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Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
Correspondence
Yan Zhou
yan.zhou{at}usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The genome consists of eight segmented RNA molecules of negative polarity. The majority of influenza A virus strains encode 11 viral proteins. Infection by the virus results in the activation of diverse cellular signalling pathways, which facilitate virus entry and replication (Ludwig et al., 1999, 2003). One such event includes the activation of the protein kinase C (PKC) pathway as a result of haemagglutinin (HA) binding to the host-cell receptor (Rott et al., 1995; Arora & Gasse, 1998). Blocking of PKC by the inhibitor bisindolylmaleimide I can block influenza virus entry and subsequent infection (Constantinescu et al., 1991; Root et al., 2000; Sieczkarski et al., 2003). Influenza virus infection also activates the mitogen-activated protein kinase (MAPK) pathway. Three members of the MAPK family, extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK) and p38, have been reported to be activated by influenza virus infection (Kujime et al., 2000; Ludwig et al., 2001; Pleschka et al., 2001). Recent studies characterized the functions of these pathways in virus infection. By using specific kinase inhibitors, p38 and JNK have been shown to regulate virus-induced RANTES in a cellular inflammatory response (Kujime et al., 2000). The Raf/MEK/ERK pathway is activated by influenza virus infection. Inhibition of this pathway by a specific inhibitor, U0126, results in nuclear retention of the ribonucleoprotein (RNP) complex in the late stage of infection and impaired function of nuclear export protein (NEP)/NS2, hence inhibiting virus propagation (Pleschka et al., 2001).
Phosphatidylinositol 3-kinases (PI3K) are a family of cellular, heterodimeric enzymes that consist of a regulatory subunit (p85) and a catalytic subunit (p110). PI3K is activated by binding of the Src homology (SH) domain in the p85 subunit to autophosphorylated tyrosine kinase receptors, non-receptor tyrosine kinases or some viral proteins in the cytoplasm (Skolnik et al., 1991; Hiles et al., 1992; Carpenter et al., 1993; Stoyanov et al., 1995; Street et al., 2004). After activation, the p110 subunit of PI3K phosphorylates the lipid substrate phosphatidylinositol 4,5-bisphosphate (PIP2) to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Toker & Cantley, 1997). This molecule serves as a lipid second messenger and is able to regulate phosphorylation of a number of kinases, including Akt. Akt is activated via phosphorylation at Thr308 and Ser473 (Alessi et al., 1996). Phosphorylated Akt plays a central role in modulating diverse downstream signalling pathways associated with cell survival, proliferation, migration, differentiation and apoptosis (Yao & Cooper, 1995; Datta et al., 1999).
Recently, an increasing amount of information has demonstrated that many viruses can trigger PI3K/Akt signalling pathway activation (Cooray, 2004). The PI3K/Akt pathway is found to be required for the efficient replication of human cytomegalovirus (Johnson et al., 2001) and coxsackievirus B3 (Esfandiarei et al., 2004). Inhibition of the PI3K/Akt pathway leads to reduction in virus yield, suggesting that cellular phosphorylation events linked to the PI3K/Akt pathway may be necessary for effective virus replication.
This study was initiated to determine whether the PI3K/Akt signalling pathway plays a role in influenza virus propagation. We report that influenza virus infection activated the PI3K/Akt pathway in the late phase of infection. Inhibition of the PI3K/Akt pathway by a specific inhibitor, LY294002, and overexpression of the general receptor for phosphoinositides-1 PH domain (Grp1-PH) resulted in reduction in virus yield; in particular, viral RNA and protein syntheses were suppressed, suggesting that PI3K/Akt plays a role in influenza virus replication.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were kindly provided by Drs E. Hoffmann and R. G. Webster, St Jude Children's Research Hospital, Memphis, TN, USA. The Grp1-PH construct (Gray et al., 1999) was a gift from Dr S. Ludwig, Westfaelische-Wilhelms-University, Muenster, Germany.
Antibodies and inhibitor.
