The Morphology and Composition of Influenza A Viru
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病菌学杂志 2005年第12期
Department of Microbiology, Mount Sinai School of Medicine, New York, New York
ABSTRACT
We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.
TEXT
Influenza A virus is an enveloped virus of the Orthomyxoviridae family with a segmented negative-strand RNA genome. Its eight viral RNA (vRNA) segments encode the three subunits of the viral RNA polymerase complex, PB1, PB2, and PA; the nucleoprotein NP; the glycoproteins HA and NA; the matrix protein M1; the ion channel protein M2; the nonstructural protein NS1; the nuclear export protein NEP; and the recently discovered facilitator of apoptosis PB1-F2 (7). The M1 protein is a major structural component of the virus particles and forms a layer underneath the lipid cell-derived envelope (43, 44). Inside the virions and in infected cells at late stages of the virus replication, the M1 protein associates with the viral ribonucleoproteins (vRNPs), which are composed of viral RNA molecules, multiple copies of the NP, and the three subunits of the viral polymerase holding the ends of the viral RNAs (3, 41, 51). Transcription and replication of the influenza virus genome take place in the nucleus of infected cells. By contrast, assembly of virus particles occurs at the apical surface of the plasma membrane where the M1 protein presumably interacts on one side with the cytoplasmic tails of the glycoproteins anchored in the membrane and on the other side with the vRNPs. Although a direct interaction of the M1 protein with the viral glycoproteins was not demonstrated, expression of HA and NA proteins stimulates membrane association of M1 protein (11). Moreover, a mutant virus with deleted cytoplasmic tails at both HA and NA proteins shows severe defects in particle morphology, genome packaging, and incorporation of NA and M1 into virions (25, 55).
Previous works show that the M1 protein plays a pivotal role in influenza A virus budding. Mutation of certain residues in the M1 protein dramatically affects the morphology of the virus particles (4, 9). Expression of the M1 protein in mammalian cells results in budding of virus-like particles (17). The M1 protein possesses a specific amino acid sequence that appears to be similar in function to the late budding motifs of the matrix proteins of retroviruses (21). A key role of matrix proteins in virus assembly and budding has also been demonstrated for other enveloped negative-strand RNA viruses. Recombinant rabies and measles viruses lacking their M genes produce significantly reduced virus titers in infected cells and release particles with altered morphology (6, 33). The M proteins from vesicular stomatitis and human parainfluenza type 1 viruses induce budding of vesicles from the plasma membrane when they are expressed alone (8, 26, 29).
In addition to the M1 protein, the M gene of influenza virus also encodes the ion channel transmembrane protein M2 (38, 47). Although the M2 protein is not essential for infectivity (48, 50), this protein appears to be needed for efficient vRNP uncoating during viral entry (18), and mutant M2 influenza viruses are highly attenuated. The M2 protein might also play a role in virus assembly and budding and, like the M1 protein, also appears to regulate virus morphology (40).
In the present study we investigated whether a reduction in the levels of M1 and M2 proteins synthesized in virus-infected cells affects virus particle assembly. We used plasmid-based reverse genetics to introduce a double mutation, C-GA-U (11-12') into the conserved region of the vRNA promoter of the M segment of influenza A/WSN/33 virus (Fig. 1) (5, 13, 19, 35). The same mutation was found to be responsible for a reduction in the levels of mRNA and protein expression when introduced into the NA, PA, and NS segments of recombinant viruses (5, 14, 46). For this purpose, the M RNA segment was PCR amplified using as template pPOLI-M plasmid (13) and primers 5'-GATCGCTCTTCGGCCAGCGAAAGCATGTAGATATTGAAAGATG-3' (positive sense; M1/M2 start codon isunderlined; inserted promoter mutation is in boldface) and 5'-GATCGCTCTTCTATTAGTAGAAACAATGTAGTTTTTTACTCC-3' (negative sense; M2 stop codon is underlined; inserted promoter mutation is in boldface). The PCR product was digested with the SapI restriction enzyme and ligated into pPOLI-SapI-RT vector (39). The resultant plasmid was used to transfect 293T/MDCK cell cocultures along with pPOLI-PB1, -PB2, -PA, -HA, -NP, -NA, and -NS (13) and pCAGGS-PB1, -PB2, -PA, and -NP plasmids (2). 293T and MDCK cells were seeded into 35-mm dishes at the proportion 5:1 and grown to approximately 70% of confluency in Dulbecco modified Eagle medium (DMEM)-10% fetal bovine serum medium. Before transfection, the medium was replaced with Opti-MEM I (Invitrogen). TransIT-LT1 transfection reagent (Mirus, Madison, WI) was used according to manufacturer's instructions. Briefly, 9 μl of TransIT-LT1 was diluted in 100 μl of Opti-MEM I medium and mixed with 1.5 μg of total plasmid DNA (containing equal amounts of each of the 12 plasmids) also diluted in 20 μl of Opti-MEM I. The DNA-TransIT complexes were allowed to form for 15 min at room temperature and were added drop by drop to the cells. Medium was replaced with DMEM-0.1% fetal bovine serum-0.35% bovine serum albumin 18 h later. Three days later supernatants were used to infect MDCK cells. Four days later, the rescue of Mmut mutant virus was confirmed by hemagglutination and plaque assays. The presence of the promoter mutations in the M segment was confirmed by sequencing of reverse transcription-PCR (RT-PCR) products derived from viral RNA. It should be noted that the 3' mutation generated an ATG in the corresponding mRNA (Fig. 1), and although this new ATG is in a bad Kozak context, it has the potential to affect translation.
