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Previous Article | Next Article 
Journal of Virology, March 2006, p. 2291-2308, Vol. 80, No. 5
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.5.2291-2308.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, David Geffen School of Medicine, UCLA, Los Angeles, California 90095
Received 31 August 2005/ Accepted 2 December 2005
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ABSTRACT |
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INTRODUCTION |
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Influenza A virus M1, a small (27.8-kDa), positively charged membrane-binding protein, is the most abundant protein in virus particles (reviewed in reference 49). The M1 protein monomer is a 60-Å-long rod (72), and the stable M1-M1 dimer is about 60 Å by 40 Å by 30 Å (76). The M1 protein consists of two globular regions (amino acids [aa] 1 to 164 and aa 165 to 252) linked by a protease-sensitive loop (Fig. 1), which often gets cleaved during expression and purification procedures (4). The structure of the N terminus portion of the M1 protein (an 18-kDa fragment; aa 1 to 164) has been determined by X-ray crystallography and shows the existence of nine 
-helices and eight loops (4, 30, 76). The C-terminal portion of the M1 protein also contains only loops and helices (78) but has not yet been resolved by X-ray diffraction. The structure of the M1 protein is divided into an N-terminal (N) domain (H1 to H4; aa 2 to 67), a middle (M) domain (H6 to H9; aa 91 to 158), and a C-terminal (C) domain (aa 165 to 252) (76) (Fig. 1).
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A majority of the cellular binding partners of PY-, PT-, and YL-type L domains belong to the components of the VPS (vacuolar protein sorting) pathway known to be involved in cellular-membrane trafficking. The PT-type motif, which mimics the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) protein, interacts with the UEV (ubiquitin-conjugating enzyme 2 [E2] variant) domain of the TSG101 (tumor susceptibility gene 101) protein of ESCRT-I (endosomal sorting complex required for transport I) (for a review, see references 3, 17, 19, 23-25, 43, and 67). The PY-type motif has been shown to bind to WW (tryptophan-rich) domains of the Nedd4 (neural precursor cell-expressed, developmentally downregulated gene 4) family of E3 ubiquitin ligase proteins (such as Yap [Yes-associated protein] binding protein, WBP-1 [WW domain binding protein-1] and WBP-2, LDI-1 [late domain interacting protein 1], and BUL1 [budding associated ubiquitin ligase 1]) (reviewed in references 19, 23, 25, 67, and 84). On the other hand, the YL-type motif on viral protein recruits the AP-50 subunit of the clathrin AP-2 (adaptor protein 2) adaptor complex (69) or interacts with AIP1/ALIX (ALG-2 [apoptosis-linked gene-2] interacting protein-1/ALG-2 interacting protein X) which in turn interacts with ESCRT-I and ESCRT-III (81, 85). However, no specific host proteins involved in influenza virus budding have been identified.
In this report, we demonstrate that the YRKL motif within H6 is a functional homologue of the L domain involved in influenza virus budding and that it represents a novel L domain. Furthermore, our data also indicate that VPS28 and Cdc42 (cell-division cycle 42) interact with the M1 protein via the YRKL motif and play an important role in the viral life cycle. These cellular partners of the M1 protein are likely to be involved in virus budding.
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MATERIALS AND METHODS |
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Antibodies.
Monoclonal anti-TSG101 antibody (clone 4A10) was obtained from GeneTex, Inc. (San Antonio, Tex.). Monoclonal anti-AP-2 (clone 100/2) and anti-E1 ubiquitin-activating enzyme (clone 2G2.3-5) antibodies were purchased from Sigma (St. Louis, Mo.). Monoclonal anti-G o antibody (clone 2A) was from Chemicon (Temecula, Calif.). Monoclonal HA probe (clone F-7), anti-Rho A (clone 26C4), anti-Rho B (clone C-5), polyclonal anti-ALG-2, anti-Nedd4, anti-VPS4B amino terminus, anti-VPS4B carboxy terminus, anti-VPS28, anti-Gi-1, anti-Gi-2, and anti-Gi-3 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Monoclonal anti-NP antibody (clone IV F8) was purchased from Abcam Inc. (Cambridge, Ma.). Polyclonal anti-M1 antibody was purchased from Biodesign (Saco, Maine). Mouse immunoglobulin G (IgG) was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.).
Generation of transfectant viruses using reverse genetics and preparation of mutant stock viruses. Mutagenesis reactions were performed by using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Transfectant viruses were generated by transfecting 293T cells with eight plasmids (1 µg each; seven Pol I-Pol II constructs of WT HA, NA, NP, NS, PA, PB1, and PB2 and either a WT or mutated M gene) as previously reported (35, 38). Individual plaques were isolated, resuspended in virus dilution buffer (36, 37), and inoculated into MDCK cells at a multiplicity of infection (MOI) of 0.001. Infected cells were incubated in virus growth medium (VGM) (38) with N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (0.5 µg/ml), and the supernatants were harvested at 48 h postinfection (p.i.) and analyzed by plaque assay (35). Mutations in each transfectant virus were checked by reverse transcription-PCR and DNA sequencing (38).
