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Journal of Virology, February 2009, p. 1611-1616, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01491-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Influenza A Virus Polymerase Is an Integral Component of the CPSF30-NS1A Protein Complex in Infected Cells

Rei-Lin Kuo and Robert M. Krug^*

Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712

Received 16 July 2008/ Accepted 25 November 2008

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The NS1A protein of influenza A virus binds the cellular CPSF30 protein, thereby inhibiting the 3'-end processing of all cellular pre-mRNAs, including beta interferon pre-mRNA. X-ray crystallography identified the CPSF30-binding pocket on the influenza virus A/Udorn/72 (Ud) NS1A protein and the critical role of two hydrophobic NS1A amino acids outside the pocket, F103 and M106, in stabilizing the CPSF30-NS1A complex. Although the NS1A protein of the 1997 H5N1 influenza A/Hong Kong/483/97 (HK97) virus contains L (not F) at position 103 and I (not M) at position 106, it binds CPSF30 in vivo to a significant extent because cognate (HK97) internal proteins stabilize the CPSF30-NS1A complex in infected cells. Here we show that the cognate HK97 polymerase complex, containing the viral polymerase proteins (PB1, PB2, and PA) and the nucleocapsid protein (NP), is responsible for this stabilization. The noncognate Ud polymerase complex cannot carry out this stabilization, but it can stabilize CPSF30 binding to a mutated (F103L M106I) cognate Ud NS1A protein. These results suggested that the viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells even when the cognate NS1A protein contains F103 and M106, and we show that this is indeed the case. Finally, we show that cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex, indicating that the NS1A protein interacts primarily with its cognate PA protein and NP in a complex that includes the cellular CPSF30 protein.

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The NS1 protein of human influenza A viruses (NS1A protein) is a small, multifunctional protein. Its N-terminal RNA-binding domain binds double-stranded RNA (dsRNA) (2, 6, 10, 21, 22), resulting in the inhibition of the alpha/beta interferon (IFN-/β)-induced oligo(A) synthetase/RNase L pathway (13). The rest of the NS1A protein, which is referred to as the effector domain, has binding sites for several cellular proteins, including CPSF30 (the 30-kDa subunit of the cellular cleavage and polyadenylation specificity factor), a cellular factor required for the 3'-end processing of all cellular pre-mRNAs (15). Because of the sequestration of CPSF30 by the NS1A protein, the substantial amount of IFN-β pre-mRNA that is synthesized in influenza A virus-infected cells is not processed to form mature IFN-β mRNA, thereby suppressing the host IFN antiviral response (3, 8, 9, 15, 16, 19, 20).

The binding interface between the NS1A protein and CPSF30 was revealed by the X-ray crystal structure of the effector domain of the NS1A protein of influenza A/Udorn/72 (Ud) virus in complex with the second and third zinc finger (F2F3) domain of CPSF30 (3). This complex is a tetramer in which each of two F2F3 molecules wraps around the two NS1A effector domains that interact with each other head-to-head. This structure identifies a largely hydrophobic CPSF30-binding pocket on the NS1A protein that is almost completely conserved among human influenza A viruses, including H5N1 viruses (3), strongly suggesting that this CPSF30 binding site is used by all human influenza A viruses to suppress the production of IFN-β mRNA. The crystal structure also shows that the interaction surface between NS1A and F2F3 in the tetrameric complex extends beyond this binding pocket. Two hydrophobic NS1A amino acids outside the binding pocket, F103 and M106, stabilize the tetrameric complex by interacting with hydrophobic amino acids in F2F3 (3). In addition, M106 in one NS1A molecule interacts with M106 in the second NS1A molecule. These two amino acids, which are highly conserved (>99%) in the NS1A proteins of human influenza A viruses (11), are required for tight binding in vitro (3). However, some binding can occur in vivo when the NS1A protein contains L instead of F at position 103 and I instead of M at position 106. Thus, the NS1A protein of the pathogenic 1997 H5N1 influenza A/Hong Kong/483/97 (HK97) virus, which has these two amino acid substitutions, binds CPSF30 to a significant, though not optimum, extent when it is expressed in a virus that also encodes the other internal HK97 (cognate) proteins (19). In contrast, little or no binding of the HK97 NS1A protein occurs when it is expressed in a virus that encodes the internal proteins of a noncognate (Ud) virus. These results indicated that one or more cognate (HK97) internal proteins stabilize the CPSF30-HK97 NS1A protein complex in infected cells.