Rabbit monoclonal phospho-Akt (Ser473) antibody, rabbit polyclonal Akt antibody and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were purchased from Cell Signaling Technology. Monoclonal NP antibody was purchased from Serotec and used in immunofluorescence staining analysis. Alkaline phosphatase (AP)-conjugated anti-rabbit IgG and Cy3-conjugated anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories.
Rabbit antisera for PR8 NP (06-3/4) and M1 (06-1/2) were generated by immunization of rabbits with purified His–NP and His–M1 proteins, respectively, and were used in Western blotting analysis. Briefly, the C-terminal portion of NP (aa 251–498) was PCR-amplified by using plasmid pHW195-NP as DNA template and primers XhoI-NP-751-Fw: 5'-CGAGCTCGAGGCTGAGTTCGAAGATCTCACT-3'/EcoRI-NP-1500-Bw: 5'-CTTCGAATTCTTAATTGTCGTACTCCTCTGCA-3'. M1 cDNA (aa 1–252) was PCR-amplified by using plasmid pHW197-M as template and primers XhoI-M1-1-Fw: 5'-CGAGCTCGAGATGAGTCTTCTAACCGAGGTC-3'/EcoRI-M1-758-Bw: 5'-CTTCGAATTCACTTGAACCGTTGCATCTG-3'. The PCR products were digested by XhoI and EcoRI and inserted individually into the XhoI/EcoRI sites of the pRSETA vector (Invitrogen). His-tagged proteins were induced in Escherichia coli BL21 cells by IPTG and purified by Ni–NTA agarose beads (Qiagen) under denaturing conditions according to the manufacturer's protocol. Purified proteins were dialysed against PBS. Each rabbit was injected subcutaneously with 500 µg antigen mixed with an equal volume of Freund's complete adjuvant (Sigma), followed by two injections with 250 µg antigen mixed with an equal volume of Freund's incomplete adjuvant at 4 week intervals. Rabbits were bled 2 weeks after the last injection. Antisera were collected, characterized, aliquotted and stored at –20 °C.
The PI3K-specific inhibitor LY294002 was purchased from Sigma. Treatment of cells with LY294002 or DMSO was usually performed at concentrations of 20 µM and 0.4 % (v/v), respectively, unless specified otherwise. Chemicals were added to the cells at 1 h post-infection (p.i.).
Transfection and MTT cell-viability assay.
MDCK cells were seeded in a six-well plate at a density of 3.5x105 cells per well and transfected with 3.5 µg Grp1-PH or empty vector by using Transit-LT 1 (Mirus) as per the manufacturer's protocol. Forty-eight hours post-transfection, cells were infected with wild-type (wt) PR8 at an m.o.i. of 0.001 p.f.u. per cell. Virus titres were determined at 16 and 24 h p.i. by plaque assay.
Cells were seeded in a 96-well plate at a density of 2.5x104 cells per well. After overnight incubation in a CO2 incubator at 37 °C, cells were treated with LY294002. Three hours before the end of the treatment, 20 µl MTT (0.5 mg ml–1; Sigma) was added to each well. The plate was incubated in a CO2 incubator at 37 °C for 3 h. Medium was then removed and 200 µl DMSO was applied to each well to dissolve the MTT crystals. A550 of each sample was measured by using a spectrophotometer. MTT activity was expressed as a percentage of that in DMSO-treated cells.
Western blot analysis.