Labeling and immunoprecipitation of M1 mutant proteins. At 18 h posttransfection (p.t.), transfected 293T cells were starved and pulse-labeled with 100 µCi 35S protein-labeling mixture (Perkin-Elmer Life and Analytical Sciences, Boston, Mass.) for 2 h (35). The cells were then lysed in 1 ml radioimmunoprecipitation assay (RIPA) buffer, immunoprecipitated with anti-M1 antibody, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%).
Indirect IF and thin-section EM. Cells were stained and observed by indirect immunofluorescence (IF) microscopy as described previously (35, 39). Thin-section electron microscopy (EM) of virus-infected cells was carried out as described previously (9).
Analysis of virus release. Virus-infected MDCK cells and cDNA-transfected 293T cells were labeled from 4 to 16 h p.i. and 4 to 60 h p.t., respectively, with 250 µCi of 35S protein-labeling mixture using nine parts Met– Cys– Dulbecco's modified Eagle's medium and 1 part VGM. At 16 h p.i. or 60 h p.t., the medium was harvested and clarified by low-speed centrifugation, and virions were pelleted by ultracentrifugation (150,000 x g; 2.5 h) through a 25% sucrose cushion. The pelleted virions were resuspended with TNE buffer (10 mM Tris [pH 7.4], 100 mM NaCl, and 1 mM EDTA) by overnight shaking at 4°C and lysed in RIPA buffer with 1% SDS at 37°C for 90 min. The virion lysate was immunoprecipitated in 1 ml of RIPA buffer (with final 0.1% SDS) by antibodies against M1 and NP and analyzed by SDS-PAGE (12%). At 16 h p.i. or 60 h p.t., infected or transfected cells were lysed in 1 ml RIPA buffer, immunoprecipitated with anti-M1 and NP antibodies, and analyzed by SDS-PAGE (12%).
CoIP. A549 cells (2 x 107/175-cm2 flask) were infected with WT virus (MOI = 5). At 4 h p.i., the cells were labeled with 2 mCi 35S protein-labeling mixture in 15 ml of VGM. After 3 h of labeling, the cells were collected by scraping and centrifugation. The cells were then lysed in ice-cold coimmunoprecipitation (CoIP) lysis buffer containing 200 mM Tris (pH 7.4), 100 mM NaCl, 0.1% NP-40, 3 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Roche Applied Science, Indianapolis, IN), protease inhibitor cocktail (Sigma), and protein phosphatase inhibitor cocktails I and II (EMD Biosciences, Inc., La Jolla, Calif.) and incubated on ice for 10 min. Insoluble material was pelleted at 13,000 x g for 10 min at 4°C. The supernatant was then precleaned by protein A- and protein G-Sepharose, and aliquots were coimmunoprecipitated with either nonspecific mouse IgG or specific antibodies (6 µg) in 1 ml CoIP lysis buffer at 4°C for 3 h. Protein A- or protein G-Sepharose was added, and the mixture was incubated at 4°C for 1 h. The immunoprecipitated complex was washed with CoIP washing buffer (200 mM Tris [pH 7.4], 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA) five times and with CoIP lysis buffer once. The immunoprecipitate was analyzed by SDS-PAGE (12%).
Immunoprecipitation-Western blot analysis. The immunocomplexes were resolved on SDS-PAGE (15%) and electroblotted onto a Trans-Blot pure nitrocellulose (NC) membrane (Bio-Rad Laboratories, Hercules, Calif.). The VPS28 and Cdc42 proteins were detected using anti-VPS28 antibody (1:100) and anti-Cdc42 antibody (1:450). NC membranes were then incubated with recombinant protein G-peroxidase conjugate (Zymed Laboratories Inc., South San Francisco, Calif.) (diluted 1:3,500), incubated with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ), and exposed to X-ray film.