The growth properties of the Mmut virus were analyzed in MDCK cells infected at low and high multiplicities of infection (MOI) (Fig. 2A and B). At different time points infectious particles present in the medium were titrated by plaque assay in MDCK cells. The Mmut virus replicated to titers approximately 2 logs lower than those of WSN virus when cells were infected at an MOI of 0.001. When cells were infected at an MOI of 1, the Mmut virus reached a maximum titer similar to that of wild-type virus but exhibited a slight decrease in growth kinetics at 4 and 6 h postinfection. It is likely that the small differences in replication between wild-type and Mmut viruses in single-cycle replication experiments (Fig. 2B) are exponentially increased during multicycle replication (Fig. 2A).
Synthesis of viral proteins in wild-type WSN and Mmut virus-infected MDCK cells was analyzed after infection at an MOI of 1. For this purpose the medium of the infected cells was replaced with DMEM supplemented with 75 μCi/ml of EXPRE35S35S protein labeling mix (Perkin-Elmer Life and Analytical Sciences, Boston, MA) at 2, 4, 6, and 8 h postinfection. Two hours later cell monolayers were washed with phosphate-buffered saline and lysed in sodium dodecyl sulfate (SDS) loading buffer. Proteins were separated on 16.5% Tris-Tricine Criterion SDS-polyacrylamide gels (Bio-Rad) and visualized by autoradiography (Fig. 2C). The levels of M1 protein synthesis in Mmut-infected cells were significantly reduced at all time points compared to those in wild-type WSN virus-infected cells. To determine differences between wild-type and Mmut viruses in the steady-state levels of M1 and M2 proteins in infected cells, we performed Western blot assays using the same cell lysates and monoclonal antibody (E10) that recognizes both M1 and M2 proteins (Fig. 2D). Sheep anti-mouse antibodies conjugated with horseradish peroxidase (Amersham Life Sciences) were used as the secondary antibodies. At all time points the levels of M1 and M2 proteins in Mmut-infected cells were significantly lower than those in cells infected with WSN virus. To quantify the differences, we used different dilutions of the lysates and developed Western blots with E10 and anti-NP monoclonal antibodies (data not shown). The intensities of bands were measured with the Gel Doc 2000 (Bio-Rad) by using Quantity One quantification software. Levels of M1 and M2 proteins in Mmut virus-infected cells were normalized to NP protein and expressed as a percentage of the corresponding proteins in wild-type virus-infected cells (Table 1). The levels of the M1 and M2 proteins in Mmut virus-infected cells were reduced approximately 20 and 10 times, respectively, compared with those in wild-type virus-infected cells. This reduction is likely to be due to a combination of reduced transcription levels from the promoter-mutated M gene and reduced translation levels of M1 and M2 proteins due to the generation of an ATG start codon (followed by a stop codon) in front of the ATG of the M1 and M2 open reading frames by the mutation inserted at the 3' end of the viral RNA, as already described for the NA gene (14).
In order to confirm that the inserted promoter mutation decreased the levels of transcription of the Mmut RNA, we quantified levels of M-specific vRNA (as indicative of replication) and of M-specific mRNA (as indicative of transcription) in virus-infected cells. MDCK cell monolayers in 60-mm dishes were infected at an MOI of 1 with wild-type and Mmut viruses. At 6 h and 11 h postinfection, cells were lysed in 2 ml of Trizol, and total RNA was extracted according to the manufacturer's instructions (Invitrogen). One μg of RNA was used for quantitative RT-PCR according to a previously published protocol for SYBR Green in an ABI7900 HT instrument (52). For mRNA quantification, RNA was reverse transcribed using a poly(T) primer. For M vRNA quantification, RNA was reverse transcribed using a primer annealing to the first nine nucleotides of the M vRNA. For NP vRNA quantification, RNA was reverse transcribed using a primer annealing to the first 12 nucleotides of the NP vRNA. cDNAs were amplified using internal M- or NP-specific primers, and the relative amount of M-specific vRNA and mRNA in Mmut virus-infected cells compared to wild-type virus-infected cells was calculated after normalization to NP vRNA and mRNA levels. The sequence of the specific primers used in the PCRs is available upon request. Different sets of M-specific primers were designed to specifically amplify M1 mRNA or to amplify both M1 and M2 mRNAs. Each transcript in each sample was assayed three times, and the median threshold cycle was used to calculate the experimental values for each RNA. We quantified RNA levels from duplicate samples. At 6 h postinfection, levels of M vRNA were similar between Mmut (114% ± 26%) and wild-type virus. At 11 h postinfection there was a slight decrease in the levels of Mmut vRNA (50% ± 18% of wild-type levels). By contrast, M mRNA levels were significantly reduced in Mmut virus-infected cells (M1 mRNA levels at 6 h: 23% ± 10%; M1 plus M2 mRNA levels at 6 h: 21% ± 10%; M1 mRNA levels at 11 h: 8% ± 5%; M1 plus M2 mRNA levels at 11 h: 9% ± 2%). These results indicate that the promoter mutation inserted in the M gene mostly affected M RNA transcription and that a reduction in mRNA levels was at least partly responsible for the decrease in M1 and M2 protein levels.
