Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center (JCCC), UCLA School of Medicine, Los Angeles, CA 90095-1747, USA1
Author for correspondence: Debi P. Nayak. Fax +1 310 2063865. e-mail dnayak{at}ucla.edu
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Abstract |
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Introduction |
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A family of heterotrimeric () guanine nucleotide-binding regulatory proteins (G proteins) are involved in the regulation of transmembrane signal transduction (for a review see Helmreich, 2001
). The G proteins operate by utilizing a guanine nucleotide-binding and -hydrolysing cycle. Agonistligand receptor interaction causes the exchange of guanosine 5'-diphosphate (GDP) for guanosine 5'-triphosphate (GTP) at the
subunit of the G protein (G
) and leads to subsequent dissociation of G
from the
subunit (G
and G
). This GTP-bound G
is the active state of the G protein, which is terminated by hydrolysis of the bound GTP by an intrinsic GTPase activity. There is increasing evidence that heterotrimeric G proteins and PKs participate in the regulation of apical membrane dynamics such as membrane ruffling, endocytosis and exocytosis (reviewed in Ceresa & Schmid, 2000
; Cavalli et al., 2001
). Moreover, G
i, G
o and GTPase activating protein GAP-43 are enriched in lipid rafts (Arni et al., 1998
; Moffett et al., 2000
), regions on the apical plasma membrane that function as the budding site of influenza virus (Scheiffele et al., 1999
; Barman & Nayak, 2000
; Zhang et al., 2000
; Barman et al., 2001
).
In the present study, we have investigated the role of G proteins and PKs in regulating influenza virus budding from the apical cell surface. Our results show that in WSN virus-infected MadinDarby canine kidney (MDCK) cells, G protein signalling stimulators (such as fluorides, compound 48/80, mastoparan or GTP analogues) increased virus budding, and that G protein signalling blockers (suramin or NF023) decreased virus budding. In addition, the introduction of the casein kinase 2 (CK2) inhibitor 5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB), or CK2-immunodepleted cytosol into permeabilized cells inhibited virus budding, whereas inhibitors of protein kinase A (PKA), protein kinase C (PKC), or phosphatidylinositol 3-kinase (PI3K) did not affect virus budding.
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Methods |
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Chemicals, drugs and peptides.
All chemicals, drugs and peptides used in these experiments were purchased from Sigma or Calbiochem. Sodium fluoride (NaF), compound 48/80, suramin, NF023 and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) were dissolved in water. H-89, LY294002 and DRB were dissolved in absolute ethanol. Ro-32-0432 and bisindolylmaleimide I (BIM) were dissolved in DMSO. The residual solvents (less than 1%) had no toxic effect on the cell monolayer. Aluminium fluoride (AlF4-) treatment on MDCK cells was performed as follows: (i) 40 µM aluminium chloride (AlCl3) plus 30 mM NaF (Brewer & Roth, 1995 ) or (ii) 50 µM aluminium ammonium sulfate [AlNH4(SO4)2] plus 10 mM potassium fluoride (KF) (Ikonen et al., 1996
). The GTP and GTP analogues guanosine 5'-O-(3-thiotriphosphate) (GTP
S) and 5' guanylylimidodiphosphate (GMP-PNP) were dissolved in serum-free DMEM just before use. G protein signalling activator mastoparan (INLKALAALAKKIL), PKA inhibitor 622 amide (TYADFIASGRTGRRNAI), PKC inhibitor peptide 1936 (RFARKGALRQKNVHEVKN) and CK2 substrate peptide (RRADDSDDDDD) were dissolved in water.
Virus infection and sample collection.
For virus infection, MDCK cells were seeded at a cell density of 1·2x106 cells/35 mm dish and maintained in 2 ml of culture medium for 30 h. Prior to infection, cells were washed with PBS+ (PBS plus 0·5 mM MgCl2 and 1 mM CaCl2). In all experiments, cells were infected with WSN virus at an input m.o.i. of 1 in virus dilution buffer (PBS+ supplemented with 0·2% BSA, 0·005% DEAE-dextran, 10 U/ml penicillin G and 10 µg/ml streptomycin). After adsorption for 1 h at 37 °C, the residual unabsorbed virus was removed and cells were washed five times with DMEM containing 0·2% BSA. The infected cells were incubated in 2 ml DMEM containing 0·2% BSA for 13 h at 37 °C. Sialidase (NA; final concentration 0·1 mU/ml) was added 2·5 h prior to removal of culture medium. Infected cells were then washed five times with 2 ml of DMEM containing 0·2% BSA to remove residual virus. The fifth wash was collected as the experimental background. The new medium, with or without the drug, was added and harvested after a 15 min or 1 h incubation for the virus assay.
LPC permeabilization of filter-grown MDCK cells.