In the present study, we show that the cognate HK97 polymerase complex, i.e., the complex containing the tripartite viral polymerase proteins (PB1, PB2, and PA) and the nucleocapsid protein (NP), is responsible for stabilizing the binding of CPSF30 to the HK97 NS1A protein that contains F103L and M106I substitutions. The HK97 matrix (M) gene products are not required. In contrast, the noncognate Ud polymerase complex cannot carry out this stabilization, whereas it can stabilize the binding of CPSF30 to a mutated (F103L M106I) cognate Ud NS1A protein (the 103/106 mutant). Conversely, the HK97 polymerase complex cannot stabilize the CPSF30 binding of this mutated Ud NS1A protein. These results suggested that a cognate viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells even when the cognate NS1A protein contains the human consensus F103 and M106 amino acids, and we show that this is indeed the case. Thus, our results demonstrate that the viral polymerase complex interacts with its cognate NS1A protein in infected cells in a complex that includes the host CPSF30 protein. Finally, we show that cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex, indicating that the NS1A protein interacts primarily with its cognate PA protein and NP in this complex. We discuss the implications of these results both for suppression of the host antiviral response and for viral RNA synthesis in infected cells.

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Generation of recombinant influenza A viruses. The pHH21 plasmids expressing the PB1, PB2, PA, NP, M, and NS genes of Ud and HK97 viruses were provided by Robert A. Lamb (18) and Yoshihiro Kawaoka (7), respectively. To generate recombinant influenza A viruses, Ud or HK97 pHH21 plasmids expressing the influenza A virus genes indicated in the text plus the four pcDNA3 plasmids encoding the Ud proteins PB1, PB2, PA, and NP were cotransfected into 293T cells. At 12 h posttransfection, the medium was changed to Opti-MEM supplemented with 3 µg/ml of N-acetyl trypsin. After an additional 24 h, the 293T cells were laid over MDCK cells for virus amplification. The recombinant viruses were amplified and their titers were determined as described previously (20). All eight genome RNA segments of the recombinant viruses were sequenced. All experiments using viruses containing any HK97 gene were carried out in a biosafety level 3 laboratory using biosafety level 3-enhanced procedures. All recombinant viruses, including those with HK97 internal genes, contained the Ud HA and NA genes.

Virus infections. Multiple-cycle growth (at a multiplicity of infection [MOI] of 0.001 PFU/cell) of and plaque assays with MDCK cells were carried out as described previously (19, 20).

Measurement of IFN-ß mRNA by real-time quantitative reverse transcription-PCR. RNA was isolated from infected cells (collected at 12 h after infection at a MOI of 2 PFU/cell) by using the TRIzol reagent, and 1 µg of total RNA, which corresponds to equal cell equivalents, was reverse transcribed using an oligo(dT) primer to generate the DNA complementary to all mRNAs. The amount of canine IFN-ß mRNA was determined using the TaqMan gene expression assay (Applied Biosystems), and real-time PCR analysis was carried out using the Perkin-Elmer/Applied Biosystems 7900HT sequence detector, as described previously (19, 20).

Assays for the binding of CPSF30 to the NS1A protein in infected cells. In one experiment (see Fig. 3), 293T cells were transfected with a pcDNA3 plasmid encoding 3X FLAG-CPSF30 or, as a control, an empty pcDNA3 plasmid. Twenty-four hours later, the cells were infected at an MOI of 2 PFU/cell with the Ud virus indicated in Fig. 3. Extracts prepared from cells at 12 h postinfection were immunoprecipitated with anti-FLAG M2 monoclonal antibody, and the immunoprecipitates were analyzed by immunoblotting using antibody directed against the Ud NS1A protein, as described previously (19). In all other experiments, 293T cells were transfected with a pcDNA3 plasmid encoding glutathione S-transferase (GST)-CPSF30 or, as a control, an empty pcDNA3 plasmid. After subsequent infection with the recombinant influenza A virus indicated in the text, cell extracts (400 µl) were mixed with 60 µl of glutathione-Sepharose beads for 18 h at 4°C, and the beads were washed two times with a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride. Specifically bound proteins were eluted by incubating the beads for 1 h at 4°C with 50 µl of the above-described wash buffer containing 10 mM glutathione. The eluted proteins were analyzed by immunoblotting using GST antibody (to detect GST-CPSF30) or NS1A antibody. Where indicated in Fig. 4, the eluted proteins were analyzed by immunoblotting to detect polymerase complex proteins by using the following antibodies: rabbit antibodies against PB1 and PA, provided by Krister Melen and Ilkka Julkunen; monoclonal antibody against PB2, provided by Jonathan Yewdell; and antibody against the major structural proteins of Ud virus, which detects the proteins HA, NP and M1, provided by Robert A. Lamb (1).

