Mutations at Alternative 5 Splice Sites of M1 mRNA Negatively
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JOURNAL OF VIROLOGY, Nov. 2008, p. 10873–10886 Vol. 82, No. 21
0022-538X/08/$08.00⫹0 doi:10.1128/JVI.00506-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Mutations at Alternative 5⬘ Splice Sites of M1 mRNA Negatively
Affect Influenza A Virus Viability and Growth Rate䌤
Chiayn Chiang,1,4 Guang-Wu Chen,1,2 and Shin-Ru Shih1,3,4*
Research Center for Emerging Viral Infections,1 Department of Computer Science and Information Engineering,2
Department of Medical Biotechnology and Laboratory Science,3 and Graduate Institute of
Biomedical Sciences,4 Chang Gung University, Taoyuan, Taiwan
Received 7 March 2008/Accepted 20 August 2008
Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However,
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the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively
studied. This study focuses on the sequences at alternative 5ⴕ splice sites on M1 mRNA. Six different mutations
at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse
genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the
intron for the mRNA3 5ⴕ splice site), C51G (at the 3ⴕ end of the exon of the M2 mRNA 5ⴕ splice site), and
G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants
with the mutation G11C (at the 3ⴕ end of exon of the mRNA3 5ⴕ splice site), G52C (for the first nucleotide of
the intron for M2 mRNA), or G145A (at the 3ⴕ end of the exon of mRNA4) were rescued, although they had
significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2
proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from
the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells
infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type
recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in
wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant
virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the
alternative 5ⴕ splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.
Influenza A virus is a pathogen of humans, birds, and other In addition to encoding the M1 protein, the M gene of
mammals for respiratory tract infections. Pandemic influenza influenza A virus also encodes the ion channel transmembrane
A virus infection causes high morbidity and mortality and has protein M2 through alternative splicing. The M2 ion channel
major social and economic impacts in the world. The virus is a protein is abundantly expressed in the plasma membrane of
member of the family Orthomyxoviridae and contains eight virus-infected cells but is significantly underrepresented in viri-
segmented, negative-stranded genomic RNAs. These eight ons because of very limited molecules that are incorporated
segments together encode viral RNA polymerase complex into virus particles (5, 22, 43, 44). M2 protein is likely needed
members PB2, PB1, and PA; glycoprotein hemagglutinin for efficient vRNP uncoating during viral entry (17), and M2
(HA); nucleoprotein (NP); neuraminidase (NA); matrix pro- mutant influenza viruses are extremely attenuated (10, 40).
tein (M1); ion channel protein (M2); nonstructural proteins Other functions of M2 include virus assembly and budding
NS1 and NS2; and an alternatively translated protein, PB1-F2 (32).
(9). The M1 matrix protein is a major structural component of During cellular mRNA maturation, introns are removed
the virus particle and forms a layer beneath the lipid cell- precisely and flanking exons are ligated. Alternative splicing of
derived envelope. Inside the virion and in infected cells during precursor mRNAs is one of the most important mechanisms
the late stages of virus replication, the M1 protein associates for introducing protein diversity in eukaryotes. Influenza A
with viral ribonucleoproteins (vRNPs) (3, 4, 39) and plays an virus M1 mRNA is colinear with viral RNA, whereas M2
important role in influenza A virus budding (19, 20, 28). Ex- mRNA is encoded by an alternative spliced transcript (23, 24,
pression of M1 protein in mammalian cells results in budding 37, 41). M1 mRNA contains three alternative 5⬘ splice sites (5⬘
of virus-like particles (16). The protein contains a specific ss): a 5⬘ ss at position 12, which produces mRNA3; a 5⬘ ss at
amino acid sequence whose function resembles the function of position 52, which produces M2 mRNA; and a 5⬘ ss at position
the late domain of retrovirus matrix proteins regarding virus 146, which produces mRNA4 (1, 23, 24, 38). Although mRNA3
budding, which evidences the role of M1 in virus budding (20). and M2 mRNA are seen in all influenza A virus strains,
The mutation of certain residues in M1 protein markedly in- mRNA4 exists only in some strains, such as A/WSN/33 (38).