About 1x106 A549 cells were plated into 35 mm dishes and were mock-infected or infected with influenza virus at a predetermined m.o.i. At the indicated times, cell monolayers were washed with PBS and lysed with RIPA buffer [0.5 M Tris (pH 8.0); 0.15 M NaCl; 0.1 % SDS; 1 % NP-40; 1 % deoxycholic acid] containing protease inhibitors (1xComplete Protease Inhibitor; Roche). The lysates were collected, homogenized by passage several times through a 1 ml syringe with a 22-gauge needle and incubated on ice for 10 min. Lysates were cleared by centrifugation for 5 min at 12 000 g. The supernatant was analysed for total protein content by using a Bradford assay (Bio-Rad). Thirty micrograms of total protein was resolved by SDS-PAGE (10 % polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for non-specific binding with Tris-buffered saline (TBS) [0.1 M Tris (pH 7.6); 0.9 % NaCl] containing 0.1 % Tween 20 and 10 % skimmed milk for 1 h at room temperature. For examination of phosphorylated Akt or total Akt, a primary antibody, either rabbit monoclonal phospho-Akt (Ser473) (1 : 1000) or rabbit polyclonal Akt antibody (1 : 1000), was applied overnight at 4 °C. A secondary antibody of HRP-conjugated anti-rabbit IgG (1 : 3000) was then added at room temperature for 1 h. Immunoblots were visualized with an enhanced chemiluminescence reagent (ECL Advance Western blotting detection kit; GE Healthcare). For examination of viral proteins, cell lysates were probed with rabbit polyclonal NP (1 : 2000) or M1 (1 : 2000) antibody followed by an incubation with AP-conjugated anti-rabbit IgG (1 : 10 000). The immunoblots were then visualized by incubating with BCIB/NBT premix solution (Sigma).
RNA analysis.
A549 cells were infected by wt PR8 virus at an m.o.i. of 0.01 p.f.u. per cell. Cells were harvested at 8, 16 and 24 h p.i. Total RNA was extracted by using an RNeasy RNA extraction kit (Qiagen) according to the manufacturer's protocol. The accumulation of viral RNAs (vRNAs) was quantified by real-time RT-PCR with primers specific for the NP gene or the M gene. vRNA was reverse-transcribed by using the Uni-12 primer (Hoffmann et al., 2001) followed by PCR using 5'-GATTGGTGGAATTGGACGAT-3'/5'-AGAGCACCATTCTCTCTATT-3' (for the NP gene) and 5'-AAGACCAATCCTGTCACCTCTGA-3'/5'-CAAAGCGTCTACGCTGCAGTCC-3' (for the M1 gene). RNA quantification was conducted in two steps by using a SuperScript III Platinum two-step qRT-PCR kit with SYBR green (Invitrogen) as per the manufacturer's instructions in an iCycler iQ-Multicolor real-time PCR detection system (Bio-Rad). The concentrations of vRNA were obtained by comparison with serially diluted plasmids pHW195-NP and pHW197-M. All RNA determinations were assayed in duplicate and repeated three times.
Immunofluorescence staining.
About 1x104 MDCK cells were plated on an eight-well chamber slide and infected with wt PR8 at an m.o.i. of 1 p.f.u. per cell in the presence of 20 µM LY294002 or 0.4 % (v/v) DMSO. At 8 h p.i., cells were fixed in a mixture of acetone and methanol (1 : 1) for 15 min at –20 °C. After rehydration with PBS, cells were incubated with monoclonal NP antibody (1 : 200) for 1 h at room temperature. Cells were rinsed three times in PBS and incubated with secondary antibody, Cy3-conjugated anti-mouse IgG (1 : 400), for 45 min at room temperature. Finally, the cells were counterstained by DAPI (Roche) for 5 min. Images were obtained on a Carl Zeiss Axiovert 200M inverted fluorescent microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Serum-starved A549 cells were mock-infected or infected with wt PR8 virus at an m.o.i. of 1 p.f.u. per cell. Cell lysates prepared at the predetermined times were subjected to Western blotting using the antibody specific for phospho-Akt (Ser473). As seen in Fig. 1(a), phosphorylated Akt was evident in virus-infected cells at 6 h p.i. and this elevated phosphorylation was sustained for the remainder of the infection. Phosphorylated Akt was not present in mock-infected cells (data not shown), eliminating the possibility that Akt phosphorylation was due to the manipulation of cells. Western blotting with total Akt antibody showed an equal amount of total Akt throughout the infection, indicating that the changes in Akt phosphorylation were not due to altered total levels of Akt. Similar patterns of Akt activation were also observed in MDCK cells following PR8 virus infection (data not shown), eliminating a possible cell line-specific phenomenon.