The lysophosphatidylcholine (LPC) permeabilization method was carried out as described previously (Hui & Nayak, 2001 ). Briefly, MDCK cells (1·2x106) were seeded on Transwells (24 mm diameter, 3·0 µm pore size; Costar), infected with WSN virus at an m.o.i. of 1 and incubated for 13 h. The Transwell filters containing the cell monolayers were permeabilized from the basal side by LPC (Avanti Polar Lipids). The Transwells were then washed and treated with DMEM with or without GTP analogues from the basal chamber for 20 min at 37 °C. Subsequently, the Transwells were incubated with 1·5 ml DMEM with 0·2% BSA and 2·5 ml DMEM in the upper and lower compartments, respectively, and virus released in the upper chamber after 15 min was assayed.
Plaque assay.
Determination of the amount of influenza virus released from infected cells into the medium was carried out by plaque assay on monolayers of MDCK cells in 35 mm tissue culture dishes using 2 ml agar overlay MEM supplemented with 0·6 % low melting point agarose, 0·075% NaHCO3 and 0·0015% DEAE-dextran. Visible plaques were counted after 3 days at 33 °C and p.f.u./ml were calculated.
Western blotting.
Proteins were resolved by SDSPAGE (10% gel) and transferred electrophoretically to Trans-Blot nitrocellulose membrane (Bio-Rad). Membranes were blocked in 1% blocking solution and probed with anti-CK2 monoclonal antibodies (Santa Cruz Biotechnology; diluted 1:500) overnight at 4 °C. The membrane was then incubated with anti-mouse monoclonal antibodies (diluted 1:1200) for 1 h at room temperature and antigens finally visualized on film by enhanced chemiluminescence (ECL; Amersham Biosciences).
Digitonin permeabilization of filter-grown MDCK cells.
Digitonin permeabilization of the basal membrane of MDCK cells was carried out as described by Esparís-Ogando et al. (1994) , and the cytosol support of permeabilized MDCK cells was carried out according to the procedures of Pimplikar et al. (1994)
and Ikonen et al. (1996)
. Briefly, MDCK cells were seeded, grown and infected on the Transwell as described previously (Hui & Nayak, 2001
). The Transwell filters containing the cell monolayers were permeabilized from the basal side with digitonin (250 µg/ml) for 15 min at room temperature. The Transwells were washed twice with PBS+ on the apical side and with transport medium [115 mM potassium acetate (KOAc), 25 mM HEPES, pH 7·4, 2·5 mM MgCl2, 1 mM DTT, 5 mM EGTA and 2·5 mM CaCO3] on the basal side. Cells were then incubated with 250 µl (2 mg) of HeLa or MDCK cytosol with or without an ATP-regenerating system (1 mM ATP, 8 mM phosphocreatine and 50 µg/ml creatine phosphokinase) in the basal chamber and with PBS+ containing 0·1 mU/ml NA in the upper compartment. After 45 min at 37 °C incubation, the upper compartment was washed five times and the fifth wash was collected for the experimental background. The Transwells were then incubated with 0·5 ml DMEM plus 0·2% BSA in the upper compartment and with cytosol in the lower compartment and virus released into the upper chamber after 15 min was assayed.
Permeabilization assessment.
The effectiveness of permeabilization was assessed by the cell viability assay (trypan blue uptake) and the release of cytosolic lactate dehydrogenase (LDH) from the basal side as previously described (Hui & Nayak, 2001 ).
Preparation of HeLa cytosol.
The preparation of HeLa cytosol was carried out as described previously (Pimplikar et al., 1994 ). Briefly, HeLa cells were grown in suspension in Eagles Minimal Essential Medium (S-MEM; Sigma) supplemented with 1 g/l glucose and 10% newborn calf serum (Gemini Bio-Products) to a density of 4x105/ml. Cells were pelleted by centrifugation, washed with ice-cold PBS and resuspended in cold HeLa swelling buffer (1 mM MgCl2, 1 mM DTT, 1 mM EGTA and 1 µM cytochalasin D). After swelling for 5 min on ice, cells were centrifuged, the supernatant removed, protease inhibitor cocktail (Sigma) added and the cells broken by a glass Dounce homogenizer with 10 strokes. Finally, 1/10 volume of 10x KOAc buffer (1x KOAc buffer: 115 mM KOAc, 25 mM HEPES, pH 7·4, 2·5 mM MgCl2 and 10 mM DTT) was added to the homogenate and further homogenized with 20 strokes. Using this procedure, >80% of the cells were broken, as judged by light microscopy. The homogenate was centrifuged at 14000 g for 10 min followed by another centrifugation at 171000 g for 90 min. The supernatant was frozen in small aliquots at -80 °C. This procedure routinely yielded
14 ml containing
12·5 mg/ml cytosolic protein from 10 l of cell suspension.
Preparation of MDCK cytosol.
The preparation of MDCK cytosol was carried out as described previously (Ikonen et al., 1996 ). Confluent MDCK monolayers from 40 150 cm2 dishes (total 7x108 cells) were trypsinized. Trypsin was then inactivated by first resuspending the cells in culture medium containing 10% FBS, pelleting and washing again with 30 ml of ice-cold PBS supplemented with 1 mg/ml soybean trypsin inhibitor. The cell pellet was resuspended in cold MDCK swelling buffer (1 mM MgCl2 and 1 mM EGTA) and incubated on ice for 10 min. After pelleting the cells, a protease inhibitor cocktail containing cytochalasin D (final concentration 1 µM) and DTT (final concentration 1 mM) was added and the cells broken by sonication. The solution containing cell fragments was then adjusted to an isotonic condition by adding 1/10 vol. of 10x KOAc stock. Following a 20 min centrifugation at 600 g at 4 °C, the supernatant was further centrifuged for 1 h at 200000 g at 4 °C. The cytosol (
2·5 ml) containing
12 mg/ml protein was aliquoted and stored at -80 °C.
