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. 2010 Oct 13;84(24):12619–12627. doi: 10.1128/JVI.01696-10

Cyclin T1/CDK9 Interacts with Influenza A Virus Polymerase and Facilitates Its Association with Cellular RNA Polymerase II▿

Junjie Zhang 1,2, Gang Li 1,2, Xin Ye 1,*
PMCID: PMC3004352  PMID: 20943989

Abstract

Influenza virus RNA-dependent RNA polymerase scavenges the 5′ cap from host pre-mRNA to prime viral transcription initiation. It is also well established that viral RNA-dependent RNA polymerase (vRNP) associates with cellular RNA polymerase II (Pol II), on which viral replication depends. Here we report that cyclin T1/CDK9 can interact with influenza virus polymerase and facilitate its association with cellular Pol II. The immunodepletion of cyclin T1/CDK9 totally abolished the association of vRNP with the C-terminal domain (CTD) Ser-2-phosphorylated form of RNA polymerase II. Further studies showed that overexpression of cyclin T1/CDK9 increased the transcription activity of vRNP, while knockdown of cyclin T1/CDK9 impaired viral replication. Our results suggest that cyclin T1/CDK9 serves as an adapter to mediate the interaction of vRNP and RNA Pol II and promote viral transcription.

The influenza A virus RNA-dependent RNA polymerase complex consists of three subunits, PB1, PB2, and PA. After infection, viral RNA-dependent RNA polymerases (vRNPs) are transported into the nucleus, where viral transcription and replication take place. The conserved noncoding sequences at the 5′ and 3′ ends of each genomic RNA segment serve as promoter elements, which are recognized by the viral polymerase. The initiation of transcription of viral mRNA requires a 5′-capped primer, which is obtained by the endonucleolytic cleavage of cellular pre-mRNAs by the viral polymerase. The PB2 subunit of the polymerase has a cap-binding function (4). The PB1 subunit is the RNA-dependent RNA polymerase and is responsible for elongation (3, 23). The PA subunit possesses endonuclease activity (7, 11, 36), together with the PB1 subunit, which is involved in the process of cap snatching from cellular pre-mRNA. It is well established that RNA polymerase II (Pol II) is involved in influenza virus replication (1, 8, 15, 18, 24). The viral polymerase binds to hyperphosphorylated forms of the large subunit of RNA Pol II, suggesting that it targets actively transcribing RNA Pol II. However, it is still uncertain whether the interaction between influenza virus polymerase and RNA Pol II is direct or is mediated by certain host factors that bind both the hyperphosphorylated C-terminal domain (CTD) and the influenza virus polymerase. It is known that cyclin T/CDK9 stimulates transcription elongation by preferentially phosphorylating Ser-2 of heptapeptide repeats of the CTD of the large subunit of RNA Pol II as well as enhances transcriptional elongation by phosphorylating and counteracting the negative elongation factors 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) (22, 38). In addition, cyclin T1/CDK9 has been reported to play a role in the transcriptional regulation of human immunodeficiency virus type 1 (HIV-1) mRNA (6, 16). We wondered if cyclin T1/CDK9 is involved in regulating the transcription of influenza A virus.

Other studies of RNA Pol II inhibitors also shed light on the coupling of Pol II activity and the influenza virus replication process. It was found that 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), which inhibits mRNA elongation, blocked influenza virus multiplication (28). Recent research showed that DRB did not affect viral primary transcription, while α-amanitin, which inhibits both mRNA initiation and elongation, totally blocked the viral transcription and replication. The preexpression of viral polymerase and NP proteins in cells prior to α-amanitin treatment could restore the levels of viral RNA (vRNA) and cRNA but not viral mRNA, suggesting that Pol II activity is required mainly for viral mRNA synthesis. α-Amanitin-resistant Pol II could rescue α-amanitin inhibition of influenza virus transcription and replication (5). Treatment with another Pol II inhibitor, H7, did not disturb viral primary mRNA transcription, while it inhibited the expression of influenza virus late genes, such as the hemagglutinin (HA) and M1 genes, but not early genes, such as the NP gene (2, 14). A series of studies showed that Pol II inhibitors, including DRB, actinomycin D, and H7, caused a nuclear accumulation of some influenza virus mRNA (2, 25, 30), implying that the efficient nuclear export of certain influenza virus mRNAs required ongoing Pol II activity.