The kinetics of protein accumulation during infection with Mmut virus was different for the M1 and M2 proteins (Table 1). The M1 protein concentration further decreased at later time points compared with those in wild-type virus-infected cells, while the M2 protein concentration slightly increased at later time points, although it stayed at levels lower than those of the wild type. The lower levels of M1 at later time points might be explained by M1 depletion from infected cells during Mmut virus budding, which could differentially change the NP/M1 ratio in the infected cells between wild-type and Mmut virus. The M2 protein is known to be incorporated into virions at low efficiency, and therefore its relative amount with respect to the NP protein is likely not affected by depletion. Nevertheless, we cannot exclude other possible explanations. For example, the mutations inserted in the M gene may have differentially affected the splicing rate of the M transcripts at early and late times postinfection, therefore explaining the different ratios between M1 and M2 proteins at different times postinfection. In any case, it appears that the inserted mutations were responsible for changes in M1 and M2 protein expression levels relative to those found in cells infected with wild-type virus.
The viral proteins NEP, NP, and M1 have been shown to regulate the export of vRNPs from the nucleus to the cytoplasm at late times of infection (10, 32, 36). Low levels of M1 expression in Mmut virus-infected cells might then result in a delayed export of vRNPs. To visualize vRNP localization, we infected MDCK cells with wild-type and Mmut viruses at an MOI of approximately 1. At different times postinfection, cells were fixed in phosphate-buffered saline containing 3% formaldehyde for 10 min, permeabilized for an additional 5 min with 0.5% Triton X-100, and incubated with a 1:500 dilution of rabbit antisera against NP, followed by a 1:250 dilution of Texas Red-conjugated donkey anti-rabbit antibody from Jackson Immunoresearch Laboratories. The location of the NP (as indicative of vRNPs) was subsequently determined by using a Leica confocal microscope (Fig. 2E). Mmut virus-infected MDCK cells showed slightly higher levels of nuclear NP at 4 h postinfection than did wild-type virus-infected cells, suggesting a delayed nucleocytoplasmic export of vRNPs in Mmut virus-infected cells. However, by 6 h postinfection, no major differences were detected between wild-type and Mmut virus-infected cells with respect to NP localization, which was mainly cytoplasmic, indicating that most of the vRNPs were already exported from the nucleus to the cytoplasm at this time of infection. Similar results were obtained at 8 h postinfection (data not shown).
Since the Mmut virus expresses wild-type M1 and M2 proteins at significantly reduced levels in virus-infected cells, this virus provided us with the unique opportunity to analyze the effects of specific reduction in M1 and M2 levels on virion assembly and morphology. We performed large-scale purification of Mmut viruses to analyze the protein and RNA composition of the particles. MDBK cells were grown in 150-mm tissue culture dishes and infected at an MOI of 0.01 with Mmut virus (15 dishes) and WSN virus (three dishes). Two days later viruses present in the supernatants were purified according to a previously described protocol (16). Mmut and WSN virions were resuspended in 100 μl and 50 μl, respectively, of NTE (NaCl, 100 mM; Tris, 10 mM; EDTA, 1 mM; pH 7.4), to obtain preparations of approximately 2 μg of protein/μl. To obtain a better resolution of the NP, HA1, and NA bands which migrate closely on SDS-polyacrylamide gel electrophoresis (PAGE), 3 μl of virions was treated with 2 μl of PNGase F (New England Biolabs, Inc.) to remove N-linked carbohydrate chains. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (Fig. 3A). There was no noticeable difference in NP, M1, HA1, and NA levels between Mmut and WSN viruses. We confirmed this observation by Western blot detection of viral proteins (Fig. 3B). Blots were first developed with E10 and anti-NP monoclonal antibodies, as described above. The membranes were then stripped and reincubated with rabbit polyclonal antiserum against WSN virus followed by donkey anti-rabbit antibody conjugated with horseradish peroxidase (Amersham Life Sciences) to detect the HA. The intensities of bands obtained using different amounts of WSN virions were compared with those produced by Mmut virions, and M1 and M2 protein levels were normalized to NP levels. The levels of M1 and M2 proteins in Mmut virions were expressed as a percentage of the corresponding proteins in WSN virions (Table 1). The amount of M1 protein incorporated into Mmut virions was similar to that in wild-type virus. The M2 content in Mmut virions was only slightly decreased (70%). In order to compare the levels of the eight different vRNAs in wild-type and Mmut virions, RNAs were extracted from purified viruses, separated by polyacrylamide gel electrophoresis, and visualized by silver staining as previously described (12, 16, 31). The results shown in Fig. 3C indicate that there are no differences in the incorporation of vRNAs into virus particles between Mmut and WSN viruses. Apparently, the two point mutations introduced into noncoding regions of the M segment did not affect its packaging signals.