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FIG. 3. Cognate Ud internal proteins do not stabilize a complex between CPSF30 and a Ud NS1A protein that contains the G184R binding pocket mutation. (A) 293T cells were transfected with either a plasmid expressing 3X FLAG-CPSF30 or an empty plasmid and were then infected with the indicated Ud virus. Extracts from cells collected at 12 h after virus infection were immunoprecipitated with anti-FLAG M2 monoclonal antibody, and the immunoprecipitates were analyzed by immunoblotting with either anti-Ud NS1A antibody or anti-FLAG antibody. wt, wild type; 103/106m, Ud-NS 103/106 mutant; G184R, Ud-NS G184R mutant; IP, immunoprecipitation; WB, Western blotting. (B) Relative amounts of IFN-β mRNA produced during single-cycle growth in MDCK cells of the Ud viruses expressing either the 103/106 or G184R mutant NS1A protein. (C) Relative plaque sizes of the Ud viruses expressing either the 103/106 or G184R mutant NS1A protein.

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FIG. 4. The viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells. 293T cells were transfected with either a plasmid expressing GST-CPSF30 or an empty plasmid and were then infected with wild-type Ud virus. Extracts from cells collected at 12 h after virus infection were selected on glutathione-Sepharose, and eluted proteins were analyzed with immunoblots probed with anti-NS1A, anti-PB1, anti-PB2, and anti-PA antibody or with an antibody (anti-Ud) against the HA, NP, and M1 proteins of Ud virus. WB, Western blotting.

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A cognate viral polymerase complex is responsible for stabilizing the binding of a cognate NS1A protein to CPSF30. Previously, we showed that cognate (HK97) internal proteins, but not noncognate (Ud) internal proteins, stabilize the binding of CPSF30 to the HK97 NS1A protein that contains F103L and M106I substitutions (19). These HK97 internal proteins included the viral polymerase complex comprised of the tripartite viral RNA polymerase proteins (PB1, PB2, and PA) and NP, plus the M1 protein (as well as the M2 integral membrane protein). To determine whether the viral polymerase complex in the absence of the HK97 M gene products is sufficient to confer this stabilization, we generated the HK-Pol/HK-NS virus, which contains the HK97 NS and polymerase complex genes and the noncognate (Ud) M gene (Fig. 1A). In addition, we generated the Ud-Pol/HK-NS virus, which contains the HK97 NS gene and the Ud rather than the HK97 polymerase complex genes.

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FIG. 1. The cognate HK97 polymerase complex, but not the noncognate Ud polymerase complex, stabilizes the CPSF30-HK97 NS1A protein complex, and this stabilization leads to reduced production of IFN-β mRNA during infection. (A) 293T cells were transfected with either a plasmid expressing GST-CPSF30 or an empty plasmid and were then infected with the indicated recombinant influenza A virus. Extracts from cells collected at 12 h after virus infection were selected on glutathione-Sepharose, and eluted proteins were analyzed with immunoblots probed with anti-NS1A or anti-GST antibody. (B) Relative amounts of IFN-β mRNA produced during single-cycle growth in MDCK cells of the HK-Pol/HK-NS and Ud-Pol/HK-NS viruses.

To determine whether the HK97 NS1A gene expressed by these two viruses binds CPSF30, we employed a modified version of our previously described method (19). We transfected 293T cells with a plasmid expressing GST-tagged CPSF30 or, as a control, an empty plasmid. Twenty-four hours later, the cells were infected at an MOI of 2 PFU/cell with either the HK-Pol/HK-NS or Ud-Pol/HK-NS virus for 12 h. Infected cell extracts were affinity selected on a glutathione-Sepharose column, followed by immunoblotting using anti-NS1A antibody (Fig. 1A). The HK97 NS1A protein expressed by the HK-Pol/HK-NS virus was coselected with GST-CPSF30 (lane 2), whereas little or none of the HK97 NS1A protein expressed by the Ud-Pol/HK-NS virus was coselected (lane 4). We conclude that the presence of coexpressed cognate (HK97) viral polymerase proteins is sufficient to enable the HK97 NS1A protein that contains L instead of F at 103 and I instead of M at 106 to bind CPSF30 in virus-infected cells. The HK97 M gene products are not required for such CPSF30 binding. The HK-Pol/HK-NS virus induced 15-fold-less IFN-β mRNA than the Ud-Pol/HK-NS virus during infection (Fig. 1B), verifying that CPSF30 binding to the NS1A protein is responsible for suppressing the production of IFN-β mRNA (3, 8, 9, 15, 16, 19, 20).

