specific immunoprecipitates contained PB1, but the control immunoprecipitates did not. Figure 1.Specificity of PB1 binding to cRNA. Cultures of COS-1 cells were infected with vaccinia–T7 virus and transfected with either pGorfB1 plasmid or pGEM3 plasmid as a control. The cultures were labeled with [35S]methionine/cysteine and soluble extracts were prepared as described in Materials and methods. (A) The extracts were used for immunoprecipitation with an anti-PB1 antiserum, and the immunoprecipitates (B1-IPP or CTRL-IPP) were incubated with 32P-labeled cNSZ probe (cRNA PROBE) or control G3N probe (CTRL PROBE) (sp. act. ∼108 c.p.m./μg) in the presence of a 250-fold excess of yeast RNA. The RNA from a fraction of the complexes was isolated and analyzed by denaturing gel electrophoresis (top panels) while the protein present was isolated and visualized by SDS–PAGE and autoradiography (bottom panels). (B) The binding assay was performed as indicated in (A), except that increasing amounts of unlabeled cNSZ probe (cRNA) (1, 10 or 100 ng; i.e. 10- to 1000-fold molar excess) or yeast RNA (yRNA) were included in the assay. The RNA retained was isolated and analyzed as indicated above (top panels). The proteins present in the immunoprecipitates are shown in the bottom panels. Numbers to the left indicate the size of protein markers in kDa. Numbers to the right indicate the length of RNA markers (MW) in nucleotides. In this and subsequent figures, the lanes indicated as Probe show one-tenth of the amount of probe that was included in the binding test. Download figure Download PowerPoint To test further the specificity of PB1–cRNA binding, competition experiments were carried out. Parallel binding tests were performed in which increasing amounts of unlabeled cNSZ RNA or unlabeled yeast RNA were added, in addition to the labeled cNSZ probe, and the retention of the latter was tested as indicated above. The results presented in Figure 1B indicate that the homologous unlabeled probe competed out the labeled probe (Figure 1B, left panel) while an equivalent excess of unrelated RNA did not (Figure 1B, right panel). The presence of equivalent quantities of PB1 in the specific immunoprecipitates was ascertained by SDS–PAGE analysis of the same samples and is presented in the lower panels of Figure 1B. To estimate the affinity of the interaction of cRNA with isolated PB1, we performed binding experiments in which increasing amounts of PB1-containing extracts were used for immunoprecipitation. The absolute quantity of PB1 present in each binding assay was determined by using as a standard total cell extract containing PB1, labeled in parallel, derived from COS-1 cells doubly infected with vaccinia–T7 and VPB1 recombinant virus, as described (González and Ortín, 1999). Such an extract contains a high proportion of PB1 that can be quantitated by Coomassie Blue staining using bovine serum albumin (BSA) as a reference. The proportion of the added cNSZ probe that was retained by the PB1-specific immunoprecipitates was determined by counting the associated Cerenkov radiation. The cumulative data from three separate experiments (Figure 2) led to an estimation of 7× 10−8 M for the Kd. This is only an estimation because the determination of the absolute concentration of PB1 is technically difficult and the maximal concentration of PB1 obtained was not sufficient to achieve saturation in the binding reaction. Figure 2.Affinity of binding of PB1 to cRNA. In vitro RNA-binding assays were carried out as described in the legend to Figure 1, using cNSZ probe at a concentration of 10−11 M. Extracts containing increasing amounts of PB1 were used, and the actual concentrations of PB1 present in each assay were determined as indicated in the text. The percentage probe retained after washing was determined by Cerenkov counting. Different symbols represent the results of three independent experiments. The curve is the best exponential fit of the data. Download figure Download PowerPoint The binding assays described above indicate a specific binding of cRNA to the PB1-containing immunoprecipitates but do not demonstrate a direct interaction of PB1 with the cNSZ probe. To address such a question directly, we carried out Northwestern assays. Extracts derived from COS-1 cells doubly infected with vaccinia–T7 and VPB1 viruses were prepared and separated by SDS–PAGE as described in Materials and methods. The separated proteins were renatured after their transfer to a nitrocellulose membrane and probed with either cNSZ or the G3N unrelated probe. After autoradiography, the filter was probed by Western blot with antibodies specific for PB1. The results are shown in Figure 3. In addition to unspecific reactive bands, a major reactive band was observed in extracts of VPB1-infected cells when the cNSZ probe was used (Figure 3A, left panel), but not when the unspecific G3N probe was used (Figure 3A, right panel). This signal had a mobility corresponding to the size of PB1 and could be superimposed on the signal of PB1 by Western blot of the same filters (Figure 3B). Figure 3.Northwestern assay with the cNSZ probe. Cultures of COS-1 cells were doubly