Next, we looked at the morphology of Mmut virus particles. MDCK cells were infected at the MOI of 1 for 24 h, and virions were purified from supernatants and negatively stained with 1% ammonium phosphotungstate according to a previously described protocol (4). The Mmut virion preparation consisted of a homogeneous population of small spherical particles similar to the WSN virions (Fig. 3D).
Our results demonstrate that a dramatic reduction in the levels of the M1 and M2 proteins in influenza A virus-infected cells results in reduced virus replication but does not significantly affect the composition and morphology of the virus particles. This is in contrast to the NA protein, whose levels in virions have been shown to correlate with those in infected cells. Thus, an influenza A virus expressing low levels of NA in infected cells also contains low levels of NA in the virions (14). Previous studies demonstrate that the M1 protein plays a crucial role in the assembly of influenza A virus. The M1 protein is a major structural component of the virus particle (15, 43); associates with vRNPs (49, 51), membranes (27, 42, 54), and HA and NA glycoproteins (1, 11); and modulates virus particle morphology (4, 9) and budding (17, 21). In the present study we show that, despite reduced levels of M1 protein in Mmut virus-infected cell, virus particles contain amounts of M1 similar to those of wild-type viruses. We therefore hypothesize that a threshold amount of M1 protein is needed for virus particle assembly and that this process is delayed when the amount of M1 protein is limited, as in the case of the Mmut virus. Interestingly, this M1 function can nevertheless be affected by specific mutations in basic amino acids that affect its levels of incorporation into virus particles (30).
Delayed replication of Mmut virus is therefore likely to reflect a delay in viral assembly. In fact, reduction of M1 expression during influenza virus infection by specific small interfering RNA silencing has recently been shown to result in reduced virus budding (22). Delayed replication might also be attributed to a delay in vRNP export, since the M1 protein has been proposed to participate in the export of vRNPs from the nucleus to the cytoplasm at late times postinfection (32). We performed immunostaining of NP antigens in virus-infected MDCK cells and detected a delay in the export of NP out of the nucleus in cells infected with Mmut virus. This observation is consistent with the previously described role of M1 protein in facilitating nuclear export of vRNPs (32).
The mechanism of incorporation of the M2 protein into virus particles is an interesting question because this protein is expressed at high levels in infected cells but it is present in virions at very low levels (28, 53). The M2 protein is a transmembrane ion channel that is required for the acidification of the inside of the virus particles during viral entry, facilitating uncoating (20, 38, 45, 47). In our studies, we found that a significant reduction in the levels of M2 protein in Mmut virus-infected cells results in only a slight reduction of M2 protein in virus particles, suggesting that this protein is specifically incorporated at low levels into virions. This is likely mediated by the ectodomain of the protein (37). Influenza B virus has recently been found to express also a protein with ion channel activity, the BM2 protein (34). Interestingly, when a recombinant influenza B virus was generated with a mutation in the initiation codon of the BM2 open reading frame, the levels of the BM2 protein were reduced in both infected cells and virus particles (23, 24). This indicates that, although the M2 of influenza A virus and the BM2 of influenza B virus are likely to play similar roles during viral entry and uncoating, their mechanisms of incorporation into virions are different.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (A.G.-S.). Microscopy was performed at the MSSM-Microscopy Shared Resource Facility, supported, in part, with funding from an NIH-NCI shared resources grant (R24 CA095823).
We express our appreciation to Valerie Williams and Paul Carman for assistance with electron and confocal microscopy and to Stuart Sealfon and Bernard Lin for their invaluable help with the quantitative RT-PCR quantifications. We thank Galina Grishina for help with quantification of Western blots. We also thank Richard Cádagan for excellent technical assistance.