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; Kandel & Hay, 1999), we then investigated the role of PI3K in Akt activation following influenza virus infection. For this purpose, we used LY294002, a specific inhibitor of PI3K. LY294002 inhibits PI3K activity via competitive inhibition of the ATP-binding site located on the p85
subunit of PI3K (Vlahos et al., 1994). A549 cells were infected by PR8 virus at an m.o.i. of 1 p.f.u. per cell, followed by treatment with increasing doses of LY294002. Cell lysates prepared at 7 h p.i. were subjected to Western blotting analysis to assess the phosphorylation of Akt on Ser473. As shown in Fig. 1(b)
, LY294002 inhibited influenza virus-induced Akt phosphorylation completely at all tested concentrations. The levels of total Akt remained constant in all samples, indicating that the changes in Akt phosphorylation were not due to changes in cellular protein levels.
PI3K/Akt pathway activation is not due to virus attachment and entry
As we observed that Akt phosphorylation occurred at 6 h p.i., we postulated that activation of the PI3K/Akt pathway by influenza virus is virus replication-dependent and may not be mediated by the initial virus–host-cell interactions. To test this hypothesis, we inactivated the virus by UV irradiation. UV irradiation blocks viral RNA transcription, mRNA synthesis and protein synthesis, but has no effect on virus receptor binding and subsequent entry into host cells (Geiss et al., 2001). To ascertain that our protocol of UV inactivation does not affect the haemagglutination activity of the virus, the activity of viral HA was examined. As seen in Fig. 2(a), twofold serial dilutions of untreated virus, PBS alone and UV-treated virus were incubated with chicken red blood cells. Whilst PBS alone did not show any HA activity, the HA activity of UV-treated virus was similar to that of untreated virus. Next, A549 cells were infected by untreated or UV-treated virus at an m.o.i. of 1 p.f.u. per cell. Cell lysates prepared at 6 h p.i. were subjected to Western blotting using anti-phospho-Akt (Ser473), anti-total Akt or anti-NP antibodies. As expected, no NP protein could be detected in UV-treated virus-infected cells. Whilst infection by either untreated or UV-treated viruses did not alter total Akt levels, replication-deficient virus failed to trigger phosphorylation of Akt (Fig. 2b).
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, after 24 h infection, virus titres from LY294002-treated samples were reduced compared with those from DMSO-treated samples. A similar degree of virus-yield inhibition by LY294002 was also seen on A549 cells (data not shown). It has been reported that inhibition of the PI3K/Akt pathway can potentially induce cell death (King et al., 1997). To determine whether inhibition of virus infection by LY294002 was due to premature cell death caused by LY294002, cell viability in response to LY294002 after 8 and 24 h incubation was determined by an MTT assay. MTT activity was calculated as a percentage of that in DMSO-treated cells. As seen in Fig. 3(b), LY294002 at concentrations of 5–20 µM did not induce detectable cell toxicity compared with DMSO-treated cells. These data suggest that the inhibitory effect of LY294002 on virus propagation is not due to premature cell death.
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). Overexpression of Grp1-PH will saturate the binding sites of PIP3, leading to blockage of the cellular PI3K/Akt pathway. As seen in Fig. 3(c), virus titre was reduced significantly in MDCK cells expressing Grp1-PH compared with those in non-transfected and vector-transfected cells.
Inactivation of the PI3K/Akt pathway inhibits vRNA synthesis
To determine whether inactivation of the PI3K/Akt pathway has any effect on vRNA synthesis, we assessed vRNA levels in wt PR8 virus-infected A549 cells (m.o.i. of 0.01 p.f.u. per cell) in the presence of LY294002 or DMSO. The amount of NP and M1 vRNA was analysed by real-time RT-PCR at 8, 16 and 24 h p.i. As seen in Fig. 4, the levels of NP and M1 vRNA obtained from wt PR8 infection were reduced in the LY294002-treated cells compared with those in DMSO-treated cells. Specifically, around 2.0- to 2.5-fold vRNA reduction for NP and 5- to 8-fold vRNA reduction for M1 were obtained. These data suggest that the PI3K/Akt pathway plays a role in the processes of vRNA synthesis.