infected with vaccinia–T7 and VPB1 viruses (VAC-B1), or singly infected with vaccinia–T7 virus (VAC-T7) as a control. Extracts prepared as indicated in Materials and methods were separated by SDS–PAGE and transferred to nitrocellulose filters. After renaturation, the filters were incubated with the cNSZ probe (cRNA) or the control G3N probe (CTRL). (A) After washing, the signal was revealed by autoradiography. (B) The same filters were developed by Western blot with an anti-PB1 serum. Numbers to the left indicate the size of protein molecular weight markers in kDa. Download figure Download PowerPoint PB1 binds both the 5′ and the 3′ arm of the cRNA panhandle We have demonstrated previously that PB1 binds preferentially to the 5′ arm of the vRNA panhandle (González and Ortín, 1999), although binding to the 3′ arm is also detectable. To study whether a similar situation exists with regard to the PB1–cRNA binding, we prepared short probes corresponding to the 5′ and 3′ arms of the cRNA panhandle and carried out in vitro binding experiments analogous to those reported above for the cNSZ probe. As shown in Figure 4, both the 5′ and 3′ arm probes were retained by PB1-specific immunoprecipitates, and binding to a 5′ + 3′ annealed probe was not enhanced as compared with the individual interactions. Under the same experimental conditions, no detectable binding was observed when an unrelated probe of similar length was used (G4S probe, 26 nucleotides, data not shown). Figure 4.Interaction of PB1 with the 5′ and the 3′ arm of the cRNA panhandle. In vitro RNA-binding assays were carried out as described in Materials and methods and in the legend to Figure 1, except that 3′ probe, 5′ probe or a mixture of both were used. The 3′ probe always contained a shorter oligonucleotide probably generated by premature termination of transcription. The top panel shows the analysis of the RNA retained. The bottom panel shows the analysis of the proteins present in the immunoprecipitates. Numbers to the left indicate the size of protein markers in kDa. Numbers to the right indicate the length of RNA markers in nucleotides. Download figure Download PowerPoint The binding of either the 5′ or the 3′ arm sequences to PB1 was also tested by Northwestern assays. Extracts obtained from cells doubly infected with vaccinia–T7 and VPB1 recombinant viruses, or only with vaccinia–T7 as a control, were separated by SDS–PAGE, renatured and tested for binding with either the 5′ arm, the 3′ arm or a control G4S probe. The results clearly indicated a direct interaction of PB1 with both 5′ and 3′ probes but not with the unrelated probe (Figure 5A). The presence of PB1 in the extracts from doubly infected cells was ascertained by Western blot and is presented in Figure 5B. Figure 5.Northwestern assay with the 5′ and the 3′ arm of the cRNA panhandle. Cultures of COS-1 cells were doubly infected with vaccinia–T7 and VPB1 viruses (VAC-B1), or singly infected with vaccinia–T7 virus (VAC-T7) as a control. Extracts were processed as indicated in Materials and methods and in the legend to Figure 3. The filters were incubated with either 5′ probe (c5′), 3′ probe (c3′) or the control G4S probe. (A) After washing, the signal was revealed by autoradiography. (B) The same filters were developed by Western blot with an anti-PB1 serum. Numbers to the left indicate the size of protein molecular weight markers in kDa. Download figure Download PowerPoint Mapping the PB1 sequences required for binding to cRNA To obtain information about the regions of PB1 involved in binding to cRNA, several deleted versions of the gene were prepared. In every case, the mutant proteins were expressed in mammalian cells as His-tagged recombinants to allow their immunoprecipitation with a common antiserum recognizing the tag present in them (see Figure 6A for a diagram). The corresponding immunoprecipitates (Figure 6D) were used for in vitro binding to vNSZ, cNSZ probe or an unrelated probe as reported above for wild-type PB1. The results of these experiments are presented in Figure 6. Similarly to the results obtained for wild-type PB1, no retention was observed for the unspecific probe (data not shown). As reported previously (González and Ortín, 1999), vNSZ RNA was retained by the N-terminal region of PB1 (mutants PB1-Δ267–757 and PB1-Δ139–757) and also by the C-terminal region (mutant PB1-Δ1–493) (Figure 6C). Binding to the cNSZ probe was also positive for mutants containing the N-terminal regions of PB1 (mutants PB1-Δ267–757 and PB1-Δ139–757) but not the C-terminal region (mutant PB1-Δ1–493). Instead, positive binding was detected with mutant PB1-267–493 (Figure 6B). The level of probe retention was similar for wild-type PB1 and for the PB1 mutants that proved positive for binding (compare Figures 1A and 6B). The results presented clearly indicate that although the N-terminal sequences of PB1 appear to bind either vNSZ or cNSZ probes, distinct regions elsewhere in the protein are involved in the recognition of vRNA and cRNA (see below). Figure 6.Binding of mutant PB1 proteins to cRNA. Cultures of COS-1 cells were infected with vaccinia–T7 virus and transfected with the