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ABSTRACT
We generated a recombinant influenza A virus (Mmut) that produced low levels of matrix (M1) and M2 proteins in infected cells. Mmut virus propagated to significantly lower titers than did wild-type virus in cells infected at low multiplicity. By contrast, virion morphology and incorporation of viral proteins and vRNAs into virus particles were similar to those of wild-type virus. We propose that a threshold amount of M1 protein is needed for the assembly of viral components into an infectious particle and that budding is delayed in Mmut virus-infected cells until sufficient levels of M1 protein accumulate at the plasma membrane.
TEXT
Influenza A virus is an enveloped virus of the Orthomyxoviridae family with a segmented negative-strand RNA genome. Its eight viral RNA (vRNA) segments encode the three subunits of the viral RNA polymerase complex, PB1, PB2, and PA; the nucleoprotein NP; the glycoproteins HA and NA; the matrix protein M1; the ion channel protein M2; the nonstructural protein NS1; the nuclear export protein NEP; and the recently discovered facilitator of apoptosis PB1-F2 (7). The M1 protein is a major structural component of the virus particles and forms a layer underneath the lipid cell-derived envelope (43, 44). Inside the virions and in infected cells at late stages of the virus replication, the M1 protein associates with the viral ribonucleoproteins (vRNPs), which are composed of viral RNA molecules, multiple copies of the NP, and the three subunits of the viral polymerase holding the ends of the viral RNAs (3, 41, 51). Transcription and replication of the influenza virus genome take place in the nucleus of infected cells. By contrast, assembly of virus particles occurs at the apical surface of the plasma membrane where the M1 protein presumably interacts on one side with the cytoplasmic tails of the glycoproteins anchored in the membrane and on the other side with the vRNPs. Although a direct interaction of the M1 protein with the viral glycoproteins was not demonstrated, expression of HA and NA proteins stimulates membrane association of M1 protein (11). Moreover, a mutant virus with deleted cytoplasmic tails at both HA and NA proteins shows severe defects in particle morphology, genome packaging, and incorporation of NA and M1 into virions (25, 55).
Previous works show that the M1 protein plays a pivotal role in influenza A virus budding. Mutation of certain residues in the M1 protein dramatically affects the morphology of the virus particles (4, 9). Expression of the M1 protein in mammalian cells results in budding of virus-like particles (17). The M1 protein possesses a specific amino acid sequence that appears to be similar in function to the late budding motifs of the matrix proteins of retroviruses (21). A key role of matrix proteins in virus assembly and budding has also been demonstrated for other enveloped negative-strand RNA viruses. Recombinant rabies and measles viruses lacking their M genes produce significantly reduced virus titers in infected cells and release particles with altered morphology (6, 33). The M proteins from vesicular stomatitis and human parainfluenza type 1 viruses induce budding of vesicles from the plasma membrane when they are expressed alone (8, 26, 29).
In addition to the M1 protein, the M gene of influenza virus also encodes the ion channel transmembrane protein M2 (38, 47). Although the M2 protein is not essential for infectivity (48, 50), this protein appears to be needed for efficient vRNP uncoating during viral entry (18), and mutant M2 influenza viruses are highly attenuated. The M2 protein might also play a role in virus assembly and budding and, like the M1 protein, also appears to regulate virus morphology (40).
In the present study we investigated whether a reduction in the levels of M1 and M2 proteins synthesized in virus-infected cells affects virus particle assembly. We used plasmid-based reverse genetics to introduce a double mutation, C-GA-U (11-12') into the conserved region of the vRNA promoter of the M segment of influenza A/WSN/33 virus (Fig. 1) (5, 13, 19, 35). The same mutation was found to be responsible for a reduction in the levels of mRNA and protein expression when introduced into the NA, PA, and NS segments of recombinant viruses (5, 14, 46). For this purpose, the M RNA segment was PCR amplified using as template pPOLI-M plasmid (13) and primers 5'-GATCGCTCTTCGGCCAGCGAAAGCATGTAGATATTGAAAGATG-3' (positive sense; M1/M2 start codon isunderlined; inserted promoter mutation is in boldface) and 5'-GATCGCTCTTCTATTAGTAGAAACAATGTAGTTTTTTACTCC-3' (negative sense; M2 stop codon is underlined; inserted promoter mutation is in boldface). The PCR product was digested with the SapI restriction enzyme and ligated into pPOLI-SapI-RT vector (39). The resultant plasmid was used to transfect 293T/MDCK cell cocultures along with pPOLI-PB1, -PB2, -PA, -HA, -NP, -NA, and -NS (13) and pCAGGS-PB1, -PB2, -PA, and -NP plasmids (2). 293T and MDCK cells were seeded into 35-mm dishes at the proportion 5:1 and grown to approximately 70% of confluency in Dulbecco modified Eagle medium (DMEM)-10% fetal bovine serum medium. Before transfection, the medium was replaced with Opti-MEM I (Invitrogen). TransIT-LT1 transfection reagent (Mirus, Madison, WI) was used according to manufacturer's instructions. Briefly, 9 μl of TransIT-LT1 was diluted in 100 μl of Opti-MEM I medium and mixed with 1.5 μg of total plasmid DNA (containing equal amounts of each of the 12 plasmids) also diluted in 20 μl of Opti-MEM I. The DNA-TransIT complexes were allowed to form for 15 min at room temperature and were added drop by drop to the cells. Medium was replaced with DMEM-0.1% fetal bovine serum-0.35% bovine serum albumin 18 h later. Three days later supernatants were used to infect MDCK cells. Four days later, the rescue of Mmut mutant virus was confirmed by hemagglutination and plaque assays. The presence of the promoter mutations in the M segment was confirmed by sequencing of reverse transcription-PCR (RT-PCR) products derived from viral RNA. It should be noted that the 3' mutation generated an ATG in the corresponding mRNA (Fig. 1), and although this new ATG is in a bad Kozak context, it has the potential to affect translation.