fluences the morphology of virus particles (6, 8, 12). The sequences of the mRNA3 5⬘ ss CAG/GUAGAU (the slash
without accompanying parentheses around the two relevant
nucleotides indicates the exon/intron boundary) and the
* Corresponding author. Mailing address: Research Center for mRNA4 5⬘ ss GAG/GUUCUC resemble that of the consensus
Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa 1st
Road, Kwei-Shan, Taoyuan, Taiwan. Phone: 886-3-2118800, ext. 5497.
5⬘ ss AAG/GUAAGU closely. The M2 mRNA 5⬘ ss sequence
Fax: 886-3-2118174. E-mail: srshih@mail.cgu.edu.tw. AAC/GUA(U/C)GU, however, does not fit well with the con-
䌤
Published ahead of print on 3 September 2008. sensus because of a C rather than a G at the 3⬘ end of the 5⬘
1087310874 CHIANG ET AL. J. VIROL.
exon. Alternative 5⬘ splicing of influenza A virus M1 mRNA is 21 of NP cRNA), and 5⬘-AGCAAAAGCAGGGTGACAAA-3⬘ (corresponding
controlled by viral polymerase and cellular splicing factors (36, to nt 1 to 20 of NS cRNA) and the KOD-plus kit (Toyobo). PCR products were
then purified with a gel extraction kit (Qiagen) and sequenced. Sequencing of the
37). Early during infection, the distal 5⬘ ss is used to produce 5⬘ and 3⬘ ends of the M vRNA was performed by rapid amplification of cDNA
mRNA3. At a relatively late stage in infection in cells, newly ends. For the sequencing of the 3⬘ end of M vRNA, purified M vRNA was first
synthesized polymerases bind to the virus mRNA 5⬘ end that transcribed by using primer 5⬘-AGCAAAAGCAGGTAGATATTGAAAGV
encompasses the first 11 or 12 nucleotides of the 5⬘ terminus, N-3⬘ (the underlined sequence is complementary to nt 1003 to 1027 of the M
vRNA). The reverse-transcribed cDNA products were amplified by using primer
thereby blocking the mRNA3 5⬘ ss located at position 12 of the
5⬘-GGATGGGGGCTGTGACCACTGAAGTGGC-3⬘ (complementary to nt
M1 mRNA (36, 37). Moreover, the cellular SF2/ASF splicing 575 to 602 of the M vRNA) and the Super SMART PCR cDNA synthesis kit
factors interact with the 3⬘ exon of the M1 mRNA and enhance (Clontech) by following the manufacturer’s instructions. For the sequencing of
activation of the M2 mRNA 5⬘ ss (36). the 5⬘ end of M vRNA, purified M vRNA was transcribed by using primer
Since only M1 mRNA and M2 mRNA encode two func- 5⬘-AAGCAGTGGTATCAACGCAGAGTACAGCAAAAGCAVN-3⬘ (the un-
derlined sequence is complementary to nt 1018 to 1027 of the M vRNA). The
tional viral M1 and M2 proteins, which are important for viral reverse-transcribed products were amplified by using primer 5⬘-GCCACTTCA
growth, several interesting questions arise. (i) Why does the GTGGTCACAGCCCCCATCC-3⬘ (corresponding to nt 575 to 602 of the M
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mRNA3 5⬘ ss exist and why has it been preserved during vRNA) and Super SMART PCR cDNA synthesis kit (Clontech) by following the
evolution? (ii) Why is the M2 mRNA 5⬘ ss weaker than manufacturer’s instructions. PCR products were then purified with a gel extrac-
mRNA3 as a signal for splicing? (iii) As the mRNA4 5⬘ ss tion kit (Qiagen) and sequenced.