Immunodepletion of CK2.
To deplete CK2, 150 µg anti-CK2 antibody was coupled to 0·1 g protein ASepharose beads (Amersham Biosciences) in PBS by mixing for 2 h at 4 °C. The beads were then washed with PBS and incubated with cytosol (10 mg) overnight on ice with intermittent mixing. After removing the beads by centrifugation, the cytosol was used in a digitonin permeabilization assay. The depletion was monitored by determining the loss of CK2 activity.
Preparation of membranes and cytosol from MDCK cells for assaying enzymatic activity.
The membranous and cytosolic fraction of MDCK cells was prepared by a modification of a previously described method (Hansen & Casanova, 1994 ). Briefly, MDCK cells were rinsed and scraped off in PBS, pelleted in a microfuge and resuspended in 1 ml of ice-cold 3 mM imidazole (pH 7·4) and 300 mM sucrose. The resuspended cells were passed at least 40 times through a 25-gauge syringe. Greater than 90% of the cells were lysed under these conditions, as assessed by trypan blue staining. The nuclei and cell debri were then pelleted by centrifugation at 14000 g for 5 min and the supernatant further centrifuged at 105000 g for 1 h. The pellet and supernatant were taken to represent membrane and cytosolic fractions, respectively.
PKC activity assay.
PKC activity assays were performed as described previously (Hui & Yung, 1992 ; Yung et al., 1994
). The reaction mixture (25 µl) containing 30 mM TrisHCl buffer, pH 7·5, 6 mM magnesium acetate, 0·25 mM EGTA, 0·4 mM CaCl2, 40 µg/ml phosphatidylserine, 8 µg/ml dioleoylglycerol, 1 mg/ml histone IIIS, 0·12 mM [
-32P]ATP (Perkin-Elmer Life Sciences) and membrane or cytosol fractions containing 10 µg of protein were incubated at 30 °C for 8 min. Phosphorylated substrates were quantified as described (Huang & Huang, 1991
). Twenty µl of the reaction mixture was spotted on to a line of origin (1·5 cm from the bottom) on an instant thin-layer chromatography strip (1x9·5 cm, type SG; Gelman Sciences), which had previously been spotted with ATP (50 mM) in 15% trichloroacetic acid (TCA). After chromatography for 6 min at room temperature in a beaker containing 5% TCA and 0·2 M KCl, the strips were air dried and the origin (1·5 cm above the line of origin) excised for counting in a scintillation counter.
CK2 activity assay.
Assays for CK2 activity were performed as described previously (Perich et al., 1992 ). Briefly, 50 µl of reaction mixtures containing 50 mM TrisHCl, pH 7·5, 12 mM MgCl2, 100 mM NaCl, 1 mM CK2 peptide substrate, 25 µM [
-32P]ATP and 10 µg isolated protein (membrane or cytosol) were incubated at 30 °C for 8 min. Reactions were stopped by adding 60 µl 10% TCA and 10 µl BSA and incubated on ice for 10 min. The precipitated protein was removed by centrifugation and 12 µl of supernatant was spotted on to pieces of P81 phosphocellulose (Whatman Int.). The phosphocellulose pieces were washed with an excess of 75 mM phosphoric acid, air dried and counted in a scintillation counter.
Statistical analysis.
The results were obtained from at least three separate experiments, each using triplicate culture plates in each experiment. Unless otherwise specified, all data were expressed as mean±standard deviation (SD) of n values (number of experiments) and where SD bars are not apparent, the SD value was less than the symbol used. The significance of the difference (P) between values was compared using the Students t-test, and P<0·05 or less was considered significant.
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Results |
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GTP analogues in LPC-permeabilized infected cells increase virus budding
The results of the above experiments strongly suggested that GTP plays an important role in influenza virus budding. To examine the role of GTP analogues, we used basolateral-surface LPC-permeabilized MDCK cells (Hui & Nayak, 2001 ), since GTP and the GTP analogues GTP
S (hydrolysis-resistant GTP analogue) and GMP-PNP (non-hydrolysable GTP analogue) cannot gain entry into intact cells (Cockcroft & Gomperts, 1985
). MDCK cells were grown and infected on a supporting microporous membrane and permeabilized by LPC from the basolateral surface (Hui & Nayak, 2001
). Permeabilized cells were then incubated with 1 mM GTP
S or GMP-PNP, which keep the G protein in an active state because they can bind but are not hydrolysed (Terry et al., 1995
). The results showed that the virus budding rate after treatment with GTP
S and GMP-PNP in LPC-permeabilized cells increased to 147±16% and 178±27%, respectively, compared with 100% in LPC-permeabilized cells with mock treatment (Fig. 2
). The enhancing effect of GTP
S and GMP-PNP could be competitively inhibited by the presence of excess GTP. A high concentration of GTP (10 mM) decreased the virus budding rate for both GTP
S and GMP-PNP to 102±3% and 99±7%, respectively (Fig. 2
). Taken together, these results suggest that G protein signalling is involved in regulating the influenza virus budding process.