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). Quantification of the density of the protein bands was performed by using Quantity One software (Bio-Rad). NP and M1 protein accumulations were normalized by total Akt level (Fig. 5b, c). As shown in Fig. 5(a), synthesis of both NP and M1 proteins was evident in DMSO-treated cells at the tested time points. In contrast, in LY294002-treated cells, synthesis of NP and M1 proteins was reduced. Interestingly, inhibition of M1 protein accumulation was more profound than that of NP protein.
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, NP was found predominantly in the cytoplasm of wt PR8-infected cells at 8 h p.i., indicating that the majority of RNP had been exported from the nucleus. However, NP accumulated in the nucleus of wt PR8-infected and LY294002-treated cells (Fig. 6b), indicating that nuclear export of RNP is disturbed. Cells were counterstained with DAPI (Fig. 6c, d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We initially examined whether the PI3K/Akt pathway is activated during influenza virus infection. We found that cellular Akt was phosphorylated at 6 h p.i. and was sustained throughout the remainder of the infection. Akt phosphorylation was abrogated by a PI3K-specific inhibitor, LY294002, suggesting that influenza virus-induced Akt phosphorylation is PI3K-dependent (Fig. 1). In addition, Akt was not phosphorylated in cells infected by UV-inactivated virus (Fig. 2), demonstrating that activation of the PI3K/Akt pathway is not due to virus binding and entry. This is consistent with the observation that Akt phosphorylation occurred in the late, rather than early, stage of infection (Fig. 1a). This also supports the idea that post-entry events, such as viral protein synthesis, are required for cellular PI3K/Akt activation. Recently, our group (Shin et al., 2007) and Hale et al. (2006) have found that the NS1 protein activates the PI3K/Akt pathway by direct interaction with the p85 subunit of PI3K. We have demonstrated that the NS1 protein interacts with the p85 subunit of PI3K via binding to C-terminal SH2 and SH3 domains of p85. Furthermore, we have shown that SH2- and SH3-binding motifs in NS1 are essential for interaction with p85 and subsequent PI3K/Akt pathway activation (Shin et al., 2007).
We then examined whether the PI3K/Akt pathway plays a role in virus replication by blocking PI3K activity with the specific inhibitor LY294002 and overexpression of Grp1-PH. We found that LY294002 treatment and Grp1-PH expression inhibited virus production in both A549 cells and MDCK cells (Fig. 3). Similar observations were reported recently by Ehrhardt et al. (2006), who found that inhibition of PI3K by wortmannin results in a reduction of virus titres. Concomitantly, both mutant virus encoding NS1 with mutations in the SH-binding motifs (Shin et al., 2007) and mutant virus expressing NS1 with the Y89F amino acid substitution (Hale et al., 2006), which failed to activate the PI3K/Akt pathway, formed small plaques and grew more slowly in tissue culture than wt virus. These results suggest strongly that activation of this pathway plays a role in supporting virus propagation. To define at which step virus replication was affected, we treated cells with LY294002 at 1 h p.i. and found that accumulations of vRNA and viral protein of both an early gene (NP) and a late gene (M1) were suppressed by LY294002 (Figs 4, 5). The degree of suppression was more significant on the late gene M1 than on the early gene NP. At this stage, the mechanism of how PI3K/Akt regulates vRNA and protein syntheses is not clear. However, there is mounting evidence suggesting that viral proteins can be phosphorylated by various protein kinases. For example, MAP kinase and PKC can phosphorylate different human immunodeficiency virus type 1 proteins, leading to augmentation of virus infectivity (Burnette et al., 1993; Yang et al., 1996; Yang & Gabuzda, 1998, 1999). MAP kinases can also phosphorylate hepatitis B virus large envelope protein (Rothmann et al., 1998). The PI3K/Akt pathway induced phosphorylation of a non-structural protein, NS5A, of Hepatitis C virus (HCV), which is associated with HCV replication (Coito et al., 2004; Mannova & Beretta, 2005). These findings lead us to postulate that the PI3K/Akt pathway may regulate influenza vRNA synthesis by direct or indirect phosphorylation of viral products. In support of this hypothesis is the finding that influenza A virus PA and NP proteins are phosphorylated proteins (Kistner et al., 1989; Sanz-Ezquerro et al., 1998). Our results of LY294002 inhibiting vRNA synthesis (Fig. 