Plaque assay. Confluent MDCK cells in 35-mm dishes were washed with
exists only in certain strains of influenza A viruses, how does it phosphate-buffered saline, and serial dilutions of the virus were adsorbed onto
affect the viability for these strains? Experimental results ob- cells for 1 h at 37°C. Unadsorbed virus was removed by washing with phosphate-
tained from a number of earlier studies may provide some buffered saline, and cells were overlaid with 3 ml of overlay Dulbecco’s modified
answers (10, 36, 38). The present study introduced a series of Eagle’s medium supplemented with 3% agarose. After incubation for 72 h at
35°C, cells were fixed with 10% formalin for 1 h. Following formalin removal,
mutations at alternative 5⬘ ss of M1 mRNA. By analyzing viral
cells were stained with crystal violet and plaques were visualized. Visible plaques
viability and growth rates of the recombinant viruses generated were counted, and concentrations of PFU/ml were determined. The plaque
by reverse genetics with 12 plasmids, we determined whether numbers and sizes were obtained from at least three independent experiments.
these mutations were lethal or whether they would reduce viral Western blotting. Transfected cells were lysed in lysis buffer (0.6 M KCl, 50
growth rates. The findings may provide a clue as to why specific mM Tris-HCl [pH 7.5], 0.5% Triton X-100) at 72 h posttransfection. Cell lysates
were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
alternative 5⬘ ss signatures in influenza virus genomes are evo- and transferred to a polyvinylidene difluoride membrane (Amersham Bio-
lutionarily preserved. sciences). The transfer membrane was first blocked for 1 h at room temperature
with Tris-buffered saline–Tween (TBS-T) containing 5% skim milk, followed by
either overnight incubation with 14C2 mouse anti-M2 immunoglobulin G (IgG)
MATERIALS AND METHODS monoclonal antibody (Ab; Affinity Bioreagents) (43) (diluted 1:1,000) or 1D6
Cell culture. Madin-Darby canine kidney (MDCK) cells and 293T human anti-M2 cytoplasmic tail monoclonal Ab (diluted 1:100) (kindly provided by
embryonic kidney cells were maintained in Dulbecco’s modified Eagle’s medium Robert A. Lamb) at 4°C or incubation with anti-M1 IgG monoclonal Ab (diluted
(Gibco) supplemented with 10% fetal bovine serum (Gibco). All cells were 1:500) (Biodesign International) for 2 h at room temperature. The membrane
maintained at 37°C under conditions of 5% CO2. was washed three times with TBS-T and then incubated for 1 h at room tem-
Construction of a mutant with a mutation at an alternative 5ⴕ ss. Mutation perature with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-
constructs were introduced into pPOLI-M-RT by using a QuikChange kit (Strat- mouse IgG Ab. Signals were detected by using the Western immunoblot ECL
agene). Sequence analysis confirmed that only the specifically introduced muta- detection system (Amersham Biosciences) and exposed to X-ray film (Kodak).