In the present study, we demonstrated that cyclin T1/CDK9 interacted with influenza A virus vRNP and mediated its association with the Ser-2-phosphorylated CTD of RNA Pol II. In addition, the depletion of cyclin T1 by RNA interference greatly inhibited viral transcription and replication, and the overexpression of cyclin T1 upregulated the transcription activity of vRNP, suggesting that cyclin T1/CDK9 plays an important role in regulating the transcription of influenza A virus.

MATERIALS AND METHODS

Cell lines, viruses, and antibodies.

Madin-Darby canine kidney (MDCK) cells, human embryo kidney 293T cells, human type II alveolar epithelial A549 cells, and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; PAA). Recombinant influenza virus A/WSN/33 was generated as previously described (37). Rabbit anti-M1 polyclonal antibody and rabbit anti-NP polyclonal antibody were generated by immunizing rabbits with purified recombinant 6His-tagged M1 or NP (37). Mouse anti-FLAG (M2) antibody was purchased from Sigma. Mouse monoclonal anti-HA antibody (26D11) was purchased from Abmart. Mouse monoclonal anti-Myc (9E10) antibody, rabbit anti-CDK9 polyclonal antibody (C-20), goat anti-cyclin T1 polyclonal antibody (T-18), goat anti-PB2 polyclonal antibody (vN-19), and rabbit anti-actin polyclonal antibody (1-19) were purchased from Santa Cruz Biotechnology. Mouse anti-PB2 polyclonal antibody and rabbit anti-PB1 polyclonal antibody were kindly provided by Yingfang Liu (Institute of Biophysics, Chinese Academy of Sciences [CAS]). Mouse anti-CTD monoclonal antibody (8WG16), anti-phosphoserine 2 CTD monoclonal antibody (H5), and anti-phosphoserine 5 CTD monoclonal antibody (H14) were purchased from Covance.

Plasmids.

CDK9 was cloned into pFLAG-CMV2 (Sigma) and pCMV-Myc (BD Clontech), a CDK9 kinase-deficient mutant (D167N) (CDK9-DN) was cloned into pFLAG-CMV2, and cyclin T1 was expressed by using pCMV-HA (BD Clontech). The cDNAs of PB1, PB2, PA, and NP were derived from strain WSN and subcloned into pcDNA3-FLAG. Plasmids for expressing nontagged PB1, PB2, PA, and NP were derived from an influenza A virus reverse-genetics system (20). The polymerase I-expressing plasmid carrying an influenza virus-like RNA encoding firefly luciferase (vNS-Luc) was kindly provided by Martin Schwemmle, University of Freiburg (10).

Luciferase assay.

293T cells in a 12-well plate were transfected with plasmids for expressing influenza A virus (A/WSN/33) PB1, PB2, PA, and NP and vNS-Luc (100 ng of each) with pCMV-β-gal (50 ng) as an internal control to normalize the transfection efficiency. The cell lysates were harvested at 30 h posttransfection, and the luciferase activity was measured according to the manufacturer's instructions (Promega) and normalized with β-galactosidase (β-gal) activity.

Immunoprecipitation.

To assess the association between FLAG-CDK9 and vRNP, 293T cells were transfected with pCMV FLAG-CDK9 or pFLAG-CMV2 as a control and then infected with A/WSN/33 (multiplicity of infection [MOI] of 0.5) for 12 h. The cells were washed with cold phosphate-buffered saline (PBS) and lysed in lysis buffer (1% Triton, 150 mM NaCl, 20 mM HEPES [pH 7.5], 10% glycerol, 1 mM EDTA) with a protease inhibitor cocktail (Roche). Cell lysates were immunoprecipitated with anti-FLAG M2 beads (Sigma), followed by immunoblotting with NP antibody.