The growth properties of the Mmut virus were analyzed in MDCK cells infected at low and high multiplicities of infection (MOI) (Fig. 2A and B). At different time points infectious particles present in the medium were titrated by plaque assay in MDCK cells. The Mmut virus replicated to titers approximately 2 logs lower than those of WSN virus when cells were infected at an MOI of 0.001. When cells were infected at an MOI of 1, the Mmut virus reached a maximum titer similar to that of wild-type virus but exhibited a slight decrease in growth kinetics at 4 and 6 h postinfection. It is likely that the small differences in replication between wild-type and Mmut viruses in single-cycle replication experiments (Fig. 2B) are exponentially increased during multicycle replication (Fig. 2A).
Synthesis of viral proteins in wild-type WSN and Mmut virus-infected MDCK cells was analyzed after infection at an MOI of 1. For this purpose the medium of the infected cells was replaced with DMEM supplemented with 75 μCi/ml of EXPRE35S35S protein labeling mix (Perkin-Elmer Life and Analytical Sciences, Boston, MA) at 2, 4, 6, and 8 h postinfection. Two hours later cell monolayers were washed with phosphate-buffered saline and lysed in sodium dodecyl sulfate (SDS) loading buffer. Proteins were separated on 16.5% Tris-Tricine Criterion SDS-polyacrylamide gels (Bio-Rad) and visualized by autoradiography (Fig. 2C). The levels of M1 protein synthesis in Mmut-infected cells were significantly reduced at all time points compared to those in wild-type WSN virus-infected cells. To determine differences between wild-type and Mmut viruses in the steady-state levels of M1 and M2 proteins in infected cells, we performed Western blot assays using the same cell lysates and monoclonal antibody (E10) that recognizes both M1 and M2 proteins (Fig. 2D). Sheep anti-mouse antibodies conjugated with horseradish peroxidase (Amersham Life Sciences) were used as the secondary antibodies. At all time points the levels of M1 and M2 proteins in Mmut-infected cells were significantly lower than those in cells infected with WSN virus. To quantify the differences, we used different dilutions of the lysates and developed Western blots with E10 and anti-NP monoclonal antibodies (data not shown). The intensities of bands were measured with the Gel Doc 2000 (Bio-Rad) by using Quantity One quantification software. Levels of M1 and M2 proteins in Mmut virus-infected cells were normalized to NP protein and expressed as a percentage of the corresponding proteins in wild-type virus-infected cells (Table 1). The levels of the M1 and M2 proteins in Mmut virus-infected cells were reduced approximately 20 and 10 times, respectively, compared with those in wild-type virus-infected cells. This reduction is likely to be due to a combination of reduced transcription levels from the promoter-mutated M gene and reduced translation levels of M1 and M2 proteins due to the generation of an ATG start codon (followed by a stop codon) in front of the ATG of the M1 and M2 open reading frames by the mutation inserted at the 3' end of the viral RNA, as already described for the NA gene (14).