mutant plasmids indicated in (A) or pRSET plasmid as a control (CTRL). The RNA-binding assay was carried out as indicated in Materials and methods and in the legend to Figure 1. The mutants were expressed as His-tagged proteins (denoted by black boxes to the left of each bar) and immunoprecipitated with anti-HisNS1 serum. (B) Analysis of the cRNA retained. (C) Analysis of the vRNA retained. (D) Analysis of the proteins present in the immuno- precipitates. Numbers indicate the size of protein molecular weight markers in kDa. Download figure Download PowerPoint Since PB1 is able to bind both the 5′ and the 3′ arm of the cRNA panhandle (Figure 4), we wished to know whether the two separate sequences of PB1 protein involved in cRNA binding, defined by mutants PB1-267–493 and PB1-Δ139–757, were involved in the recognition of either arm of the panhandle or, alternatively, if each arm was recognized by both binding regions of PB1. Hence, in vitro binding experiments were carried out using either 5′ or 3′ arm probes and the various deletion mutants described above. The results presented in Figure 7 further substantiate the conclusion derived from Figure 6 with regard to the mapping of the two independent regions of PB1 involved in cRNA binding and indicate that they both interact with each arm of the cRNA panhandle. Thus, N-terminal and internal sequences of PB1 (mutants PB1-Δ139–757, PB1-Δ267–757 and PB1-267–493) are able to retain either probe, while the C-terminal sequences (mutant PB1-Δ1–493) are not (Figure 7A and B). Figure 7.Binding of mutant PB1 proteins to the 5′ and the 3′ arm of the cRNA panhandle. Cultures of COS-1 cells were infected with vaccinia–T7 virus and transfected with the mutant plasmids indicated in Figure 6A or pRSET plasmid as a control (CTRL). The RNA-binding assay was carried out as indicated in Materials and methods and in the legend to Figure 1. Analysis of the RNA retained when using (A) the 5′ probe and (B) the 3′ probe. (C) Analysis of the proteins present in the immunoprecipitates. Numbers to the right indicate the length of RNA markers in nucleotides. Numbers to the left indicate the size of protein molecular weight markers in kDa. Download figure Download PowerPoint The PB1-binding sites for vRNA and cRNA are distinct and partially overlap The mapping of the binding of vNSZ and cNSZ probes on PB1 indicated that the N-terminal region of the protein interacts with both RNAs while distinct sequences elsewhere in the protein contribute to the binding of vRNA and cRNA (González and Ortín, 1999; Figures 6 and 7). To ascertain whether the N-terminal region contains a single RNA-binding site, common to both vRNA and cRNA, or two distinct interaction sites, competition experiments were carried out. Each of the PB1 mutant proteins was used in binding experiments in which a labeled probe corresponding to the 5′ or the 3′ arm of either positive or negative polarity RNA (v5′, v3′, c5′ or c3′ probes) (Figure 8A) was competed with excess unlabeled homologous or heterologous probes. In every case, the homologous unlabeled probe was able to compete out binding (data not shown), in a way similar to the data presented in Figure 1B for the cNSZ probe. When the N-terminal sequences of PB1 were used for binding (mutant PB1-Δ267–757), the binding of labeled v5′ or v3′ probes was competed with excess unlabeled c5′ or c3′ probes, respectively (Figure 8A). Likewise, the binding of labeled c5′ or c3′ probes was competed with unlabeled v5′ or v3′ probes, respectively (Figure 8B). These results indicate that a binding site common to both vRNA and cRNA exists at the N-terminal end of the PB1 molecule. In contrast, the binding of v5′ or v3′ labeled probes to the C-terminal sequence of PB1 (mutant PB1-Δ1–493) was not competed with excess unlabeled c5′ or c3′ unlabeled probes (Figure 8C). Similarly, no competition of c5′ or c3′ binding to the internal sequences of PB1 protein (mutant PB1-267–493) was observed when excess unlabeled v5′ or v3′ probes were used, respectively (Figure 8D). These results are in agreement with the binding of vRNA or cRNA panhandle arms to these regions of the protein (González and Ortín, 1999; Figures 6 and 7). Figure 8.Competition assays. Cultures of COS-1 cells were infected with vaccinia–T7 virus and transfected with the mutant plasmids pRPB1Δ267–757 (N-terminal), pRPB1-267–493 (internal) or plasmid pRPB1Δ1–493 (C-terminal). The RNA-binding assay was carried out as indicated in Materials and methods and in the legend to Figure 1 using the labeled probes indicated in the figure. (A) Diagram indicating the probes used in the competition experiments. Results obtained for binding to the (B) PB1Δ267–757 mutant, (C) the PB1-267–493 mutant and (D) the PB1Δ1–493 mutant. In each case, the indicated amounts (in ng) of competing unlabeled probes were included in the binding tests. Download figure Download PowerPoint Discussion The process of replication of influenza virus RNA implies first the synthesis of a complementary ribonucleoprotein (cRNP) that serves as a replication intermediate (Hay, 1982; reviewed in Krug et al., 1989; Portela et al., 1999). This involves the shift of