Analysis by siRNA. To perform small interfering RNA (siRNA)-mediated cellular protein depletion, a double-stranded siRNA with a 19-nucleotide duplex RNA and 2-nucleotide 3' dTdT overhangs targeting the specific sequences was designed. The siRNAs for Cdc42 (sense, 5'-CUAUGCAGUCACAGUUAUGdTdT-3'; antisense, 5'-CAUAACUGUGACUGCAUAG-dTdT-3') (75), TSG101 (sense, 5'-CCUCCAGUCUUCUCUCGUCdTdT-3'; antisense, 5'-GACGAGAGAAGACUGGAGGdTdT-3') (26), and VPS28 (sense, 5'-GGCCUACAUCAAGGACUGUdTdT-3'; antisense, 5'-ACAGUCCUUGAUGUAGGCCdTdT-3') were synthesized by Dharmacon Inc. (Chicago, Ill.). The siRNA for luciferase GL2 (Luc) (sense, 5'-CGUACGCGGUUTUCTTCGUdTdT-3'), which does not affect influenza virus budding (40), was used as a negative control. A549 cells (1.5 x 105/35-mm dish) or 293T cells (6.5 x 105/well in a six-well plate) were transfected twice with 25 nM siRNA using the TransIT-TKO transfection reagent (Panvera, Madison, WI) at 24-h intervals. At 48 h p.t., the cells were infected with WT virus at an MOI of 0.1, and at 16 h p.i., the media were collected and released viruses were titrated by plaque assay.
Protein expression in 293T cells. Plasmids expressing the HA-tagged wild-type Cdc42, dominant-negative Cdc42-T17N, and constitutively active Cdc42-G12V were purchased from cDNA Resource Center, University of Missouri—Rolla (Rolla, MO). 293T cells (6.5 x 105 cells/well in a six-well plate) were transfected with 4 µg of expression vectors in a BD BioCoat poly-D-lysine/laminin cellware six-well plate (BD Biosciences, Bedford, Mass.) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. The medium was changed 24 h p.t. to OPTI-MEM-I medium supplemented with 5% FBS and 1 mCi/ml 35S protein-labeling mixture without antibiotics. After 48 h, cells were harvested and lysed in RIPA buffer, and expressed tagged Cdc42 proteins were immunoprecipitated by anti-HA-tagged antibody. Immunoprecipitates were resolved by SDS-PAGE (12%).
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RESULTS |
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1 log unit less) and small plaque size (Table 1, set II). When these residues were mutated individually, mutant viruses were rescued and exhibited the WT phenotype (35). Lack of virus rescue from R101A/K102A, R102A/K104A, and K104A/R105A (Table 1, set II) confirmed the results reported previously (35). The leucine residues proximal to the YXXL motif on equine infectious anemia virus (EIAV) p9gag have been shown to contribute significantly to optimal virus budding (68) and AIP1 interaction (81). There are two leucine residues 99-LYRKL-103 in influenza virus M1, neither of which, when individually mutated, affected virus replication significantly (35). To analyze further the roles of these leucine residues in H6, we made three double mutations (L99A/Y100A, L99A/L103A, and Y100A/L103A) (Table 1, set III). These three mutant proteins, when expressed in cDNA-transfected cells, were present at similar levels (Fig. 2A). Infectious virus was rescued from each of the three mutants. Two mutant viruses (L99A/Y100A and L99A/L103A) exhibited lower titers and small plaque size (Table 1, set III). The other mutant virus (Y100A/L103A) exhibited normal virus titer and plaque size (Table 1, set III), confirming earlier results (35). These data show that although leucine residues of 99-LYRKL-103 individually do not contribute significantly to virus replication, they cooperate with other residues, as evidenced by the lethality of double mutations (K98A/L99A and R101A/L103A) and by lower virus titer (L99A/Y100A and L99A/L103A).
Our previous study (35) showed that R105A could not be rescued and that R105K mutant virus exhibited reduced virus growth without affecting the spherical morphology of virus particles. We postulated that in R105K-infected cells, fewer particles were produced on the cell surface because of the defective exit of M1 from the nucleus (35). To further investigate the function of Arg105, we mutated Arg105 to a negatively charged glutamic acid residue (R105E) (Table 1). Although the mutant protein was expressed in cDNA-transfected cells at a level similar to that of the WT M1 (Fig. 2A), the R105E mutant virus grew to a lower titer (100 times less) and produced significantly smaller plaques than the WT virus (Table 1). To further investigate the nature of the R105E defect, we examined virus particle budding on the apical plasma membranes of infected MDCK cells by transmission EM. Polarized MDCK cells, grown on the polycarbonate filters, were infected with WT or R105E virus at an MOI of 3, and at 12 h p.i. they were examined by thin-section EM. The results (Fig. 3) showed that the R105E mutant virus had a morphogenesis defect. R105E particles (Fig. 3B) were elongated and partly filamentous in shape, as was observed for R101A (Fig. 3C). Often, some mutant virus particles were empty, lacking vRNP cores, whereas WT virus particles were mostly spherical and had a dense core (Fig. 3A). These results were different from those with the R105K mutant, which also exhibited reduced growth and a small-plaque phenotype but had a spherical particle morphology (35).