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PKC inhibitors do not alter virus budding
PKC is a large superfamily of related PKs, which carry out diverse regulatory roles in many cellular processes (for a review, see Pears, 1996 ). PKC has been shown to be involved in plasma membrane dynamics such as membrane ruffling and endocytosis (reviewed in Ceresa & Schmid, 2000
). Therefore, we treated the infected MDCK cells with the highly specific membrane-permeable PKC inhibitors such as BIM or Ro-32-0432. However, neither BIM (50 and 200 nM) nor Ro-32-0432 (50 and 200 nM) exhibited any significant effect on virus budding (Table 1
). The cytosolic and membranous PKC activity decreased in cells treated with the PKC inhibitors (data not shown). Although PKC has been shown to play a role in influenza virus mRNA translation (Kurokawa et al., 1990
) and virus entry (Root et al., 2000
), our data indicate that, like PKA, PKC does not play a significant role in influenza virus budding.
PI3K inhibitors do not alter virus budding
Besides PKC, PI3K has been found to regulate various steps in endocytic trafficking (for a review, see Ridley, 2001 ). Therefore, we treated the infected MDCK cells with the highly specific membrane-permeable PI3K inhibitors, LY294002 and wortmannin. However, neither LY294002 (5 and 20 µM) nor wortmannin (25 and 50 nM) had any significant effect on virus budding (Table 1
). These data indicate that PI3K does not play a significant role in influenza virus budding.
CK2 inhibitor decreases virus budding
Previous studies have indicated that CK2 activity is associated with influenza virus particles (Tucker et al., 1990 ; for a review, see Hui, 2002
) and that the viral PA protein in the influenza polymerase complex is phosphorylated by CK2 (Sanz-Ezquerro et al., 1998
; Perales et al., 2000
). Therefore, we wanted to know whether the CK2 plays any role in virus budding. When virus-infected cells were treated with the membrane-permeable CK2 inhibitor DRB at different concentrations (5, 10 and 50 µM) for 15 min or 1 h, the virus budding rate from MDCK cells decreased with increasing concentrations of DRB (Fig. 3
, Table 1
). CK2 activities in infected and uninfected cells after treatment with 50 µM DRB were determined in parallel and the results indicated that both the cytosolic and membranous CK2 activities decreased in virus-infected cells after DRB treatment (data not shown) and corresponded to the decrease in virus budding.
We next examined whether CK2 activity changed during the virus infectious cycle. The CK2 activity assay indicated that both the cytosolic and membranous CK2 activities increased during influenza virus infection (Fig. 4A, B
). It should be noted that the maximum increase in CK2 activity was observed around 15 h p.i., which is consistent with the time of maximum virus budding (Hui & Nayak, 2001
). CK2 is a tetramer composed of two types of subunit with the general structure
2
2 or
'
2 (reviewed in Allende & Allende, 1995
). The
(4244 kDa) and
' (38 kDa) subunits are catalytic subunits, whereas the
(2641 kDa depending on cell type) subunit is a regulatory subunit. By Western blot analysis of the CK2
subunit, we did not find any significant change in the amount of CK2
subunit in infected MDCK cells (Fig. 4C
). However, we failed to detect CK2
' subunit in MDCK cells by Western blotting (data not shown), suggesting that MDCK cells do not express the CK2
' subunit. These data indicate that the enzymatic activity, but not expression of the CK2 protein, was stimulated during influenza virus infection.
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To determine the effect of CK2 peptide on virus budding, MDCK cells were grown on the membrane and infected with WSN virus at an m.o.i. of 1. At 13 h p.i., cells were permeabilized by digitonin and incubated with HeLa cytosol with CK2 substrate peptide or PKA inhibitor 622 amide or PKC inhibitor peptide 1936. For studying virus budding in digitonin-permeabilized MDCK cells, we routinely used HeLa cytosol because it was easy to prepare. The HeLa cytosol was comparable with MDCK cytosol, since the virus budding efficiency was similar between these two cytosols (Table 2). The results showed that the 15 min virus budding rate after incubation with 20 or 100 µM CK2 substrate peptides in digitonin-permeabilized cells decreased to 65±5% and 56±4%, respectively, compared with 80±5% after mock treatment (Fig. 5A
, Table 2
). Again, treatment with 20 or 200 nM PKA and 2 or 20 µM PKC inhibitor peptides did not significantly affect virus budding (79±7%, 78±11%, 83±8% and 81±6%, respectively; Fig. 5A
, Table 2
), supporting the results observed early (Table 1
).