template and the recognition of the cRNA by the newly synthesized polymerase and NP. Since we showed previously that the isolated PB1 subunit binds a vRNA template with high affinity (González and Ortín, 1999), a binding that might reflect in part the interaction that takes place in the vRNP, we set out to study the interaction of the isolated PB1 protein with the alternative template, cRNA. The PB1 subunit of the polymerase interacts with cRNA The interaction experiments described demonstrate that the PB1 subunit of the polymerase is able to bind specifically a cRNA probe. The specificity of the interaction was validated by using an unrelated probe and by competition experiments with homologous and heterologous RNAs (Figure 1). Furthermore, a PB1–cRNA direct interaction was established by Northwestern assays (Figures 3 and 5). The specificity of binding was confirmed further by the interaction of PB1 with short probes corresponding to the 5′ and 3′ arms of the cRNA panhandle (Figure 4), but not with an unrelated probe of similar length (data not shown). A substantial binding affinity could be inferred for the PB1–cRNA interaction, as the estimated Kd was ∼7× 10−8 M (Figure 2), in the range of a typical RNA-binding protein (10−7–10−9 M; Draper, 1995) (González and Ortín, 1999). Since a dramatic shift from cRNA to vRNA synthesis occurs early in the influenza virus infection (Enami et al., 1985), the simplest explanation for this template shift would be that the viral polymerase would have a higher affinity for cRNA than for vRNA binding, but this situation is not reflected in the interaction of these RNAs with isolated PB1 (González and Ortín, 1999; this study). It is tempting to speculate that the other subunits of the polymerase make a more important contribution to the polymerase–cRNA interaction than to the polymerase–vRNA interaction. In addition to the vRNA to cRNA template shift, it is remarkable that the newly synthesized vRNA is not used for cRNA synthesis (Enami et al., 1985). This fact suggests that other viral or cellular factor(s) might alter the newly synthesized vRNPs so as to direct their utilization for transcription and avoid further formation of cRNPs. In this context, it is worth mentioning that NS1 protein has been found associated with transcription–replication complexes in the infected cell (Marión et al., 1997a), but is absent from virions. Another difference between vRNA and cRNA binding to PB1 protein is reflected in the different interaction of the 5′ and 3′ arms of the vRNA and cRNA panhandles. For vRNA, a preferential interaction with the 5′ arm was found, whereas binding of the 3′ arm was dependent on the previous interaction of the 5′ sequences (González and Ortín, 1999). This situation might be the basis of the activation of capped primer binding mediated by the interaction with the 5′ arm (Hagen et al., 1994; Cianci et al., 1995). In the case of cRNA, either arm of the panhandle was equally able to interact with PB1, and no synergistic effect was observed when both arms were used for interaction in vitro (Figure 4). Suggestion for a conformational change in PB1 protein upon interaction with either vRNA or cRNA The mapping of the PB1 sequences involved in the recognition of a vRNA probe indicated that two regions of the protein were active in binding in vitro: the N-terminal 83 amino acids and the C-terminal 264 amino acids (González and Ortín, 1999). These results are partly contradictory to those presented in another report (Li et al., 1998), in which the interaction of the polymerase complex with vRNA was tested by cross-linking of thio-U-substituted RNA. These authors could cross-link the 5′ probe to a sequence located at around position 250 in the PB1 molecule. This discrepancy could be attributed to the fact that the positions modified with thio-U (position 15 in the 5′ arm and position 10 in the 3′ arm) are a few nucleotides away from the site of interaction mapped in the vRNA panhandle (Tiley et al., 1994; Klumpp et al., 1997). The evidence presented here indicates that an analogous N-terminal region of the protein interacts with cRNA (positions 1–139) (Figures 6 and 7) but downstream sequences different from those reported for vRNA binding are involved in cRNA recognition (positions 267–493 instead of positions 493–757). Furthermore, the competition experiments presented in Figure 8 indicate that the binding sites for cRNA downstream of position 267 do not overlap, i.e. no competition is observed when the heterologous probes are used, but the binding site close to the N-terminus is common for vRNA and cRNA binding, i.e. compet" @default.
• W2078796009 created "2016-06-24" @default.
• W2078796009 creator A5049122951 @default.
• W2078796009 date "1999-07-01" @default.
• W2078796009 modified "2023-10-18" @default.
• W2078796009 title "Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates" @default.
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• W2078796009 doi "https://doi.org/10.1093/emboj/18.13.3767" @default.
• W2078796009 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1171453" @default.
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