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The YRKL motif can function in a position-independent manner. One unique characteristic of the L domains of a number of viruses is that they can function in a position-independent manner (1, 18, 51, 62, 64, 89, 94). Furthermore, although the heterologous L-domain motifs have limited ability to promote release of HIV-1 (60), some L domains are functionally interchangeable among different viruses (51, 57, 62, 77, 94). Our previous study (35) showed that the mutation of 100-YRKL-103 to the 4A mutant or 101-RK-102 to the 2A mutant (i.e., R101A/K102A) were lethal and could not be rescued to infectious virus by reverse genetics after repeated attempts. However, insertion of a foreign heterologous L domain (PTAP or YPDL) could rescue the function of the mutated YRKL (the 4A mutant), producing infectious virus with the WT phenotype (35). From these results, we hypothesized that YRKL and possibly the adjacent region contain a functional L domain. Therefore, we wanted to determine if the YRKL motif can function in virus replication when inserted in different locations of M1. Accordingly, the YRKL sequence was inserted in six different locations of the M1 protein containing the 4A mutation of YRKL (Table 2). These positions were located on the N terminus (4A:YRKL^1-2), loop 3 (L3) (i.e., the H3-H4 junction; 4A:YRKL^51-52), just before 4A (4A:YRKL^99-100), right after 4A (4A:YRKL^103-104), on loop 6 (L6) (i.e., the H6-H7 junction; 4A:YRKL^107-108), or after H9 (4A:YRKL^160-161) (Table 2). The two numbers on the names of the mutants indicate the insertion location of YRKL between these two aa sequences. Three of these insertion positions were in the loop structure (4A:YRKL^51-52 in L3, 4A:YRKL^107-108 in L6, and 4A:YRKL^160-161 after H9), but other insertions were made in H6 either before or after the 4A mutation and one (4A:YRKL^1-2) was at the extreme N terminus of H1. One insertion at aa 103 to 104 (4A:YAAL^103-104) contained YAAL, in which the 101-RK-102 residues were mutated (Table 2). Again, all proteins were expressed in cDNA-transfected cells to levels similar to that of the WT protein (Fig. 2B). Virus rescue experiments showed that all insertions of YRKL at different positions yielded infectious virus titer and plaque size in MDCK cells like those of the WT virus (Table 2). Again, infectious virus could not be rescued after repeated attempts with the 4A mutant, as reported before (35). Furthermore, YAAL, when inserted, could not restore the function of YRKL (4A:YAAL^103-104) (Table 2). Earlier, we showed that the YAAL mutation in its normal position was lethal and did not yield infectious virus (35). It should be noted that YRKL insertions at distant locations, such as the extreme N terminus (4A:YRKL^1-2) or H9 (4A:YRKL^160-161), also yielded infectious virus with a WT phenotype. Electron microscopic analysis showed that 4A:YRKL^160-161 exhibited a WT spherical morphology (Fig. 3A and D). Therefore, insertion of the YRKL motif at six atopic locations of the 4A mutant M1 protein restored normal budding (Table 2), and virus particles exhibited spherical morphology (Fig. 3D). These results showed that YRKL can act in a position-independent manner, as has been reported for other L domains (1, 18, 51, 62, 64, 89, 94).
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Effects of mutations on virus release. The results presented above showed that some mutations in YRKL and neighboring sequences reduced virus titer by 1 to 2 log units (Table 1) and that the insertion of YRKL in the 4A lethal mutant restored the WT virus titer (Table 2 and Fig. 4). To determine if reduced virus titers in mutant viruses were due to defects in virus release and budding, MDCK cells were infected with WT or mutant viruses at an MOI of 1 and metabolically labeled with 35S protein-labeling mixture for 12 h at 4 h p.i. At 16 h p.i., the cell lysates and released virus particles in medium were used for immunoprecipitation with anti-NP and -M1 antibodies. Virus particles in the medium were pelleted by centrifugation (150,000 x g; 2.5 h) through a sucrose (25%) cushion, and the pellet was used for immunoprecipitation. For lethal mutation 4A, 293T cells were transfected with the 4A M1 mutant and seven WT cDNAs (as used for generating transfectant viruses) and labeled from 4 to 60 h p.t. The cell lysate and the medium from transfected cells were processed as described above. The results show that, as expected, neither M1 nor NP could be detected in the media of mutant 4A and R101A/K102A cDNA-transfected cells (Fig. 5 and Table 3), even after longer exposure (data not shown). Except in R105E, all other virus-infected cell lysates had essentially similar amounts of NP and M1. To avoid the potential difference in protein expression in different virus-infected cells, the amounts of proteins were quantified by densitometry, and the percentage of release of NP or M1 was calculated as follows: the amount of NP or M1 in the virus pellet over total NP or M1 (i.e., NP or M1 in the pellet plus the NP or M1 in the cell lysate). It should be noted that in WT virus-infected cells, about 32% of the total M1 was released, whereas only 1.4% of the total NP was present in virus particles in the medium (Table 3). In 4A:YRKL^51-52 virus-infected cells, which exhibited a WT phenotype (Table 2), the percentages of both NP and M1 released in the medium were essentially the same as those for the WT virus (1.2% and 32%, respectively) (Table 3). The infection of 4A:YRKL^160-161 virus exhibited similar percentages (data not shown). For the mutant viruses yielding lower virus titers, the numbers of particles released in the medium were markedly reduced (more than 10-fold),whereas the percentages of M1 released in the medium were essentially the same as for the WT virus (i.e., 30 to 38%) (Table 3). It is likely that NP reduction in these mutant virus particles was much greater, but it could not be accurately estimated because of the low densitometric readings. Therefore, mutant viruses exhibiting reduced infectivity had reduced NP/RNP content without much difference in M1 content. It has been reported that, compared to spherical particles, filamentous particles exhibited reduced NP/M1 ratios (71).