To determine whether a similar requirement for cognate internal proteins is also the case for the Ud NS1A protein, we generated two recombinant viruses expressing a mutant Ud NS1A protein containing the same substitutions (F103L and M106I) found in the wild-type HK97 NS1A protein. One recombinant expressed cognate Ud internal genes, and the second recombinant expressed noncognate HK97 internal genes (Fig. 2). The cognate Ud internal genes enabled the mutant Ud NS1A protein to bind CPSF30, whereas the noncognate HK97 internal genes did not confer this activity to the mutant NS1A protein (Fig. 2A). This CPSF30 binding resulted in a 10-fold decrease in IFN-β mRNA production during infection (Fig. 2B). The Ud viral polymerase genes were sufficient to confer this activity to the mutant NS1A protein (data not shown). These results verify that a cognate viral polymerase complex enables a cognate NS1A protein that contains the F103L and M106I substitutions to bind CPSF30 in virus-infected cells.

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FIG. 2. Cognate Ud internal proteins, but not noncognate HK97 internal proteins, stabilize a complex containing CPSF30 and a Ud NS1A protein that has F103L and M106I substitutions, and this stabilization leads to reduced production of IFN-β mRNA during infection. (A) 293T cells were transfected with either a plasmid expressing GST-CPSF30 or an empty plasmid and were then infected with the indicated recombinant influenza A virus. Analysis of infected cell extracts was carried out as described in the legend of Fig. 1. (B) Relative amounts of IFN-β mRNA produced during single-cycle growth in MDCK cells of the Ud/Ud-NS 103/106 and HK/Ud-NS 103/106 viruses.

An NS1A protein that contains a mutation in its CPSF30-binding pocket does not bind CPSF30 even in the presence of its cognate viral polymerase complex. Whereas the function of F103 and M106 of the NS1A protein is to stabilize the tetrameric CPSF30-NS1A protein complex, CPSF30 binding per se is mediated by the NS1A amino acids comprising the largely hydrophobic binding pocket (3). Mutagenesis of individual Ud NS1A amino acids comprising the CPSF30 pocket eliminates detectable F2F3 binding in vitro, and mutation of one of these pocket amino acids, G184 to R, has been shown to attenuate virus replication and, coupled with efficient processing of IFN-β pre-mRNA, to produce mature IFN-β mRNA (3). Here we determined whether there is residual CPSF30 binding to a G184R mutant NS1A protein, possibly via stabilization provided by the cognate viral polymerase complex (Fig. 3A). In this experiment, we transfected a plasmid expressing CPSF30 with a 3X FLAG tag, and infected cell extracts were immunoprecipitated with anti-FLAG antibody, as described previously (19). No detectable NS1A protein with the G184R mutation coimmunoprecipitated with 3X FLAG-CPSF30 (lane 6), whereas a significant amount of the Ud 103/106 mutant NS1A protein coimmunoprecipitated with 3X FLAG-CPSF30 (lane 4). The latter amount of coimmunoprecipitated NS1A was approximately 15% of the amount of the wild-type NS1A protein that coimmunoprecipitated with 3X FLAG-CPSF30 (compare lanes 2 and 4). Consequently, a wild-type CPSF30-binding site is required for detectable CPSF30 binding even in the presence of the cognate viral polymerase complex.

The ability of the 103/106 mutant NS1A protein, but not the G184R mutant NS1A protein, to bind to a certain extent to CPSF30 in infected cells leads to predicted differences in virus-induced production of IFN-β mRNA and hence in the rates of virus replication. Fivefold-more IFN-β mRNA is produced in cells infected by the Ud virus expressing the G184R mutant NS1A protein than in cells infected by the Ud virus expressing the 103/106 mutant NS1A protein (Fig. 3B). In addition, the Ud virus expressing the G184R mutant NS1A protein is attenuated relative to the virus expressing the 103/106 mutant NS1A protein, as shown by smaller plaque size (Fig. 3C) and by 10-fold-slower replication during multiple-cycle growth (at a MOI of 0.001 PFU/cell); e.g., at 24 h postinfection, the titers of the G184R mutant and 103/106 mutant viruses are 3.4 x 10⁵ and 3.8 x 10⁶ PFU/ml, respectively.

The viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells. The ability of a cognate viral polymerase complex to stabilize the binding of a cognate 103/106 mutant NS1A protein to CPSF30 strongly suggested that the viral polymerase complex is a component of the CPSF30-NS1A protein complex in infected cells even when the NS1A protein contains the optimum consensus F103 and M106 amino acids. To determine whether this is in fact the case, 293T cells were transfected with a plasmid expressing GST-CPSF30, followed by infection with Ud virus expressing the wild-type Ud NS1A protein, which contains F103 and M106. Infected cell extracts were affinity selected on glutathione-Sepharose, and the eluates were analyzed by immunoblotting to determine whether the proteins of the viral polymerase complex were coselected with the NS1A protein. As shown in Fig. 4, immunoblots showed that PB1, PB2, PA, and NP were coselected. Other viral proteins were not coselected: the immunoblot with the antibody directed against the major Ud structural proteins showed that only NP, and not the HA and M1 proteins were coselected. The same results were obtained using the Ud mutant virus expressing an NS1A protein that lacks only dsRNA-binding activity (with an R38A mutation) (data not shown), demonstrating that dsRNA binding by the NS1A protein does not have a role in the association of the viral polymerase complex with the CPSF30-NS1A protein complex.

Cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex. To determine whether a subset of the cognate viral polymerase complex proteins are sufficient for stabilizing the binding of the HK97 NS1A protein to CPSF30, we generated a series of recombinant viruses containing the HK97 NS gene and various combinations of cognate HK97 and noncognate Ud polymerase complex genes. Cells transfected with a plasmid expressing GST-CPSF30 were infected with one of these recombinant viruses, and infected cell extracts were affinity selected on a glutathione-Sepharose column, followed by immunoblotting using anti-NS1A antibody. The recombinant Ud-Pol/HK-NS virus, which contains only the HK97 M gene in addition to the HK97 NS gene, served as the negative control (Fig. 5, lanes 1 and 2). The recombinant virus containing the cognate HK97 PA and NP genes and the noncognate Ud PB1 and PB2 genes led to substantial coselection of the NS1A protein (lanes 3 and 4). Consequently, cognate PA protein and NP are sufficient to stabilize the CPSF30-HK97 NS1A protein complex, and cognate PB1 and PB2 proteins are not required. Other combinations of cognate and noncognate polymerase complex genes did not lead to coselection of the NS1A protein (data not shown).

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FIG. 5. Cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex. 293T cells were transfected with either a plasmid expressing GST-CPSF30 or an empty plasmid and were then infected with the indicated recombinant influenza A virus. Extracts from cells collected at 12 h after virus infection were selected on glutathione-Sepharose, and eluted proteins were analyzed with immunoblots probed with anti-NS1A or anti-GST antibody. WB, Western blotting.

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We used a genetic approach to demonstrate that the influenza A virus NS1A protein interacts with cognate proteins of the viral polymerase complex in infected cells. This approach capitalized on insights obtained from the X-ray crystal structure of the complex of the Ud effector domain with the F2F3 domain of CPSF30 (3). This complex is a tetramer that contains two NS1A effector domains and two F2F3 domains. Each of the two NS1A effector domains contains a pocket that binds the F2F3 domain of CPSF30, and the tetrameric structure is stabilized by two NS1A amino acids that are outside the binding pocket, F103 and M106. These two amino acids are conserved in greater than 99% of the NS1A proteins of influenza A viruses isolated from humans (3, 11) and are required for binding F2F3 in vitro (3). Nonetheless, in infected cells, an NS1A protein that contains different hydrophobic amino acids at these positions, specifically L at 103 and I at 106, does bind CPSF30 to a significant extent (19), but as shown in the present study, binding occurs only when this NS1A protein is expressed in conjunction with cognate proteins of the viral polymerase complex. Expression of such an NS1A protein in infected cells in the presence of noncognate viral polymerase complex proteins does not result in the binding of the NS1A protein to CPSF30. The requirement for cognate proteins clearly shows that there are specific interactions between the NS1A protein and cognate proteins of the viral polymerase complex that lead to the stabilization of the CPSF30-NS1A complex. In fact, we were able to use this genetic approach to demonstrate that the primary, if not the only, interaction occurs between the NS1A protein and its cognate PA protein and NP. Thus, the HK97 NS1A protein, which contains L103 and I106, binds to CPSF30 in cells infected by a recombinant virus that expresses only the HK97 PA protein and NP, but not the HK97 PB1 and PB2 proteins, of the viral polymerase complex. It should be possible to use this genetic approach to identify the interacting sequences of the NS1A protein and its cognate PA protein and NP.