tions were present in the plasmids. RNA extraction and reverse transcription-PCR (RT-PCR). Total RNA from
Plasmid-based reverse genetics. Mutant viruses (A/WSN/33 strain) were gen- transfected 293T cells transfected via reverse genetics with 12 plasmids or virus-
erated using the 12-plasmid-based reverse genetics system described by Fodor et infected MDCK cells were collected using an RNeasy mini kit (Qiagen) following
al. (15). Plasmids were kindly provided by George G. Brownlee. For virus rescue, the manufacturer’s instructions. The cDNA was made using a SuperScript II
106 293T cells were cotransfected with four protein expression plasmids reverse transcription kit (Invitrogen) by following the manufacturer’s instruc-
(pcDNA-PB2, pcDNA-PB1, pcDNA-PA, and pcDNA-NP) and eight viral RNA tions. The specific primers for viral M mRNAs and M vRNA were described
(vRNA) transcription plasmids (pPOLI-PB2-RT, pPOLI-PB1-RT, pPOLI-PA- previously by Cheung et al. (10). Primers for mRNA4 were sense primer 5⬘-G
RT, pPOLI-HA-RT, pPOLI-NP-RT, pPOLI-NA-RT, pPOLI-M-RT, and AACACCGATCTTGAGGCCTAT-3⬘ (the underlined sequence corresponds to
pPOLI-NS-RT) by using Lipofectamine 2000 (Invitrogen). After 24 h, the trans- nt 130 to 145 of the M cRNA; the italicized sequence corresponds to nt 740 to
fection medium was removed from the cells and replaced with fresh medium 744 of M cRNA) and antisense primer 5⬘-CTGTTCCTTTCGATATTCTTC-3⬘
containing 0.5% fetal bovine serum, penicillin, and streptomycin. The trans- (corresponding to nt 95 to 75 of the M vRNA). Primers for NP vRNA were
fected cells were maintained for 2 to 3 days after transfection. The medium from described previously by Liang et al. (26). Amplified products were further ana-
transfected cells was collected daily and assayed for the presence of the influenza lyzed by 2% agarose gel electrophoresis.
virus by attempting to create plaques with a 0.5-ml aliquot on MDCK cells by Real-time quantitative PCR. The specific primers used for M1 mRNA, M2
using standard methods. The remaining medium was transferred into 25-cm2 mRNA, and mRNA3 were described previously by Cheung et al. (10). The
flasks containing subconfluent MDCK cells for amplification of any rescued specific primers for mRNA4 are described in the previous paragraph. Values
virus. The rescued virus showed a specific property characteristic of the influenza were normalized with the -actin mRNA level. The primers for -actin mRNA
A/WSN/33 virus (i.e., it formed plaques on MDCK cells in the absence of were sense primer 5⬘-GCTCGTCGTCGACAACGGCTC-3⬘ and antisense
trypsin). The plaques formed by the rescued virus were comparable in size to primer 5⬘-CAAACATGATCCTGGGTCATCTTCTC-3⬘. The PCR amplifica-
those formed by an authentic wild-type influenza A/WSN/33 virus grown on the tion yielded a product of 352 bp. The cDNA was amplified using Sybr green
same MDCK cells. real-time PCR master mix (Bio-Rad), 5 mM of each primer and 0.5 l of the
Sequencing of recombinant viruses. M, NP, and NS vRNA from transfected or cDNA product in a total volume of 50 l. Forty cycles of PCR (one cycle consists
infected supernatants was reverse transcribed by using primers 5⬘-AGTAGAA of 10 min at 95°C, 15 s at 95°C, and 1 min at 60°C) were performed using the
ACAAGGTAGTTTTT-3⬘ (corresponding to nucleotides [nt] 1007 to 1027 of M Bio-Rad iQ5 system. A melting curve analysis was performed to verify the
cRNA), 5⬘-AGTAGAAACAAGGGTATTTTT-3⬘ (corresponding to nt 1545 to specificity of the products; the relative values were calculated using the ⌬⌬CT
1565 of NP cRNA), and 5⬘-AGTAGAAACAAGGGTGTTTT-3⬘ (corresponding method. Each experiment was performed in triplicate.