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The M1 protein is essential for influenza virus budding and is structurally similar to the HIV-1 Gag protein (31). The YRKL sequence on the M1 protein can be functionally replaced by selected L domains from other viruses (35). Therefore, it raises a question of whether influenza virus, like other viruses, uses the VPS machinery for budding. We therefore wanted to identify the L-domain-related host protein partners of M1 by CoIP. Accordingly, human type II alveolar epithelial cells (A549) were infected with WT virus (MOI = 5) and pulse-labeled at 4 h p.i. for 3 h (i.e., pulse-labeling from 4 to 7 h p.i.). The cells were then lysed in CoIP lysis buffer and coimmunoprecipitated with a number of host protein-specific antibodies.
Antibodies against a number of proteins, including AP-2, TSG101, Nedd4, and VPS4B, which have been shown to interact with the L domains of a number of viruses, failed to coimmunoprecipitate the radiolabeled M1 protein from the virus-infected cell lysate (Fig. 7A). Similarly, antibodies against proteins such as ALG-2 and the Ub-activating enzyme E1, which are involved in retroviral budding, were negative as well (Fig. 7A). However, CoIP analysis with anti-VPS28 antibody showed the presence of an M1 band (Fig. 7A, lane 12), indicating interaction between VPS28 and M1. To further verify these results, WT virus-infected A549 cells were lysed at 7 h p.i., and the lysates were incubated with either anti-VPS28, anti-M1, or anti-NP antibody. The immunocomplexes were resolved by SDS-PAGE (15%), electroblotted onto NC membranes, and probed with anti-VPS28 antibody. Immunoblots showed the presence of a protein band at the same position as VPS28 (28 kDa) upon coimmunoprecipitation with anti-M1 antibody (Fig. 7B, lanes 2 and 3; compare the band immunoprecipitated with anti-VPS28 antibody). These results further support the idea that VPS28 interacts with M1.
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To investigate the involvement of VPS28 in influenza virus budding, we depleted the intracellular VPS28 using a synthesized siRNA targeting VPS28 mRNA. Accordingly, A549 cells were transfected with 25 nM siRNA twice at an interval of 24 h in order to deplete intrinsic VPS28. The Western blot indicated that VPS28 protein expression was reduced to 32% (Fig. 7D, inset). The VPS28-depleted cells were then infected with influenza virus (MOI = 0.1). At 16 h p.i., viruses released into the medium were collected and analyzed by PFU assay. The results (Fig. 7D) show that the transfection of VPS28 siRNA inhibited influenza virus replication to 41%, whereas Luc and TSG101 siRNAs did not affect virus replication significantly (94% and 93%, respectively). These results indicate that VPS28 siRNA exhibited an inhibitory effect on influenza virus replication.
Then, we examined viral-particle release efficiency by radioimmunoprecipitation of the NP and M1 proteins from the cell lysate and pelleted viral particles of siRNA-treated 293T cells (Fig. 7E). The intracellular expression levels of both NP and M1 were similar to those of the control (Luc siRNA transfection) (Fig. 7E, lanes 2 to 4). However, the number of particles released from VPS28 siRNA-transfected cells was reduced to half (Fig. 7E, bottom), essentially confirming the PFU data (Fig. 7D).
Cdc42 interacts with influenza virus M1.