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Discussion |
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However, relatively little information is available on the involvement of host components in the budding process of enveloped viruses in general and influenza viruses in particular. Recently, a number of elegant studies have shown that the regulatory role of host components in the release of virus by budding in retroviruses (HIV, Rous sarcoma virus and equine infectious anaemia virus), vesicular stomatitis virus (VSV), rabies virus and Ebola viruses. The AP-50 subunit (medium chain) of cellular AP-2 clathrin-associated adaptor protein complex, the Yes-associated protein (Yap), the Nedd4-like family of E3 ubiquitin protein ligase and Tsg101 have been shown to be actively involved in the release of virus by budding. The majority of these viruses contain specific late domains (L domains) in the matrix proteins, which interact with cellular components (reviewed in Luban, 2001 ; Freed, 2002
). However, similar information is not yet available for influenza virus budding. Towards this goal, we have initiated studies for defining the role of different host components in influenza virus budding. We have developed a system to reconstitute the budding process by permeabilizing the polarized MDCK cells from the basal side, exchanging and introducing the cytosolic components from the basal side and assaying virus budding from the intact apical side (Hui & Nayak, 2001
). Using such a system, we have recently shown that ATP is actively required for influenza virus budding and that ATP hydrolysis (and not just ATP binding) is required for the budding and release of virus particles.
In the present paper, we show that the G protein and CK2 also play a critical role in influenza virus budding. G proteins are known to play important roles in a number of cellular signals and the basic mechanism of G protein-mediated signal transduction is well understood. Different classes of G protein have been shown to be involved in apical versus basolateral transport in polarized epithelial cells. In virus-infected MDCK cells, the Gs class of G proteins has been shown to be involved in the apical transport of HA, whereas Gi proteins affected the basolateral transport of VSV G proteins (Pimplikar & Simons, 1993 ). Our data show that GTP binding, which activates G protein signalling, is required for influenza virus budding. However, at present, the precise role of the G protein in influenza virus budding is unclear. Initiation of virus budding may involve processes similar to membrane ruffling, which is affected by cytoskeletal components and lipid rafts, as well as signal transduction by G proteins (see Ridley, 1994
). Furthermore, it is likely that the M1 protein can directly or indirectly interact with G proteins in initiating the budding process or releasing virus particles, since the expression of M1 protein alone can lead to the formation and release of virus-like particles (Gómez-Puertas et al., 2000
; Latham & Gularza, 2001
).
Host PKs are also actively involved in different steps of influenza virus replication and many studies on the effect of PK inhibitors during influenza virus replication have been carried out (Kurokawa et al., 1990 ; Martin & Helenius, 1991
; Vogel et al., 1994
; Neumann et al., 1997
; Bui et al., 2000
; Root et al., 2000
; Pleschka et al., 2001
). It appears that six of the ten influenza A virus proteins can be phosphorylated. These are NP (Petri & Dimmock, 1981
; Almond & Felsenreich, 1982
; Kistner et al., 1985
, 1989
), NS1 (Petri et al., 1982
), M1 (Gregoriades et al., 1984
, 1990
), NEP/NS2 (Richardson & Akkina, 1991
), M2 (Holsinger et al., 1995
) and PA (Sanz-Ezquerro et al., 1998
). Although phosphorylated M1 and NP have been found in virus particles (Gregoriades et al., 1984
), there is no data showing a specific requirement for phosphorylation of any viral protein in the budding process. Among the viral proteins, M1 and PA have been shown to be phosphorylated by PKC (Reinhardt & Wolff, 2000
) and CK2 (Perales et al., 2000
), respectively. Kinases involved in the phosphorylation of other viral proteins are as yet uncharacterized. However, our studies show that neither PKA nor PKC, which have been shown to affect different phases of virus replication (Kurokawa et al., 1990
; Neumann et al., 1997
; Root et al., 2000
), are involved in the budding process. CK2, on the other hand, appears to be actively involved in the budding process, since the CK2 activity was stimulated during the virus replication cycle, reaching a maximum during the budding phase, and specific inhibitors of CK2 decreased virus budding significantly.
CK2 is a ubiquitous eukaryotic Ser/Thr kinase present in the plasma membrane, cytoplasm, mitochondria and nucleus (reviewed in Faust & Montenarh, 2000 ). This enzyme is cyclic-nucleotide-independent and insensitive to calcium. CK2 is unusual among the PKs since it can use both ATP (Km 10 µM) and GTP (Km 2030 µM) as phosphate donors. CK2 is known to phosphorylate more than a hundred cellular substrates, many of which are involved in regulating signal transduction pathways. Since virus budding is regulated by ATP, the membrane physical state, the G protein and PKs, as well as the cytoskeleton, the substrate(s) of CK2 during virus budding could be one or more of these components, including the cytoskeletal proteins, which interact with RNP/M1 complexes. Both membrane-bound and cytosolic CK2 phosphorylate many cytoskeletal proteins, such as spectrin, ankyrin, adducin, MAP-1B, tau and E-cadherin (reviewed in Allende & Allende, 1995
; Faust & Montenarh, 2000
). Moreover, CK2 is also known to play a role in the regulation of membrane proximal signalling events (for a review, see Allende & Allende, 1995
).