to nt 871 to 890 of NS cRNA) and SuperScript II reverse transcriptase (Invitro- Primer extension assay. Primer extension reactions were performed by using
gen). The reverse-transcribed cDNA was amplified by PCR by using specific the primer extension system–AMV reverse transcriptase kit (Promega) by fol-
primers 5⬘-AGCAAAAGCAGGTAGATATT-3⬘ (corresponding to nt 1 to 20 of lowing the manufacturer’s instructions. Briefly, 5 g total RNA was mixed with
M cRNA), 5⬘-AGCAAAAGCAGGGTAGATAA-3⬘ (corresponding to nt 1 to M vRNA-specific 32P-labeled primer and positive-sense RNA-specific 32P-la-VOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10875
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FIG. 1. Structures of M1 mRNA in influenza A virus and its three alternative spliced mRNA products, namely, mRNA3, M2 mRNA, and
mRNA4, of influenza virus A/WSN/33. Solid squares at the 5⬘ end represent the 5⬘ cap from host cells. Gray, hatched, and white rectangles
represent coding regions. Bold lines at the 5⬘ and 3⬘ ends of the mRNAs represent noncoding regions. The N terminus of the M2 protein and the
segment of mRNA4 protein corresponding to the coding region each overlap that of M1 protein by 9 residues and 40 residues, respectively. The
C terminus of the mRNA4 protein segment corresponding to the coding region overlaps that of M1 protein by 14 residues. Italic letters represent
the introns of the splice site sequences. aa, amino acids.
beled primer described by Mullin et al. (27). Primer extensions were performed spliced mRNAs, including M2 mRNA, mRNA3, and mRNA4,
at 42°C for 90 min. Transcription productions were denatured at 90°C for 10 min, were detected in the wild type and in the G11C mutant (Fig.
separated on 6% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA
buffer, and detected by autoradiography.
2B, lanes 2, 6, 10, and 14 and lanes 3, 7, 11, and 15, respec-
tively). However, for the G12C mutant, M1 mRNA was barely
detected and M2 mRNA, mRNA3 and mRNA4 were not
RESULTS found (Fig. 2B, lanes 4, 8, 12, and 16). Expression levels of the
Genomic signatures for mRNA3 5ⴕ ss are required for M M1 and M2 proteins in the G11C mutant were moderately
gene synthesis. The mRNA3 5⬘ ss is located at nt 11 and 12 of lower than those in wild-type-transfected cells (Fig. 2C, com-
M1 mRNA, which can produce mRNA3 with only a coding pare lanes 1 and 2). Neither M1 nor M2 protein was detected
potential of nine amino acids (Fig. 1). The sequences AG/GU in the 12-plasmid-transfected 293T cells in which the M gene
of mRNA3 5⬘ ss in all influenza A viruses were analyzed and had the G12C mutation (Fig. 2C, lane 3). The expression level
were highly conserved without variation. To determine the of NP was used as the control for transfection efficiency, and
impact of the conserved mRNA3 5⬘ ss on influenza A viruses, the actin level served a loading control (Fig. 2C, lower panels).
mutations at this splice site were introduced and recombinant When the sequence at the mRNA3 5⬘ ss was changed,
viruses were generated by using reverse genetics with 12 plas- change in the promoter sequence in 3⬘ end of M vRNA was
mids. Nucleotide changes were made at 3⬘ end of the 5⬘ exon also introduced accordingly. It has been reported that positions
(position 11) or intron donor site (position 12) of the M1 11 and 12 are within the promoter region of the 3⬘ end of
mRNA (Fig. 2A). The plasmid carrying mutated M vRNA and vRNA and the 5⬘ end of cRNA (Fig. 3A). We hypothesized
the remaining seven genomic vRNA expression plasmids were that G11C and G12C would decrease M vRNA synthesis and
cotransfected with PB1, PB2, PA, and NP protein expression reduce the expression of M1 mRNA, which consequently affect
plasmids into 293T cells. The G-to-C substitution was made at the production of M1 and M2 proteins. To test this hypothesis,
position 11 (G11C) such that the mRNA3 5⬘ ss sequence primer extension and RT-PCR were conducted to detect M
AC/GU did not fit the consensus splicing sequence AG/GU gene synthesis. Significant decreases in the M vRNA, M1
(wild type). The resulting mutant (the G11C mutant) was res- mRNA, and M cRNA levels were seen for the G11C mutant
cued, although it had a reduced growth rate (Fig. 2A; see Fig. (Fig. 3B, lane 3, and C, lane 3), and little M vRNA was
7A). The G-to-C substitution was also made at position 12 detected in the G12C mutant (Fig. 3B, lane 4, and C, lane 4).