4) also support this notion. Another possibility is that the PI3K/Akt pathway phosphorylates some cellular factors that contribute to efficient virus replication and protein synthesis. In line with this notion, a couple of cellular proteins, such as HSP90 and RAF-2, have been identified as factors involved in influenza vRNA synthesis (Momose et al., 2001, 2002). Furthermore, it has been reported that the heat-shock protein HSP90 is linked with PI3K/Akt activity (Sato et al., 2000), whereas RAF-2p48 and RAF-2p36 are phosphoproteins (Momose et al., 2001). Hence, these cellular factors could be potential targets for PI3K/Akt signalling and contribute to vRNA synthesis. In terms of a possible association of cellular-factor phosphorylation with influenza viral protein synthesis, it was found that a protein kinase inhibitor, H7, interferes specifically with the synthesis of the late influenza viral proteins, including M1 (Kistner et al., 1989; Vogel et al., 1994; Bui et al., 2000). Our experiment also showed that the PI3K-specific inhibitor LY294002 has a more profound inhibitory effect on M1 protein synthesis than on that of the early gene NP (Fig. 5). One of the downstream effectors of PI3K/Akt signalling is mammalian target of rapamycin (mTOR). mTOR phosphorylates the eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) and positively modulates translation initiation (Gingras et al., 1998). Thus, the LY294002 inhibitory effect on influenza viral protein synthesis may be attributed to the reduced activity of PI3K/Akt–mTOR–4E-BP signalling by LY294002. Further study in this regard is being performed in our laboratory.
A study by Ehrhardt et al. (2006) reported a biphasic activation of the PI3K/Akt pathway upon influenza virus infection. In this study, we observed PI3K/Akt activation in the late phase of infection, which is virus replication-dependent. One of the reasons for this discrepancy might be that they used a higher m.o.i. (5) than we did (1). Additionally, different sources of cell lines and different virus' passage history in different laboratories may contribute to the different observations. With regard to at which step virus infection was enhanced by PI3K pathway, the data of Ehrhardt et al. (2006) suggested that early activation of PI3K seems to be required for efficient virus uptake. As we did not detect early-phase activation of the PI3K/Akt pathway, we did not investigate any early events that could be regulated by the PI3K/Akt pathway. Our results suggested a mechanism by which PI3K/Akt-associated direct or indirect phosphorylation of viral or cellular factors might be involved in modulating influenza vRNA and protein syntheses. Taking all of these results together, it is reasonable to speculate that influenza virus may have developed different mechanisms through manipulating the PI3K/Akt pathway at early and late phases during infection to enhance virus replication.
We also observed that nuclear export of RNP was blocked in LY294002-treated cells (Fig. 6). There is evidence that specific inhibitors of cell-signalling pathways can block influenza viral RNP nuclear export. Pleschka et al. (2001) proposed that inhibition of the Raf/MEK/ERK signalling pathway by U0126 impairs NEP/NS2 nuclear RNP export through some cellular factors. A study by the same group also reported that inhibition of caspase 3 activity results in blockage of influenza virus propagation due to nuclear RNP retention (Wurzer et al., 2003). However, caspase-dependent RNP export probably occurs through a different mechanism, in which active caspases increase the diffusion limit of nuclear pores directly or indirectly. As inhibition of NP and M1 protein synthesis by LY294002 was seen at 8 h p.i., this effect might result from a reduced amount of NP protein and/or may be attributed to the reduced amount of M1 protein, as one of the functions of M1 is to associate with vRNP and participate in vRNP export from the nucleus to the cytoplasm at the late phase of infection (Martin & Helenius, 1991).
In summary, our investigation demonstrates an important role of the PI3K/Akt pathway in regulating influenza virus replication, which strengthens our previous finding and the findings by Ehrhardt et al. (2006) and Hale et al. (2006). Understanding of this process can greatly enhance our knowledge on influenza virus pathogenesis and provide insights into antiviral therapeutic design.
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ACKNOWLEDGEMENTS |
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Received 17 August 2006;
accepted 21 November 2006.
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