The presence of CK2 in the influenza virion (Tucker et al., 1990 ; reviewed in Hui, 2002
) suggests the possibility of CK2 presence in the vicinity of the budding area of influenza virus and its active involvement in the budding process. Some viruses (such as HIV and herpes simplex virus) have been shown to encapsidate cellular PKs, which play an important role in virus infectivity and virulence (for a review, see Hui, 2002
). However, the only known function of CK2 in influenza virus replication is to phosphorylate viral polymerase PA (Perales et al., 2000
), which is likely to occur during the RNA replication phase of the influenza virus life-cycle. Furthermore, this function of CK2 should be restricted to cell nuclei, which contain high levels of CK2 and where the virus replication/transcription occurs. Therefore, the encapsidation of CK2 into influenza virus particles is not essential for PA phosphorylation and suggests a different role for CK2 function in the assembly and budding processes.
In summary, we have demonstrated that both G protein activity and CK2 function are critically involved in the budding process of influenza viruses. Influenza virus causes a serious worldwide health problem. Understanding more about viruscell interactions during the budding process could provide new targets for therapy and intervention in the disease process.
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Acknowledgments |
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References |
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---|
Almond, J. W. & Felsenreich, V. (1982). Phosphorylation of the nucleoprotein of an avian influenza virus. Journal of General Virology 60, 295-305.[Abstract]
Arni, S., Keilbaugh, S. A., Ostermeyer, A. G. & Brown, D. A. (1998). Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues. Journal of Biological Chemistry 273, 28478-28485.
Avalos, R. T., Zhang, Y. & Nayak, D. P. (1997). Association of influenza virus NP and M1 proteins with cytoskeletal elements in influenza virus-infected cells. Journal of Virology 71, 2947-2958.[Abstract]
Barman, S. & Nayak, D. P. (2000). Analysis of the transmembrane domain of influenza virus neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft association. Journal of Virology 74, 6538-6545.
Barman, S., Ali, A., Hui, E. K.-W., Adhikary, L. & Nayak, D. P. (2001). Transport of viral proteins to the apical membranes and interaction of matrix proteins with glycoproteins in the assembly of influenza viruses. Virus Research 77, 61-69.[Medline]
Beindl, W., Mitterauer, T., Hohenegger, M., Ijzerman, A. P., Nanoff, C. & Freissmuth, M. (1996). Inhibition of receptor/G protein coupling by suramin analogues. Molecular Pharmacology 50, 415-423.[Abstract]
Bigay, J., Deterre, P., Pfister, C. & Chabre, M. (1985). Fluoroaluminates activate transducin-GDP by mimicking the -phosphate of GTP in its binding site. FEBS Letters 191, 181-185.[Medline]
Brewer, C. B. & Roth, M. G. (1995). Polarized exocytosis in MDCK cells is regulated by phosphorylation. Journal of Cell Science 108, 789-796.
Bui, M., Wills, E., Helenius, A. & Whittaker, G. R. (2000). The role of influenza virus M1 in nuclear export of viral RNPs. Journal of Virology 74, 1781-1786.
Cavalli, V., Corti, M. & Gruenberg, J. (2001). Endocytosis and signaling cascades: a close encounter. FEBS Letters 498, 190-196.[Medline]
Ceresa, B. P. & Schmid, S. L. (2000). Regulation of signal transduction by endocytosis. Current Opinion in Cell Biology 12, 204-210.[Medline]
Cheng, H. C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patter, S. M. & Walsh, D. A. (1986). A potent synthetic peptide inhibitor of the camp-dependent protein kinase. Journal of Biological Chemistry 261, 989-992.
Cockcroft, S. & Gomperts, B. D. (1985). Role of guanine nucleotide regulatory binding proteins in the activation of polyphosphoinositide phosphodiesterase. Nature 314, 534-536.[Medline]
Dowrick, P., Kenworthy, P., McCann, B. & Warn, R. (1993). Circular ruffle formation and closure lead to macropinocytosis in hepatocyte growth factor/scatter factor treated cells. European Journal of Cell Biology 61, 44-53.[Medline]
Eker, P., Holm, P. K., van Deurs, B. & Sandvig, K. (1994). Selective regulation of apical endocytosis in polarized Madin-Darby canine kidney cells by mastoparan and cAMP. Journal of Biological Chemistry 269, 18607-18615.
Esparís-Ogando, A., Zurzolo, C. & Rodriguez-Boulan, E. (1994). Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency. American Journal of Physiology 267, C166-C176.
Faust, M. & Montenarh, M. (2000). Subcellular localization of protein kinase CK2: a key to its function? Cell and Tissue Research 301, 329-340.[Medline]
Freed, E. O. (2002). Viral late domains. Journal of Virology 76, 4679-4687.