To analyze which subunit of vRNP interacted with CDK9, 293T cells were transfected with Myc-CDK9 and FLAG-tagged NP, PA, PB1, or PB2, respectively. Cell lysates were precipitated with anti-FLAG M2 beads, followed by immunoblotting with Myc antibody.

To assess the interaction between vRNP and cyclin T1/CDK9 or RNA Pol II, 293T cells were infected with A/WSN/33 at an MOI of 0.5 or mock infected for 12 h. The cell lysates were incubated with 1 μg of rabbit anti-CDK9 polyclonal antibody or normal rabbit IgG for 4 h at 4°C and incubated with 10 μl of protein A-Sepharose beads for 4 h at 4°C. Proteins bound to the beads were then washed extensively with lysis buffer and applied onto the immunoblot with NP and PB2 antibodies. For NP immunoprecipitation, the cell lysates were incubated with 1 μg of rabbit anti-NP polyclonal antibody or normal rabbit IgG for 4 h at 4°C followed by incubation with protein A-Sepharose beads. Proteins bound to the beads applied to the immunoblot with cyclin T1 antibody or RNA Pol II antibody (8WG16).

Immunofluorescence.

HeLa cells were infected with influenza A virus (A/WSN/33) at an MOI of 1 for 8 h. The cells were washed with PBS and fixed for 10 min with 4% paraformaldehyde, followed by permeabilization with 1% Triton X-100 for 5 min. The samples were then blocked with 10% normal goat serum in PBS for 2 h at room temperature, followed by incubation with mouse anti-PB2 and rabbit anti-CDK9 antibodies for 1 h at 37°C. Slides were then washed with 0.5% NP-40-PBS and incubated with goat anti-mouse tetramethyl rhodamine isocyanate (TRITC) conjugates and goat anti-rabbit fluorescein isothiocyanate (FITC) conjugates (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The cells were observed with a Leica confocal microscope.

RNA interference and virus infection.

A549 or HeLa cells were transfected with cyclin T1-specific small interfering RNA (siRNA) (6) or control siRNA (50 nM for A549 and 100 nM for HeLa cells) for 36 h. The cells were then infected with influenza A virus (A/WSN/33) at an MOI of 0.5 (A549 cells) or 0.1 (HeLa cells) for 10 h. The medium was collected, the virus titer was measured by a plaque assay, and the cell extracts were harvested for immunoblotting.

Immunodepletion.

293T cells were transfected with influenza A virus minireplicon plasmids as described above except that PB1 was replaced with PB1-FLAG. Cell lysates were incubated with 1 μg of CDK9 antibody or rabbit IgG for 4 h at 4°C and then incubated with 20 μl of protein A-Sepharose beads overnight at 4°C. After centrifugation, the supernatants were collected and immunoprecipitated with FLAG beads. The immunoprecipitated proteins were analyzed by immunoblotting with the indicated antibodies.

Quantitative RT-PCR.

A549 cells were transfected with 50 nM cyclin T1-specific siRNA or control siRNA for 36 h followed by infection with influenza A virus (A/WSN/33) at an MOI of 0.5 for 8 h. The total RNA of cells was then extracted by using TRIzol reagent (Invitrogen). Reverse transcription (RT) was conducted by using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega). Primers used for reverse transcription of vRNA, cRNA, and mRNA were 5′-AGCGAAAGCAGG-3′, 5′-AGTAGAAACAAGG-3′, and oligo(dT), respectively. Real-time PCR was performed by using SYBR green Realtime PCR Master mix (Toyobo). The PCR conditions were 95°C for 3 min and 50 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The amounts of viral RNA were normalized by 18S RNA.

RESULTS

Cyclin T1/CDK9 interacts with the vRNP of influenza A virus.