Since earlier studies suggested the involvement of GTP-binding protein (G protein) in influenza virus budding (37), we also tested G protein antibodies by CoIP assay (Fig. 8A). The CoIP results showed that M1 protein was coimmunoprecipitated by antibodies against the monomeric Cdc42 G protein (Fig. 8A, lane 7), but not the other monomeric small G proteins, such as Rho A and Rho B (Fig. 8A, lanes 5 and 6), or heterotrimeric G proteins (Go or Gi class subunit, such as G o, G i-1, G i-2, and G i-3) (data not shown).
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To elucidate the interaction of M1 with Cdc42, the CoIP assays were performed using lysates from the WT virus-infected A549 cells (Fig. 8C, lanes 2 to 4), 4A cDNA-transfected 293T cells (Fig. 8C, lanes 5 to 7), and 4A:YRKL^160-161 virus-infected A549 cells (Fig. 8C, lanes 8 to 10). The anti-Cdc42 antibody coimmunoprecipitated the M1 protein from both WT and 4A:YRKL^160-161 virus-infected cells (Fig. 8C, lanes 4 and 10), but not from 4A cDNA-transfected cells (Fig. 8C, lane 7). These data support the idea that the M1 and Cdc42 interaction also involves the H6 domain containing the YRKL motif.
To investigate the involvement of Cdc42 in influenza virus replication, we depleted the intracellular Cdc42 using a synthesized siRNA targeting Cdc42 mRNA. 293T cells were transfected with 25 nM siRNA twice at an interval of 24 h, and the Western blot indicated the depletion of the Cdc42 protein level to 10% (Fig. 9A, inset). The Cdc42-depleted cells were then infected with influenza virus (MOI = 0.1). At 16 h p.i., viruses released into the medium were collected and analyzed by plaque assay. The results (Fig. 9A) show that the transfection of Cdc42 siRNA inhibited influenza virus replication to 28%, whereas Luc siRNA did not affect virus replication significantly (87%). These results indicated that Cdc42 siRNA exhibited an inhibitory effect on the influenza virus life cycle.
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To further determine the effect of Cdc42 activity on influenza virus budding, 293T cells were transfected with the HA-tagged WT Cdc42, dominant-negative Cdc42-T17N, or constitutively active Cdc42-G12V and then infected with influenza virus (MOI = 0.1) at 48 h p.t. The immunoprecipitation experiment confirmed the expression of those HA-tagged Cdc42 proteins (Fig. 9C, inset). As seen in Fig. 9C, the dominant-negative Cdc42-T17N inhibited the infectious-virus titer to half that of the control (51%) by PFU assay. In contrast, the overexpression of the constitutively active Cdc42-G12V increased virus budding. These results demonstrated that Cdc42 activity was involved in the influenza virus life cycle.
Next, we examined viral-particle release by radioimmunoprecipitation of cell lysates and pelleted viral particles from cDNA-transfected 293T cells infected with WT virus (Fig. 9D). The intracellular expression levels of NP and M1 were similar (Fig. 9D, lanes 2 to 5), whereas virus particle release from Cdc42-T17N cDNA-transfected cells was reduced significantly. NP and M1 in the medium of cells transfected with Cdc42-T17N cDNA were reduced to 18% and 25%, respectively (Fig. 9D, lane 8). On the other hand, collected viral particles from Cdc42-G12V cDNA-transfected cells exhibited increased release (Fig. 9D).
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DISCUSSION |
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Data presented in this report indicate that 100-YRKL-103 present in H6 of the influenza virus M1 protein functions as the L domain in influenza virus budding. This conclusion is based on the effect of mutation of the YRKL sequence on virus replication (35); the ability of the foreign heterologous L domains, such as PTAP and YPDL, to restore the function of the YRKL mutation (35); and finally, the ability of the YRKL motif to restore virus budding in a position-independent manner when inserted in atopic positions, as shown here (Table 2). Furthermore, YRKL is a novel motif of the L domain, since it does not contain any proline residues but contains two positive charges. Since Y and L residues can be replaced (35), it does not represent the YXXL motif found in EIAV (i.e., YPDL) (68, 69). However, the YRKL motif can be replaced by motifs such as YPDL (35) or YPKL (R101P) (Table 1), suggesting that 101-RK-102 residues can be replaced by some but not other residues, such as YAAL (Table 1, set II) (35). Also, the results reported here, along with previous data (35), show that the insertion of 100-YRKL-103 does not function by restoring the NLS function of 101-RKLKR-105 and that the NLS function of M1 is not required for the replication and budding of influenza virus. It has been reported that M1, defective in NLS function, can be transported into the nucleus when expressed with other viral components (34, 66).