Freissmuth, M., Boehm, S., Beindl, W., Nickel, P., Ijzerman, A. P., Hohenegger, M. & Nanoff, C. (1996). Suramin analogues as subtype-selective G protein inhibitors. Molecular Pharmacology 49, 602-611.[Abstract]
Garcia, J. G. N., Dominguez, J. & English, D. (1991). Sodium fluoride induces phosphoinositide hydrolysis, Ca2+ mobilization and prostacyclin synthesis in cultured human endothelium: further evidence for regulation by a pertussis toxin-insensitive guanine nucleotide-binding protein. American Journal of Respiratory Cell and Molecular Biology 5, 113-124.[Medline]
Geiss, G. K., An, M. C., Bumgarner, R. E., Hammersmark, E., Cunningham, D. & Katze, M. G. (2001). Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. Journal of Virology 75, 4321-4331.
Gómez-Puertas, P., Albo, C., Pérez-Pastrana, E., Vivo, A. & Portela, A. (2000). Influenza virus matrix protein is the major driving force in virus budding. Journal of Virology 74, 11538-11547.
Gregoriades, A., Christie, T. & Markarian, K. (1984). The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein. Journal of Virology 49, 229-235.[Medline]
Gregoriades, A., Guzman, G. G. & Paoletti, E. (1990). The phosphorylation of the integral membrane (M1) protein of influenza virus. Virus Research 16, 27-41.[Medline]
Hansen, S. H. & Casanova, J. E. (1994). Gs stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A. Journal of Cell Biology 126, 677-687.[Abstract]
Helmreich, E. J. M. (2001). Signal transduction pathways through heterotrimeric G proteins: transmission of hormonal and sensory signals. In The Biochemistry of Cell Signalling , pp. 76-101. Edited by E. J. M. Helmreich. Oxford:Oxford University Press.
Higashijima, T., Burnier, J. & Ross, E. M. (1990). Regulation of Gi and Go by mastoparan, related amphiphilic peptides and hydrophobic amines. Journal of Biological Chemistry 265, 14176-14186.
Holsinger, L. J., Shaughnessy, M. A., Micko, A., Pinto, L. H. & Lamb, R. A. (1995). Analysis of the posttranslational modifications of the influenza virus M2 protein. Journal of Virology 69, 1219-1225.[Abstract]
House, C. & Kemp, B. E. (1987). Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 238, 1726-1728.[Medline]
Huang, K.-P. & Huang, F. L. (1991). Purification and analysis of protein kinase C isozymes. Methods in Enzymology 200, 241-252.[Medline]
Hui, E. K.-W. (2002). Virion-associated protein kinases. Cellular and Molecular Life Sciences 59, 920931.[Medline]
Hui, E. K.-W. & Nayak, D. P. (2001). Role of ATP in influenza virus budding. Virology 290, 329-341.[Medline]
Hui, E. K.-W. & Yung, B. Y.-M. (1992). Protein kinase C activity during sphinganine potentiation of retinoic acid-induced differentiation in a human leukemia cell line (HL-60). Life Sciences 51, 415-422.[Medline]
Ikonen, E., Parton, R. G., Lafont, F. & Simons, K. (1996). Analysis of the role of p200-containing vesicles in post-Golgi traffic. Molecular Biology of the Cell 7, 961-974.[Abstract]
Kistner, O., Muller, H., Becht, H. & Scholtissek, C. (1985). Phosphopeptide fingerprints of nucleoproteins of various influenza A virus strains grown in different host cells. Journal of General Virology 66, 465-472.[Abstract]
Kistner, O., Muller, K. & Scholtissek, C. (1989). Differential phosphorylation of the nucleoprotein of influenza A viruses. Journal of General Virology 70, 2421-2431.[Abstract]
Koch, G., Haberman, B., Mohr, C., Just, I. & Aktories, K. (1991). Interaction of mastoparan with the low molecular mass GTP-binding proteins rho/rac. FEBS Letters 291, 336-340.[Medline]
Kurokawa, M., Ochiai, H., Nakajima, K. & Niwayama, S. (1990). Inhibitory effect of protein kinase C inhibitor on the replication of influenza type A virus. Journal of General Virology 71, 2149-2155.[Abstract]
Latham, T. & Galarza, J. M. (2001). Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. Journal of Virology 75, 6154-6165.
Luban, J. (2001). HIV-1 and Ebola virus: the getaway driver nabbed. Nature Medicine 7, 1278-1280.[Medline]
Ludwig, S., Pleschka, S. & Wolff, T. (1999). A fatal relationship influenza virus interactions with the host cell. Viral Immunology 12, 175-196.[Medline]
Marin, O., Meggio, F. & Pinna, L. A. (1994). Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinase-1 and -2. Biochemical and Biophysical Research Communication 198, 898-905.
Martin, K. & Helenius, A. (1991). Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67, 117-130.[Medline]
Moffett, S., Brown, D. A. & Linder, M. E. (2000). Lipid-dependent targeting of G proteins into rafts. Journal of Biological Chemistry 275, 2191-2198.
Mousli, M., Bronner, C., Landry, Y., Bockaert, J. & Rouot, B. (1990). Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80. FEBS Letters 259, 260-262.[Medline]
Nayak, D. P. (1996). A look at assembly and morphogenesis of orthomyxo- and paramyxoviruses. ASM News 62, 411-414.