In order to confirm that the inserted promoter mutation decreased the levels of transcription of the Mmut RNA, we quantified levels of M-specific vRNA (as indicative of replication) and of M-specific mRNA (as indicative of transcription) in virus-infected cells. MDCK cell monolayers in 60-mm dishes were infected at an MOI of 1 with wild-type and Mmut viruses. At 6 h and 11 h postinfection, cells were lysed in 2 ml of Trizol, and total RNA was extracted according to the manufacturer's instructions (Invitrogen). One μg of RNA was used for quantitative RT-PCR according to a previously published protocol for SYBR Green in an ABI7900 HT instrument (52). For mRNA quantification, RNA was reverse transcribed using a poly(T) primer. For M vRNA quantification, RNA was reverse transcribed using a primer annealing to the first nine nucleotides of the M vRNA. For NP vRNA quantification, RNA was reverse transcribed using a primer annealing to the first 12 nucleotides of the NP vRNA. cDNAs were amplified using internal M- or NP-specific primers, and the relative amount of M-specific vRNA and mRNA in Mmut virus-infected cells compared to wild-type virus-infected cells was calculated after normalization to NP vRNA and mRNA levels. The sequence of the specific primers used in the PCRs is available upon request. Different sets of M-specific primers were designed to specifically amplify M1 mRNA or to amplify both M1 and M2 mRNAs. Each transcript in each sample was assayed three times, and the median threshold cycle was used to calculate the experimental values for each RNA. We quantified RNA levels from duplicate samples. At 6 h postinfection, levels of M vRNA were similar between Mmut (114% ± 26%) and wild-type virus. At 11 h postinfection there was a slight decrease in the levels of Mmut vRNA (50% ± 18% of wild-type levels). By contrast, M mRNA levels were significantly reduced in Mmut virus-infected cells (M1 mRNA levels at 6 h: 23% ± 10%; M1 plus M2 mRNA levels at 6 h: 21% ± 10%; M1 mRNA levels at 11 h: 8% ± 5%; M1 plus M2 mRNA levels at 11 h: 9% ± 2%). These results indicate that the promoter mutation inserted in the M gene mostly affected M RNA transcription and that a reduction in mRNA levels was at least partly responsible for the decrease in M1 and M2 protein levels.
The kinetics of protein accumulation during infection with Mmut virus was different for the M1 and M2 proteins (Table 1). The M1 protein concentration further decreased at later time points compared with those in wild-type virus-infected cells, while the M2 protein concentration slightly increased at later time points, although it stayed at levels lower than those of the wild type. The lower levels of M1 at later time points might be explained by M1 depletion from infected cells during Mmut virus budding, which could differentially change the NP/M1 ratio in the infected cells between wild-type and Mmut virus. The M2 protein is known to be incorporated into virions at low efficiency, and therefore its relative amount with respect to the NP protein is likely not affected by depletion. Nevertheless, we cannot exclude other possible explanations. For example, the mutations inserted in the M gene may have differentially affected the splicing rate of the M transcripts at early and late times postinfection, therefore explaining the different ratios between M1 and M2 proteins at different times postinfection. In any case, it appears that the inserted mutations were responsible for changes in M1 and M2 protein expression levels relative to those found in cells infected with wild-type virus.
The viral proteins NEP, NP, and M1 have been shown to regulate the export of vRNPs from the nucleus to the cytoplasm at late times of infection (10, 32, 36). Low levels of M1 expression in Mmut virus-infected cells might then result in a delayed export of vRNPs. To visualize vRNP localization, we infected MDCK cells with wild-type and Mmut viruses at an MOI of approximately 1. At different times postinfection, cells were fixed in phosphate-buffered saline containing 3% formaldehyde for 10 min, permeabilized for an additional 5 min with 0.5% Triton X-100, and incubated with a 1:500 dilution of rabbit antisera against NP, followed by a 1:250 dilution of Texas Red-conjugated donkey anti-rabbit antibody from Jackson Immunoresearch Laboratories. The location of the NP (as indicative of vRNPs) was subsequently determined by using a Leica confocal microscope (Fig. 2E). Mmut virus-infected MDCK cells showed slightly higher levels of nuclear NP at 4 h postinfection than did wild-type virus-infected cells, suggesting a delayed nucleocytoplasmic export of vRNPs in Mmut virus-infected cells. However, by 6 h postinfection, no major differences were detected between wild-type and Mmut virus-infected cells with respect to NP localization, which was mainly cytoplasmic, indicating that most of the vRNPs were already exported from the nucleus to the cytoplasm at this time of infection. Similar results were obtained at 8 h postinfection (data not shown).
Since the Mmut virus expresses wild-type M1 and M2 proteins at significantly reduced levels in virus-infected cells, this virus provided us with the unique opportunity to analyze the effects of specific reduction in M1 and M2 levels on virion assembly and morphology. We performed large-scale purification of Mmut viruses to analyze the protein and RNA composition of the particles. MDBK cells were grown in 150-mm tissue culture dishes and infected at an MOI of 0.01 with Mmut virus (15 dishes) and WSN virus (three dishes). Two days later viruses present in the supernatants were purified according to a previously described protocol (16). Mmut and WSN virions were resuspended in 100 μl and 50 μl, respectively, of NTE (NaCl, 100 mM; Tris, 10 mM; EDTA, 1 mM; pH 7.4), to obtain preparations of approximately 2 μg of protein/μl. To obtain a better resolution of the NP, HA1, and NA bands which migrate closely on SDS-polyacrylamide gel electrophoresis (PAGE), 3 μl of virions was treated with 2 μl of PNGase F (New England Biolabs, Inc.) to remove N-linked carbohydrate chains. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (Fig. 3A). There was no noticeable difference in NP, M1, HA1, and NA levels between Mmut and WSN viruses. We confirmed this observation by Western blot detection of viral proteins (Fig. 3B). Blots were first developed with E10 and anti-NP monoclonal antibodies, as described above. The membranes were then stripped and reincubated with rabbit polyclonal antiserum against WSN virus followed by donkey anti-rabbit antibody conjugated with horseradish peroxidase (Amersham Life Sciences) to detect the HA. The intensities of bands obtained using different amounts of WSN virions were compared with those produced by Mmut virions, and M1 and M2 protein levels were normalized to NP levels. The levels of M1 and M2 proteins in Mmut virions were expressed as a percentage of the corresponding proteins in WSN virions (Table 1). The amount of M1 protein incorporated into Mmut virions was similar to that in wild-type virus. The M2 content in Mmut virions was only slightly decreased (70%). In order to compare the levels of the eight different vRNAs in wild-type and Mmut virions, RNAs were extracted from purified viruses, separated by polyacrylamide gel electrophoresis, and visualized by silver staining as previously described (12, 16, 31). The results shown in Fig. 3C indicate that there are no differences in the incorporation of vRNAs into virus particles between Mmut and WSN viruses. Apparently, the two point mutations introduced into noncoding regions of the M segment did not affect its packaging signals.