Although 100-YRKL-103 has been shown to function as the L domain motif of influenza virus, the presence of other sequences or motifs in influenza virus proteins affecting virus budding cannot be ruled out. Recently, we showed that a mutation in the cytoplasmic tail of NA caused a budding defect with the generation of elongated particles (8). Multiple (closely spaced or overlapping) motifs are present in many viruses, such as HIV-1 Gag protein (26, 57, 81), human T-cell leukemia virus type 1 Gag protein (13, 32, 86), Mason-Pfizer monkey virus Gag protein (29), Ebola virus VP40 (41, 52, 57, 90), Lassa virus Z protein (82), and prototype foamy virus Gag protein (65, 80). Recent data also support the existence of a bipartite L domain in HIV (54). These multiple L domains have been shown to function independently (42, 52), as well as cooperatively (13), in enhancing virus budding.
However, we did not observe the accumulation of virus particles at the cell surface in virions in the cases of 4A and R101A/K102A. One possible explanation is that the block affects not only the late step of pinching off, but also an earlier step(s) of virus assembly. For example, the YEIL motif on prototypic foamy virus is required at an assembly step prior to budding (65). Another possibility is that mutant buds are unstable and rapidly collapse back into the cytoplasm rather than accumulate. Alternatively, it is possible that a late step of budding has been affected, but the budding defect in the pinching-off process of influenza virus is difficult to detect by EM because very few cells are transfected by all eight plasmids.
Although YRKL is probably the minimum sequence motif that can restore budding, neighboring sequences can also aid in the function of the L domain. This is evident from the effects of multiple dual mutations in this region, which were lethal or reduced the virus titer significantly (Table 1, sets II and III). A hexapeptide, a heptapeptide, an octapeptide, and a nonapeptide containing YRKL and neighboring sequences had various effects on restoring virus replication, depending on the site of insertion. When introduced into H6, between aa 100 and 101, only a tetrapeptide (YRKL) was tolerated. On the other hand, when introduced in the loop region (L3), between aa 51 and 52, all inserts containing YRKL produced a WT phenotype (Fig. 4B), suggesting that loop structures are more tolerant of peptide insertion than helices. Therefore, loop structures may be useful for the introduction of foreign epitopes.
Analysis of virus release into the supernatant by metabolic labeling of cells infected with several mutant viruses revealed an interesting aspect of influenza virus budding. Mutant viruses exhibiting reduced virus titers contained reduced NP compared to M1 (Fig. 5 and Table 3). Reduced virus titer with elongated particle morphology could be due to either incomplete closure of multiple particles present in the same filament, as seen in HIV with a budding defect (19, 20, 24-26, 28, 67, 81, 85), or inability to pinch off, leading to the formation of elongated particles due to continuing incorporation of M1 and the release of fewer infectious particles. Reduced incorporation of vRNP, as measured by NP (Fig. 5), in released virus particles with an elongated morphology and reduced PFU titer supports the latter model. This would also suggest that the elongated particles do not contain multiple sets of vRNPs. It has been shown recently that reduction in M1 and M2 synthesis in mutant virus-infected cells causes reduction in virus titer but does not lead to any abnormal defect in virus morphology and produces spherical particles possessing the same NP/M1 ratio as WT spherical virus particles (15).
By definition, L-domain sequences are required for the final pinching off of virus particles from the membrane. This process requires fusion of the apposing cellular and viral membranes, leading to the fission of the bud and release of virus particles into the extracellular environment. A number of host proteins are recruited by the L domains and brought to the budding site, which then facilitate the pinching-off process. However, the mechanism of this process and the roles of host proteins in this process are as yet unknown. Although a number of host proteins, such as TSG101, Nedd4, AP-2, and AIP1, have been found to interact with different L domains and to affect virus budding, no host protein has been shown to interact with the influenza virus YRKL motif. Unlike the majority of L domains, YRKL, the influenza virus L domain, does not contain any proline residues and therefore is unlikely to interact with the WW domain of host proteins. In this report, we show for the first time that two host proteins, VPS28 (a member of the ESCRT family) and Cdc42 (a member of the Rho family G proteins), form a complex with M1 and that both of these proteins are involved in virus replication. The siRNA-mediated reduction of both VPS28 and Cdc42 (Fig. 7D and E and 9A and B) inhibited influenza virus release. Furthermore, the expression of dominant-negative Cdc42 reduced virus production, whereas constitutively active Cdc42 stimulated virus yield (Fig. 9C). It should also be noted that TSG101 did not interact with WT M1 (Fig. 7A and C), nor did the TSG101 siRNA interfere with influenza virus production (Fig. 7D and E). However, as expected, TSG101 did interact with PTAP M1, as shown by coimmunoprecipitation (Fig. 7C).