Nayak, D. P. (2000). Virus morphology, replication and assembly. In Viral Ecology , pp. 64-123. Edited by C. J. Hurst. London:Academic Press.
Nayak, D. P. & Barman, S. (2002). Role of lipid rafts in virus assembly and budding. Advances in Virus Research 58, 128.[Medline]
Nayak, D. P. & Hui, E. K.-W. (2002). Assembly and morphogenesis of influenza viruses. Recent Research Developments in Virology 4, 3554.
Neumann, G., Castrucci, M. R. & Kawaoka, Y. (1997). Nuclear import and export of influenza virus nucleoprotein. Journal of Virology 71, 9690-9700.[Abstract]
Patnaik, A., Chau, V. & Wills, J. W. (2000). Ubiquitin is part of the retrovirus budding machinery. Proceedings of the National Academy of Sciences, USA 97, 13069-13074.
Pears, C. J. (1996). Role of the protein kinase C gene family in the regulation of cell function. In Protein Phosphorylation in Cell Growth Regulation , pp. 111-133. Edited by M. J. Clemens. New York & London:Harwood Academic Publishers.
Perales, B., Sanz-Ezquerro, S., Gastaminza, P., Ortega, J., Santarén, J. F., Ortín, J. & Nieto, A. (2000). The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis. Journal of Virology 74, 1307-1312.
Perich, J. W., Meggio, M., Reynolds, E. C., Marin, O. & Pinna, L. A. (1992). Role of phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1. Biochemistry 31, 5893-5897.[Medline]
Petri, T. & Dimmock, N. J. (1981). Phosphorylation of influenza virus nucleoprotein in vivo. Journal of General Virology 57, 185-190.[Abstract]
Petri, T., Patterson, S. & Dimmock, N. J. (1982). Polymorphism of the NS1 protein of type A influenza virus. Journal of General Virology 61, 217-231.[Abstract]
Pimplikar, S. W. & Simons, K. (1993). Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature 362, 456-458.[Medline]
Pimplikar, S. W., Ikonen, E. & Simons, K. (1994). Basolateral protein transport in streptolysin O-permeabilized MDCK cells. Journal of Cell Biology 125, 1025-1035.[Abstract]
Pleschka, S., Wolff, T., Ehrhardt, C., Hobom, G., Planz, O., Rapp, U. R. & Ludwig, S. (2001). Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. Nature Cell Biology 3, 301-305.[Medline]
Portela, A. & Digard, P. (2002). The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. Journal of General Virology 83, 723-734.
Reinhardt, J. & Wolff, T. (2000). The influenza A virus M1 protein interacts with the cellular receptor of activated C kinase (RACK) 1 and can be phosphorylated by protein kinase C. Veterinary Microbiology 74, 87-100.[Medline]
Richardson, J. C. & Akkina, R. K. (1991). NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Archives of Virology 116, 69-80.[Medline]
Ridley, A. J. (1994). Membrane ruffling and signal transduction. BioEssays 16, 321-327.[Medline]
Ridley, A. J. (2001). Rho protein, PI 3-kinase and monocyte/macrophage motility. FEBS Letters 498, 168-171.[Medline]
Root, C. N., Wills, E. G., McNair, L. L. & Whittaker, G. R. (2000). Entry of influenza viruses into cells is inhibited by a highly specific protein kinase C inhibitor. Journal of General Virology 81, 2697-2705.
Sanz-Ezquerro, J. J., Férnandez Santarén, J., Sierra, T., Aragón, T., Ortega, J., Ortín, J., Smith, G. L. & Nieto, A. (1998). The PA influenza virus polymerase subunit is a phosphorylated protein. Journal of General Virology 79, 471-478.[Abstract]
Scheiffele, P., Rietveld, A., Wilk, T. & Simons, K. (1999). Influenza viruses select ordered lipid domains during budding from the plasma membrane. Journal of Biological Chemistry 274, 2038-2044.
Terry, N., van Montagu, M. & Inze, D. (1995). GTP-binding proteins in plant. In Guidebook to Small GTPase , pp. 32-38. Edited by M. Zerial & L. A. Huber. Oxford:Oxford University Press.
Tucker, S. P., Penn, C. R. & McCauley, J. W. (1990). Characterisation of the influenza virus associated protein kinase and its resemblance to casein kinase II. Virus Research 18, 243-262.
Vogel, U., Kunerl, M. & Scholtissek, C. (1994). Influenza A virus late mRNAs are specifically retained in the nucleus in the presence of a methyltransferase or a protein kinase inhibitor. Virology 198, 227-233.[Medline]
Yung, B. Y.-M., Hsiao, T.-F., Wei, L. L.-L. & Hui, E. K.-W. (1994). Sphinganine potentiation of dimethyl sulfoxide-induced granulocytic differentiation, increase of alkaline phosphatase activity and decrease of protein kinase C activity in a human leukemia cell line (HL-60). Biochemical and Biophysical Research Communications 199, 888-896.[Medline]
Zhang, J., Pekosz, A. & Lamb, R. A. (2000). Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins. Journal of Virology 74, 4635-4644.
Received 5 June 2002;
accepted 18 July 2002.