(G12C) to knock out the splicing at the mRNA3 5⬘ ss. The Neither M1 mRNA nor M cRNA was detected in the G12C
G12C mutant could not be rescued (Fig. 2A). These results mutant (Fig. 3B, lane 4). However, due to the superior sensi-
indicate that mutations at the alternative 5⬘ splice junctions for tivity of the RT-PCR assay, the weak band for M1 mRNA
mRNA3 affect the production of the influenza A virus. Neither remained detectable in the G12C mutant (Fig. 2B, lane 4). The
the G11C mutation nor the G12C mutation alters any amino level of NP vRNA, the control, did not change in the G11C or
acid, because positions 11 and 12 are not within the regions G12C mutant (Fig. 3C, right panel, lanes 6 to 8). These results,
coding for M1 and M2 proteins (Table 1). therefore, suggest that the G-to-C substitutions at positions 11
This study further investigated M1 mRNA splicing in the and 12 of the M gene affect promoter activity during M gene
293T cells transfected with the indicated 12 plasmids. The pro- synthesis.
duction of the spliced products and expression levels of the M1 The conserved 3⬘ and 5⬘ terminal nucleotides on vRNA form
and M2 proteins were examined. The M1 mRNA and all a corkscrew secondary structure through partially complemen-10876 CHIANG ET AL. J. VIROL.
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FIG. 2. (A) Mutations introduced into mRNA3 5⬘ ss at positions 11 and 12 and virus rescue of these mutants. Underlined nucleotides are the
ones changed in the plasmid used to generate mutant virus. Viruses rescued by reverse genetics, those not rescued, and those rescued yet with a
reduced growth rate are described by ⫹, ⫺, and ⫹*, respectively. (B) Detection of M1 mRNA splicing from mRNA3 5⬘ ss mutants 72 h after
transfection of 293T cells with 12 plasmids via reverse genetics. The upper panels show the relative proportions of M1 mRNA, M2 mRNA,
mRNA3, and mRNA4 of G11C and G12C mutants in comparison with those of the wild type as detected by real-time RT-PCR. The lower panels
show the results of RT-PCR (sizes listed in bp). The RNAs were 123 (M1 mRNA), 276 (M2 mRNA), 204 (mRNA3), and 230 (mRNA4) bp long.
(C) Expression of M1 and M2 proteins from mRNA3 5⬘ ss mutants 72 h after transfection of 293T cells with 12 plasmids via reverse genetics. The
M1 and M2 proteins were detected by mouse anti-M1 Ab and 14C2 mouse anti-M2 Ab, respectively.
tary base-pairing, and this is required for the promoter activity end of M cRNA of G11C and G12C, were made to restructure
(7, 13, 14). The cRNA promoter is complementary to the the base-pairing of the promoter regions (Fig. 4A). While the
vRNA promoter and structures a corkscrew base-pairing be- newly made G11C-CM12 mutant still presented a growth rate
tween the 5⬘ nt 10⬘ to 12⬘ and the 3⬘ nt 11 to 13 (Fig. 4A) (2). as low as that of the G11C mutant, the new G12C-CM13
Two mRNA3 5⬘ ss mutations at position 11 (G11C) and 12 mutant was successfully rescued, in contrast to the lethal G12C
(G12C) of M1 mRNA, as seen here, inevitably disrupted the mutant (Fig. 4A). Significant levels of M vRNA, M1 mRNA,
potential base-pairing in M cRNA promoter (Fig. 4A). To and M cRNA were restored in G12C-CM13 (Fig. 4B, lane 4).