Although CoIP data demonstrate that VPS28 and Cdc42 form a complex with the M1 protein via the YRKL motif (Fig. 7C and 8C), it remains to be determined whether both VPS28 and Cdc42 interact directly with the sequence containing the YRKL motif of M1 or via another protein. Human VPS28 is a poorly characterized 28-kDa cytoplasmic protein and is a component of the human ESCRT-I complex, along with TSG101 and VPS37 (7, 11, 47). ESCRT-I plays a crucial role in the initial steps of MVB formation and cargo sorting. VPS28 normally binds tightly to TSG101 (26, 67). The PT-type L domain interacts with an ESCRT-I component, and TSG101 is necessary for budding of virus containing a PT-type L domain. The PY-type L domain first associates with Nedd4 and subsequently binds TSG101 (12). The YL-type L domain interacts with AIP1/ALIX, which subsequently binds ESCRT-I and ESCRT-III (56, 81, 85). Although different L domains interact with different host proteins, these data together suggest that all known classes of L domains, including YRKL of influenza virus M1, access a common pathway involving ESCRT complexes.
We have also shown that G-protein inhibitors inhibit influenza virus budding (37). However, unlike VPS28, Cdc42 has not been implicated in the VPS pathway, and therefore, how Cdc42 is functionally required for influenza virus budding is as yet unknown. Cdc42 was first identified as a factor required for polarization of the budding yeast Saccharomyces cerevisiae (46). Cdc42, a member of the Rho family of small GTPase proteins, regulates multiple cell functions, including motility, proliferation, apoptosis, and cell morphology (reviewed in references 21 and 45). Cdc42 plays a central role in establishing cell polarity in all eukaryotic cells and is particularly important in cytoskeletal remodeling (for a review, see references 5 and 58). Cdc42 localizes at the leading edge of the cell and forms a complex with polarity proteins (such as PAR proteins) and protein kinases (such as protein kinase C) (22). Cdc42 also induces membrane ruffling (48). Our observations raise the possibility that Cdc42 may be important during the process of influenza virus assembly and budding. Since previous findings indicate that RNP-M1 associates with actin (6) and actin depolymerization affects virus budding and virus morphology (70), it is possible that the interaction between Cdc42 and M1 is involved in cytoskeletal rearrangement during budding. However, the detailed mechanism of Cdc42 in the influenza virus life cycle awaits further investigation.
Interestingly, the influenza virus budding mechanism exhibits similarity to that of the retrovirus EIAV. First, both the EIAV YPDL sequence on p9gag (68) and influenza virus YRKL on the M1 protein (35) are tyrosine- and leucine-containing L-domain motifs. Second, although the release of retroviruses utilizing either PY- or PT-type L domains are blocked by agents that inhibit ubiquitin modification, EIAV Gag release is insensitive to ubiquitin-blocking agents (i.e., proteasome inhibitors) (61, 63). Similarly, influenza virus budding is also insensitive to proteasome inhibitors, such as MG-132 and lactacystin, as well (36). Third, EIAV Gag utilizes the host VPS machinery, since an L-domain mutant of EIAV Gag, when fused with VPS28, is released efficiently in the medium (83). In this study, we demonstrated that VPS28 is a binding partner of the influenza virus M1 protein and is involved in virus replication (Fig. 7). Fourth, TSG101 does not appear to participate in the release of the EIAV Gag particle (83). EIAV budding was not affected by the expression of a C-terminal fragment of TSG101 (TSG-3') (77). In this study, we demonstrated that influenza virus M1 was not associated with TSG101, since the M1 protein could not be coimmunoprecipitated with TSG101 (Fig. 7A and C) and TSG101 depletion by siRNA did not have any effect on influenza virus budding (Fig. 7D and E).
However, the differences between influenza virus YRK and the EIAV YPDL late-domain motif are notable, as well. First, in the YPDL motif, the Y and L residues are critical for virus budding (69) and interaction with AIP1/ALIX (81). In contrast, our results showed that the Y and L positions are not important for YRKL function; rather, the two central basic residues R and K are necessary. Second, although the EIAV Gag-VPS28 fusion protein showed that EIAV can be directed to the MVB machinery (83), it does not show that VPS28 is normally involved in EIAV release. Third, EIAV release is broadly blocked by dominant-negative VPS4A and VPS4B expression, with budding reduced to about 1 to 10% of the control level (77, 81, 83, 85). However, dominant-negative VPS4B did not affect influenza virus release significantly (data not shown).
In conclusion, we have shown that influenza virus M1 possesses a novel YRKL motif and forms complexes with VPS28 and Cdc42. Identification of other cellular binding partners of this novel L domain and their roles in virus budding may provide additional targets for therapeutic intervention against influenza virus infection.
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ACKNOWLEDGMENTS |
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This work was supported by USPHS grants RO1 AI16348 and RO1 AI41681.
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FOOTNOTES |
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. Cell 106:489-498.[CrossRef][Medline]
, elongated virus particles; 
, villi.