Next, we looked at the morphology of Mmut virus particles. MDCK cells were infected at the MOI of 1 for 24 h, and virions were purified from supernatants and negatively stained with 1% ammonium phosphotungstate according to a previously described protocol (4). The Mmut virion preparation consisted of a homogeneous population of small spherical particles similar to the WSN virions (Fig. 3D).
Our results demonstrate that a dramatic reduction in the levels of the M1 and M2 proteins in influenza A virus-infected cells results in reduced virus replication but does not significantly affect the composition and morphology of the virus particles. This is in contrast to the NA protein, whose levels in virions have been shown to correlate with those in infected cells. Thus, an influenza A virus expressing low levels of NA in infected cells also contains low levels of NA in the virions (14). Previous studies demonstrate that the M1 protein plays a crucial role in the assembly of influenza A virus. The M1 protein is a major structural component of the virus particle (15, 43); associates with vRNPs (49, 51), membranes (27, 42, 54), and HA and NA glycoproteins (1, 11); and modulates virus particle morphology (4, 9) and budding (17, 21). In the present study we show that, despite reduced levels of M1 protein in Mmut virus-infected cell, virus particles contain amounts of M1 similar to those of wild-type viruses. We therefore hypothesize that a threshold amount of M1 protein is needed for virus particle assembly and that this process is delayed when the amount of M1 protein is limited, as in the case of the Mmut virus. Interestingly, this M1 function can nevertheless be affected by specific mutations in basic amino acids that affect its levels of incorporation into virus particles (30).
Delayed replication of Mmut virus is therefore likely to reflect a delay in viral assembly. In fact, reduction of M1 expression during influenza virus infection by specific small interfering RNA silencing has recently been shown to result in reduced virus budding (22). Delayed replication might also be attributed to a delay in vRNP export, since the M1 protein has been proposed to participate in the export of vRNPs from the nucleus to the cytoplasm at late times postinfection (32). We performed immunostaining of NP antigens in virus-infected MDCK cells and detected a delay in the export of NP out of the nucleus in cells infected with Mmut virus. This observation is consistent with the previously described role of M1 protein in facilitating nuclear export of vRNPs (32).
The mechanism of incorporation of the M2 protein into virus particles is an interesting question because this protein is expressed at high levels in infected cells but it is present in virions at very low levels (28, 53). The M2 protein is a transmembrane ion channel that is required for the acidification of the inside of the virus particles during viral entry, facilitating uncoating (20, 38, 45, 47). In our studies, we found that a significant reduction in the levels of M2 protein in Mmut virus-infected cells results in only a slight reduction of M2 protein in virus particles, suggesting that this protein is specifically incorporated at low levels into virions. This is likely mediated by the ectodomain of the protein (37). Influenza B virus has recently been found to express also a protein with ion channel activity, the BM2 protein (34). Interestingly, when a recombinant influenza B virus was generated with a mutation in the initiation codon of the BM2 open reading frame, the levels of the BM2 protein were reduced in both infected cells and virus particles (23, 24). This indicates that, although the M2 of influenza A virus and the BM2 of influenza B virus are likely to play similar roles during viral entry and uncoating, their mechanisms of incorporation into virions are different.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (A.G.-S.). Microscopy was performed at the MSSM-Microscopy Shared Resource Facility, supported, in part, with funding from an NIH-NCI shared resources grant (R24 CA095823).
We express our appreciation to Valerie Williams and Paul Carman for assistance with electron and confocal microscopy and to Stuart Sealfon and Bernard Lin for their invaluable help with the quantitative RT-PCR quantifications. We thank Galina Grishina for help with quantification of Western blots. We also thank Richard Cádagan for excellent technical assistance.
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