To examine whether CDK9 can interact with influenza A vRNP, 293T cells were transfected with pFLAG-CMV-CDK9 or pFLAG-CMV-2 as a control and then infected with A/WSN/33 at an MOI of 0.5 for 12 h. Cell extracts were immunoprecipitated with FLAG antibody and immunoblotted with NP antibody. As shown in Fig. 1A, CDK9 was found to bind with vRNP. We next examined whether endogenous cyclin T1/CDK9 could interact with vRNP. 293T cells were infected with A/WSN/33 at an MOI of 0.5 for 12 h, and cell lysates were immunoprecipitated with CDK9 antibody and immunoblotted with NP and PB2 antibodies, and the results showed that NP and PB2 could interact with CDK9 (Fig. 1B). The cell lysates were also subjected to immunoprecipitation with NP antibody and immunoblotted with cyclin T1 antibody. The data showed that cyclin T1 could interact with NP in infected cells (Fig. 1C). PB1, the subunit of vRNP, could also be coimmunoprecipitated with cyclin T1 (Fig. 1D), which further confirmed that cyclin T1/CDK9 interacted with vRNP. Furthermore, immunofluorescence data indicated that CDK9 was partially colocalized with PB2 in virally infected HeLa cells (Fig. 1E). These data demonstrated that CDK9 could interact with the viral polymerase complex during influenza A virus infection.

FIG. 1.

FIG. 1.

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CDK9 interacted with influenza A virus polymerase. (A) 293T cells were transfected with FLAG-CDK9 or pFLAG-CMV2 as a control and then infected with A/WSN/33. Cell lysates were immunoprecipitated (IP) with FLAG antibody followed by immunoblotting (IB) with NP antibody. (B, C, and D) 293T cells were infected with A/WSN/33, and cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (E) HeLa cells were infected with A/WSN/33 and then immunostained with mouse anti-PB2 antibody and rabbit anti-CDK9 antibody. (F) 293T cells were transfected with Myc-CDK9 and FLAG-tagged NP, PA, PB1, and PB2. The cell lysates were applied for immunoprecipitation with FLAG antibody and immunoblotted with the indicated antibodies.

To determine which subunit of vRNP mediates the interaction with CDK9, binary protein-protein interactions were examined. pCMV-Myc-CDK9 was introduced together with FLAG-tagged PB1, PB2, PA, or NP into 293T cells. The cell extracts were immunoprecipitated with anti-FLAG M2 beads and immunoblotted with Myc antibody. The results indicated that PA, PB1, and PB2 could interact with CDK9, while NP could not (Fig. 1F).

The cyclin T1/CDK9 complex serves as an adaptor for influenza virus vRNP targeting RNA polymerase II.

Cyclin T1/CDK9 is responsible for the phosphorylation of RNA Pol II at the carboxy-terminal domain (CTD) heptapeptide repeats, which stimulates it to elongate the RNA transcripts (22). Several lines of evidence indicate that RNA Pol II is involved in influenza viral transcription. Influenza virus vRNP interacts with hyperphosphorylated RNA Pol II (8). The drugs that interfered with RNA Pol II transcription activity impaired influenza virus propagation (5, 17). These observations prompted us to ask if vRNP interacts with RNA Pol II and how vRNP is associated with Pol II. To address this question, 293T cells were infected with influenza virus, and cell lysates were harvested and subjected to immunoprecipitation with NP antibody and immunoblotted with RNA Pol II antibody. The data showed that vRNP can bind with the hyperphosphorylated form of RNA Pol II specifically in virus-infected cells (Fig. 2A), which is consistent with data from a previous report (8). As we found that cyclin T1/CDK9 could interact with the vRNP complex (Fig. 1), it implied that cyclin T1/CDK9 may serve as an adaptor protein between vRNP and RNA Pol II. To verify this model and determine whether cyclin T1/CDK9 mediated the interaction of vRNP and RNA Pol II, we took the approach of an influenza A virus minireplicon assay except that PB1 was replaced by PB1-FLAG; therefore, vRNP could be immunoprecipitated more efficiently with FLAG antibody. 293T cells were transfected with influenza A virus minireplicon plasmids, and the cell lysates were prepared and divided equally into 2 aliquots. One set was immunodepleted with CDK9 antibody, and the other was immunodepleted with rabbit IgG as a control. The samples were then subjected to immunoprecipitation with anti-FLAG M2 beads. As shown in Fig. 2B (left), levels of CDK9 and cyclin T1 were reduced significantly in the CDK9-depleted extract in comparison to the control, but the amounts of RNA Pol II and phosphorylated Pol II did not change. As expected, vRNP-bound cyclin T1/CDK9 decreased to a very low level. Levels of RNA Pol II and the ser-5-phosphorylated form of RNA Pol II [RNA Pol II(pS-5)] bound with vRNP were slightly reduced, while RNA Pol II(pS-2) almost could not be detected in the vRNP complex (Fig. 2B). These results indicate that vRNP is unable to bind to RNA Pol II(pS-2) in the absence of cyclin T1/CDK9 complexes, strongly suggesting that cyclin T1/CDK9 served as an adaptor for vRNP to bind with RNA Pol II(pS-2).