The stabilization provided by the cognate viral polymerase complex operates in the interaction surface between NS1A and CPSF30 that is outside the CPSF30-binding pocket itself. The cognate viral polymerase complex cannot stabilize CPSF30 binding to an NS1A protein that has a mutated CPSF30-binding pocket. It will be of great interest to determine the mechanism of stabilization, specifically, how the interaction of the PA and NP subunits of the viral polymerase complex with its cognate NS1A protein that contains nonconsensus hydrophobic amino acids at positions 103 and 106 (L and I, respectively) enables this NS1A protein to bind to CPSF30, albeit suboptimally. The finding of the stabilization by a cognate viral polymerase complex led to the prediction that the viral polymerase complex is an integral part of the CPSF30-NS1A complex, a prediction that was verified in the present study. The macromolecular complex containing the viral polymerase complex and the NS1A protein likely includes other host cell proteins in addition to CPSF30. Because we showed previously that the binding of the NS1A protein to CPSF30 does not disrupt the interaction between CPSF30 and other CPSF subunits (15), it is likely that these other CPSF subunits are also part of this macromolecular complex containing the viral polymerase complex and the NS1A protein. This macromolecular complex may also contain other host factors that bind directly to the viral polymerase rather than to the NS1A protein. A well-documented example of such a host factor is the large subunit of the cellular RNA polymerase II, which binds to the tripartite viral polymerase (PB1, PB2, PA) via its C-terminal domain (4).

Our results verify that the NS1A protein interacts with the viral polymerase complex in infected cells. Previous studies provided evidence for an NS1A protein-polymerase interaction and for a role of the NS1A protein in viral RNA synthesis (5, 12, 14, 17). For example, we recently found that the NS1A protein regulates the time course of viral RNA synthesis during infection: a recombinant Ud virus that expresses an NS1A protein in which only two amino acids (123 and 124) are changed to alanines deregulates the normal time course of viral RNA synthesis that occurs in cells infected by wild-type Ud virus (14). The NS1A-viral polymerase interaction described in the present study occurs in the context of a macromolecular complex that includes the host CPSF30 protein. It is possible that this is the primary, if not the only, context in which the NS1A protein interacts with the viral polymerase complex in infected cells. Alternatively, interaction between the NS1A protein and the viral polymerase complex may occur on its own, and the resulting multipartite complex may then interact with CPSF30 and/or with other factors. No matter how it is formed, the macromolecular complex containing the host CPSF30 protein, the viral NS1A protein, and the viral polymerase complex may have an important role in the regulation of viral RNA synthesis in infected cells. Future research will determine whether this is the case.

 
This work was supported by Public Health Service grant AI-11772 from the National Institute of Allergy and Infectious Diseases.

We thank Chen Zhao for helpful discussions.

 
* Corresponding author. Mailing address: Institute for Cellular and Molecular Biology, MBB 2.122, 2500 Speedway, University of Texas at Austin, Austin, TX 78712. Phone: (512) 232-5563. Fax: (512) 232-5565. E-mail: rkrug{at}mail.utexas.edu

Published ahead of print on 3 December 2008.