FIG. 2.

FIG. 2.

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Cyclin T1/CDK9 was an adaptor between vRNP and RNA Pol II(pS-2). (A) 293T cells were infected with A/WSN/33 for 12 h. The cell lysates were immunoprecipitated with rabbit anti-NP polyclonal antibody or rabbit IgG as a control and immunoblotted with RNA Pol II monoclonal antibody. (B) 293T cells were transfected with influenza A virus minireplicon plasmids as described in the text except that PB1 was replaced by PB1-FLAG. The cell lysates were immunodepleted with CDK9 antibody or rabbit IgG as a control and then immunoprecipitated with anti-FLAG M2 beads and immunoblotted with the indicated antibodies.

Cyclin T1/CDK9 is involved in influenza A virus transcription.

To determine the role of cyclin T1/CDK9 in replication, we asked if cyclin T1/CDK9 was involved in regulating viral transcription. An influenza A virus minireplicon assay was employed to measure viral polymerase activity (10). Using this assay, we found that overexpression of either wild-type CDK9 or the dominant negative form of CDK9 (CDK9-DN) (29) did not affect viral transcription activity, while overexpression of cyclin T1 promoted the transcription activity of vRNP significantly (Fig. 3A). Immunoblotting analysis indicated that the expression of either CDK9 or cyclin T1 did not affect the expression of NP. These results suggest that cyclin T1 may be a limiting factor to form the cyclin T1/CDK9 complex to promote influenza virus polymerase activity. Both CDK9 and CDK9-DN can bind vRNP at similar levels, indicating that the kinase activity of CDK9 was not required for its interaction with vRNP (Fig. 3B).

FIG. 3.

FIG. 3.

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Cyclin T1/CDK9 promoted influenza A virus transcription. (A) 293T cells were transfected with plasmids for expressing influenza A virus (A/WSN/33) PB1, PB2, PA, and NP; vNS-Luc; and pCMV β-gal for 30 h. The luciferase activity was determined and normalized with β-gal activity. Error bars represent standard deviations determined from triplicate samples. (B) 293T cells were transfected with FLAG-CDK9 or FLAG-CDK9-DN and then infected with A/WSN/33. Cell lysates were immunoprecipitated with FLAG antibody and then immunoblotted with PB2 antibody. (C) 293T cells were transfected with influenza A virus minireplicon plasmids and other plasmids, as indicated, for 30 h. Cell lysates were applied for a luciferase assay, normalized with β-gal expression. Error bars represent the standard deviations of data from triplicate samples. (D) 293T cells were transfected with influenza A virus minireplicon and HA-cyclin T1 (4 μg), FLAG-CDK9 (1 μg), or FLAG-CDK9 (4 μg). Cell lysates were applied for immunoprecipitation with HA agarose and immunoblotted with HA and FLAG antibodies. The free CDK9 in supernatants after immunoprecipitation was also examined by immunoblotting with FLAG antibody.

Previous studies indicated that free CDK9 is present in cells (12), suggesting that cyclin T1 could be the restriction factor for the interaction between the cyclin T1/CDK9 complex and vRNP. It is reasonable to hypothesize that the overexpression of CDK9 may generate excess CDK9, which could reverse cyclin T1 promotion activity. To test this hypothesis, 293T cells were transfected with two different doses (0.1 and 0.5 μg) of FLAG-CDK9 with 0.5 μg of HA-cyclin T1 together with influenza A virus minireplicon plasmids. As shown in Fig. 3C, the level of vRNP transcription activity in the cells transfected with 0.1 μg of FLAG-CDK9 was much higher than that in the cells transfected with 0.5 μg of CDK9 and was comparable to that in cells transfected with cyclin T1 alone. The total amounts of CDK9, both cyclin T1 bound and unbound, in cell lysates were examined. As shown in Fig. 3D, the levels of cyclin T1-bound CDK9 were similar in the cells transfected with both doses of FLAG-CDK9, while there was more free CDK9 in cells transfected with the higher dose of FLAG-CDK9. These data indicated that excess CDK9 expression could reverse cyclin T1/CDK9 promotion activity toward viral transcription.

To further investigate whether cyclin T1/CDK9 affects the replication of influenza A virus, we took the approach of RNA interference to knock down cyclin T1 (6). Previous reports demonstrated that the knockdown of cyclin T1 with siRNA or short hairpin RNA (shRNA) did not affect cell viability (6, 35). A549 cells were transfected with small RNA duplexes targeting cyclin T1 and then infected with influenza A virus. Cell lysates and supernatants were harvested and analyzed by immunoblotting and plaque assay, respectively. As shown in Fig. 4A, cyclin T1 was specifically knocked down in the cells transfected with siRNA. In these cells, the level of CDK9 was also decreased, which was consistent with a previous report showing that CDK9 stability depends on the presence of cyclin T1 (6). A quantitative analysis of proteins indicated that cyclin T1 and CDK9 levels were reduced to about 30% (Fig. 4C). As the level of cyclin T1/CDK9 decreased, the levels of expression of influenza virus M1 and NP proteins were significantly decreased in infected cells (Fig. 4A and C), and the amounts of virus particles released into the medium were also significantly reduced (Fig. 4B). Similar results were observed for HeLa cells as cyclinT1 was knocked down (Fig. 4D and E). Taken together, these results demonstrated that cyclin T1/CDK9 was an important regulator for influenza virus propagation.

FIG. 4.

FIG. 4.

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Knockdown of cyclin T1 impaired influenza virus replication. A549 and HeLa cells were transfected with cyclin T1-specific siRNA or control siRNA for 36 h and infected with influenza A virus (A/WSN/33) for 8 h. (A and D) The cell extracts were harvested for immunoblotting. (C) The band intensity of proteins was quantified from four independent experiments with A549 cells by using ImageJ (NIH). (B and E) The medium from A549 (B) or HeLa (E) cells was collected to measure the virus titer by plaque assay. (F) A549 cells were transfected with cyclin T1-specific RNA for 36 h and infected with influenza A virus (A/WSN/33) for 6 h. Total RNAs were extracted and subjected to quantitative RT-PCR for the RNA of the M segment, with 18S RNA as an internal control.

The data described above showed that the knockdown of cyclin T1/CDK9 impaired influenza virus propagation and that cyclin T1/CDK9 promoted vRNP activity, suggesting that cyclin T1/CDK9 is involved in regulating virus transcription. To examine whether cyclin T1/CDK9 regulates influenza virus transcription, A549 cells were transfected with siRNA targeting cyclin T1 and then infected with influenza virus. Total RNA was prepared and used to analyze the amount of influenza virus M segment RNA by quantitative RT-PCR. As shown in Fig. 4F, the M1 mRNA level was significantly reduced in cyclin T1 knockdown cells. vRNA and cRNA syntheses were also impaired, probably due to reduced viral protein synthesis. These data suggest that cyclin T1/CDK9 facilitates influenza virus transcription.