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1
1. Chen, B. J., G. P. Leser, E. Morita, and R. A. Lamb. 2007. Influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles. J. Virol. 81:7111-7123.[Abstract/Free Full Text]
2
2. Chien, C. Y., Y. Xu, R. Xiao, J. M. Aramini, P. V. Sahasrabudhe, R. M. Krug, and G. T. Montelione. 2004. Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode. Biochemistry 43:1950-1962.[CrossRef][Medline]
3
3. Das, K., L.-C. Ma, R. Xiao, J. Aramini, J. Marklund, R.-L. Kuo, E. Arnold, R. M. Krug, and G. T. Montelione. 2008. Structural basis for suppression by influenza A virus of a host antiviral response. Proc. Natl. Acad. Sci. USA 105:13093-13098.[Abstract/Free Full Text]
4
4. Engelhardt, O. G., M. Smith, and E. Fodor. 2005. Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J. Virol. 79:5812-5818.[Abstract/Free Full Text]
5
5. Falcon, A. M., R. M. Marion, T. Zurcher, P. Gomez, A. Portela, A. Nieto, and J. Ortin. 2004. Defective RNA replication and late gene expression in temperature-sensitive influenza viruses expressing deleted forms of the NS1 protein. J. Virol. 78:3880-3888.[Abstract/Free Full Text]
6
6. Hatada, E., and R. Fukuda. 1992. Binding of influenza A virus NS1 protein to dsRNA in vitro. J. Gen. Virol. 73:3325-3329.[Abstract/Free Full Text]
7
7. Hatta, M., P. Gao, P. Halfmann, and Y. Kawaoka. 2001. Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293:1840-1842.[Abstract/Free Full Text]
8
8. Kim, M. J., A. G. Latham, and R. M. Krug. 2002. Human influenza viruses activate an interferon-independent transcription of cellular antiviral genes: outcome with influenza A virus is unique. Proc. Natl. Acad. Sci. USA 99:10096-10101.[Abstract/Free Full Text]
9
9. Li, Y., Z. Y. Chen, W. Wang, C. C. Baker, and R. M. Krug. 2001. The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo. RNA 7:920-931.[Abstract]
10
10. Lu, Y., M. Wambach, M. G. Katze, and R. M. Krug. 1995. Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214:222-228.[CrossRef][Medline]
11
11. Macken, C., H. Lu, J. Goodman, and L. Boykin. 2001. The value of a database in surveillance and vaccine selection, p. 103-106. In A. Osterhaus, N. Cox, and A. W. Hampson (ed.), Options for the control of influenza, IV. Elsevier Science, Amsterdam, The Netherlands.
12
12. Marion, R. M., T. Zurcher, S. de la Luna, and J. Ortin. 1997. Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo. J. Gen. Virol. 78:2447-2451.[Abstract]
13
13. Min, J.-Y., and R. M. Krug. 2006. The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: inhibiting the 2'-5' OAS/RNase L pathway. Proc. Natl. Acad. Sci. USA 103:7100-7105.[Abstract/Free Full Text]
14
14. Min, J.-Y., S. Li, G. C. Sen, and R. M. Krug. 2007. A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363:236-243.[CrossRef][Medline]
15
15. Nemeroff, M. E., S. M. Barabino, Y. Li, W. Keller, and R. M. Krug. 1998. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. Mol. Cell 1:991-1000.[CrossRef][Medline]
16
16. Noah, D. L., K. Y. Twu, and R. M. Krug. 2003. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS. Virology 307:386-395.[CrossRef][Medline]
17
17. Shimizu, K., H. Handa, S. Nakada, and K. Nagata. 1994. Regulation of influenza virus RNA polymerase activity by cellular and viral factors. Nucleic Acids Res. 22:5047-5053.[Abstract/Free Full Text]
18
18. Takeda, M., A. Pekosz, K. Shuck, L. H. Pinto, and R. A. Lamb. 2002. Influenza A virus M2 ion channel activity is essential for efficient replication in tissue culture. J. Virol. 76:1391-1399.[Abstract/Free Full Text]
19
19. Twu, K. Y., R. L. Kuo, J. Marklund, and R. M. Krug. 2007. The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells. J. Virol. 81:8112-8121.[Abstract/Free Full Text]
20
20. Twu, K. Y., D. L. Noah, P. Rao, R.-L. Kuo, and R. M. Krug. 2006. The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J. Virol. 80:3957-3965.[Abstract/Free Full Text]
21
21. Wang, W., K. Riedel, K. Lynch, C. Chien, G. T. Montelione, and R. M. Krug. 1999. RNA-binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5:195-205.[Abstract]
22
22. Yin, C., J. A. Khan, G. V. Swapna, A. Ertekin, R. M. Krug, L. Tong, and G. T. Montelione. 2007. Conserved surface features form the double-stranded RNA-binding site of nonstructural protein 1 (NS1) from influenza A and B viruses. J. Biol. Chem. 282:20584-20592.[Abstract/Free Full Text]

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Journal of Virology, February 2009, p. 1611-1616, Vol. 83, No. 4
0022-538X/09/$08.00+0     doi:10.1128/JVI.01491-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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