DISCUSSION

Cyclin T1/CDK9 complexes are responsible for controlling the elongation process of transcription by RNA polymerase II (Pol II). Its activity is regulated negatively by associations with Hexim1 and a small nuclear RNA called 7SK RNA (19, 21, 33, 34). It was reported that the cyclin T1/CDK9 complex can serve as an important factor for HIV transcription. The HIV Tat protein binds the trans-activating response region (TAR), recruits cyclin T1/CDK9 to nascent viral transcripts, and stimulates full-length viral transcription by phosphorylating Spt5, RD, and serine 2 of the RNA Pol II CTD (22, 38). The kinase activity of CDK9 is very crucial to HIV transcription, and dominant negative CDK9 strongly reduces HIV transcription (9). Unlike HIV, influenza virus transcription was not strictly dependent on CDK9 kinase activity, since CDK9-DN expression did not reduce influenza virus transcription activity (Fig. 3A). Our data showed that CDK-DN can also bind with viral vRNP (Fig. 3B), which suggests that its association with vRNP, rather than its kinase activity, may be involved in the virus transcription process. Surprisingly, we found that CDK9 interacted with three components of influenza virus vRNP, PB1, PB2, and PA, when they are expressed in cells. It was reported previously that the host protein RuvB-like protein 2 (RBL2) could interact with three different vRNP components, PB1, PB2, and NP (13). The interaction between RBL2 and NP was pivotal and involved in restricting virus replication. Thus, it seems possible that some host proteins can interact with several components of vRNP. Which component of viral polymerase plays a dominant role in interacting with cyclin T1/CDK9 during influenza virus infection needs further investigation. When CDK9 is overexpressed, it may partially drive cyclin T1/CDK9 out of Hexim1 and 7SK RNA contraction, which may enhance viral transcription. Free CDK9 that did not associate with cyclin T1 could still interact with vRNP, but these vRNPs could not be guided to RNA Pol II, while the overexpression of cyclin T1 increases cyclin T1/CDK9 complex formation and promotes influenza virus transcription. Furthermore, cyclin T1/CDK9 knockdown inhibited viral mRNA synthesis, and cyclin T1/CDK9 immunodepletion nearly blocked vRNP binding with RNA Pol II(pS-2). Taken together, these results demonstrated that cyclin T1/CDK9 mediated the interaction between vRNP and RNA Pol II and improved viral transcription (Fig. 5). Consistently, the levels of viral mRNA, vRNA, and cRNA were reduced as cyclin T1/CDK9 was knocked down in virus-infected cells. We proposed that the reduction in levels of viral mRNA in cyclin T1 knockdown cells may be due to poor vRNP recruitment to RNA Pol II(pS-2), while the reduction in levels of vRNA and cRNA may be caused by reduced viral protein expression, as shown in Fig. 4A and C.

FIG. 5.

FIG. 5.

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Schematic diagram of cyclin T1/CDK9 in recruiting vRNP onto active transcribing Pol II. In normal cells, DNA-dependent RNA polymerase II was recruited to the promoter region during transcription initiation. Next, TFIIH, a complex containing CDK7 and cyclin H, was recruited to phosphorylate Ser-5 of the Pol II CTD and initiate transcription. Shortly after initiation, mRNA elongation is arrested by the negative elongation factors DSIF and NELF, which ensure the capping of the mRNA. P-TEFb (CDK9/T1) then further phosphorylated Ser-2 of the Pol II CTD to promote the transcription elongation process (top). In virus-infected cells, cyclin T1/CDK9 can recruit vRNP to hyperphosphorylated Pol II for cap snatching and facilitate viral mRNA transcription (bottom).

As a cyclinT1/CDK9 inhibitor (32), DRB can reduce mRNA, vRNA, and cRNA synthesis, and it is worthwhile to note that DRB treatment did not totally inhibit viral mRNA synthesis, while α-amanitin, which blocks both Pol II initiation and elongation, abolished viral mRNA synthesis (5). The above-mentioned data were consistent with our model that Pol II(pS-2) and Pol II(pS-5) may serve as two independent bridges for vRNP to snatch the cap of host mRNA, and DRB inhibited Pol II(pS-2), leading to a partial inhibition of viral mRNA synthesis, while α-amanitin inhibited both Pol II(pS-2) and Pol II(pS-5) and therefore totally abolished viral mRNA synthesis.

It was reported previously that RNA Pol II was involved in the nuclear export of influenza virus mRNA (2, 25). Those researchers showed that DRB treatment inhibited the nuclear export of the transcript of viral late genes (the M gene, for example), while the drug showed little effect on early viral gene transcripts (the NP gene, for example). Although our data showed that the cyclin T1/CDK9 knockdown impaired viral mRNA synthesis, the possibility that viral mRNA transcripts may be regulated at the posttranscriptional level, especially its nuclear export process, could not be ruled out. Actually, as shown in Fig. 4C, the knockdown of cyclin T1/CDK9 reduced M1 expression by 80% and NP expression by 50%, which suggests that the transcriptions of viral late genes such as the M1 gene were more sensitive to cyclin T1/CDK9 knockdown, and this could probably be due to additional inhibition during their mRNA nuclear export process.

It is shown in Fig. 2B (right) that the depletion of CDK9 resulted in a failure of the interaction of vRNP with RNA Pol II(pS-2) but did not affect the association of vRNP with RNP Pol II(pS-5). This finding implies that there might be some other adaptor proteins that facilitate vRNP targeting RNA Pol II(pS-5). The unidentified adaptor may also be pivotal for the association of vRNP and Pol II, since cyclin T1/CDK9 knockdown only slightly reduced vRNP binding with total Pol II (Fig. 2B). CDK7, which is the kinase responsible for phosphorylating Ser-5 of the RNA Pol II CTD, could be a candidate for such an adaptor. However, we did not detect the binding of CDK7 with vRNP in influenza A virus-infected 293T cells (data not shown). Increasing evidence suggests that Pol II plays multiple roles in the influenza virus infection process. It is well established that Pol II is involved in the cap-snatching process and viral mRNA nuclear export. Previous studies found that influenza virus infection causes the specific degradation of the hypophosphorylated form of the largest subunit of Pol II. The degradation was dependent on viral polymerase and the proteolytic activity of PA (26, 27). A recent study reported that the hyperphosphorylated form of Pol II was also degraded during influenza virus infection and that the proteasome pathway was involved in the degradation. It was proposed that Pol II degradation may represent a viral strategy to subvert the host antiviral system (31). Other studies pointed out that attenuated influenza virus strains did not cause Pol II degradation, suggesting that the Pol II degradation may be linked with virulence (26). We also found that the level of the hypophosphorylated form of Pol II was decreased in A/WSN/33-infected cells compared to that in uninfected cells (data not shown), but the mechanisms underlying the regulation of Pol II degradation by virus need to be further elucidated.

Finally, CDK9 activity may be modified during influenza virus infection. We demonstrated that CDK9 activity was not necessary for virus transcription. It was reported previously that the level of Pol II(pS-2) was reduced during A/WSN/33 infection (31) and that there was a significant reduction in Pol II densities in the coding region of Pol II-transcribing genes in infected cells (5), which implied that influenza virus polymerase may inhibit Pol II(pS-2) and cellular transcription elongation. Although Vreede et al. provided experimental evidences that the ubiquitin system may be involved in the degradation of Pol II (31), it is reasonable to hypothesize that influenza virus polymerase may target CDK9 and inhibit its phosphorylation of Pol II. On the contrary, if serine 2 of the Pol II CTD can be freely phosphorylated by CDK9 during influenza virus infection, then host transcription elongation would leave the cap structure spatially away from vRNP. As mentioned above, all three subunits of viral polymerase could interact with CDK9. We proposed that viral polymerase complexes could interact with cyclin T1/CDK9 to favor its association with Pol II and the cap-snatching process, while the individual subunit of viral polymerase could bind with CDK9 to reduce levels of Pol II(pS-2).

In conclusion, cyclin T1/CDK9 interacted with influenza A virus polymerase and served as an adaptor for vRNP targeting of RNA Pol II(pS-2) and therefore facilitated viral transcription.

Acknowledgments

We thank Martin Schwemmle for providing us the influenza A virus minireplicon plasmids. We thank Yingfang Liu for providing PB1 and PB2 antibodies.

This work was supported by grants from the Ministry of Science and Technology of China (grants 2007DFC30240, 2004BA519A64, 2009ZX10004-101, and 2008ZX10002-009) and Chinese Academy of Sciences Innovation projects (KSCX2-YW-R-198 and KSCX2-YW-N-054). Xin Ye is the principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (NSFC) (grant 81021003).

Footnotes

▿

Published ahead of print on 13 October 2010.

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