avoid the structure disruption, two compensatory mutations, G11C-CM12, on the other hand, did not recover the M RNA
G11C-CM12 and G12C-CM13, at positions 12 and 13 in the 3⬘ synthesis (Fig. 4B, lane 3). We further examined the splicing byVOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10877
TABLE 1. Mutations introduced into alternative 5⬘ splice junctions and virus rescue of these mutants
Recombinant virus Nucleotide changea Amino acid change Virus
mutation mRNA3 5⬘ ss M2 mRNA5 ss mRNA4 5⬘ ss M1 M2 rescueb
None (wild type) CAG/GUAGAU AAC/GUACGU GAG/GUUCUC ⫹
G11C CAC/GUAGAU ⫹
G12C CAG/CUAGAU ⫺
C51G AAG/GUACGU Thr9Arg Thr9Arg ⫺
G52C AAC/CUACGU ⫹
G145A GAA/GUUCUC ⫹
G146C GAG/CUUCUC Val41Leu ⫺
a
The underlined letters indicate the nucleotides changed in the plasmid used to generate mutant virus.
b
⫹, virus was capable of being rescued by reverse genetics; ⫺, virus was incapable of being rescued by reverse genetics.
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real-time RT-PCR and found that M1 mRNA level of G11C- The G12C-CM13 mutant, on the other hand, produced levels
CM12 was decreased (Fig. 4B, lane 3), followed by the reduc- of M1 mRNA, M2 mRNA, and mRNA4 (Fig. 4C, lanes 4, 8,
tion of spliced M2 mRNA, mRNA3, and mRNA4 (Fig. 4C, and 16), but not mRNA3 (Fig. 4C, lane 12), that were compa-
lanes 3, 7, 11, and 15). In terms of protein synthesis levels, M1 rable to those of the wild type. Expression of the M1 and M2
and M2 of G11C-CM12 were also decreased (Fig. 4D, lane 2). proteins were barely affected by this mutation (Fig. 4D, lane 3).
FIG. 3. Effect of mutations in mRNA3 5⬘ ss on M gene synthesis. (A) Schematic diagram of M cRNA, M vRNA, and M1 mRNA synthesized
during viral transcription and replication in infected cells. Underlined letters represent the conserved nucleotides at the 5⬘ and 3⬘ ends of the vRNA
and cRNA promoter regions. The gray rectangle marks the mRNA3 5⬘ splice junction at positions 11 and 12 of the M1 mRNA, and the open
rectangle represents the coding region. The cap structure and the 10 to 13 heterologous nucleotides at the 5⬘ end of M1 mRNA are derived from
host cells. (B) M gene synthesis was analyzed by a primer extension assay. Size standards (in bp) of the 32P-labeled DNA ladder are shown in lane
1. Positions of M vRNA, M1 mRNA, and M cRNA signals are indicted on the right. (C) The upper panels show the relative proportions of the
M vRNA and NP vRNA detected by real-time RT-PCR. The lower panels show the results by RT-PCR. The size of the M vRNA was 123 bp and
that of the NP vRNA was 161 bp.Downloaded from http://jvi.asm.org/ on December 24, 2020 by guest
FIG. 4. (A) Influenza virus M cRNA promoter shown in a corkscrew structure. The gray rectangle marks the mRNA3 5⬘ splice junction at
positions 11 and 12 of the M1 mRNA, which correspond to the nt 11⬘ and 12 pair and the nt 12⬘ and 13 pair of M cRNA promoter. The prime
notation (⬘) is used to identify nucleotides of the 5⬘ end of the promoter. Underlined letters represent the nucleotides mutated in each of these
four mutants. Viruses rescued by reverse genetics, those not rescued, and those rescued yet with a reduced growth rate are labeled with ⫹, ⫺, and
⫹*, respectively. (B) Effect of compensatory mutations in the M cRNA promoter of G11C and G12C mutants. RNA was analyzed by a primer
extension assay. Size standards (in bp) of the 32P-labeled DNA ladder are shown in lane 1. Positions of M vRNA, M1 mRNA, and M cRNA signals
are indicted on the right. (C) Detection of M1 mRNA splicing from G11C-CM12 and G12C-CM13 mutants at 72 h after transfection of 293T cells
with 12 plasmids via reverse genetics. The upper panels show the relative proportions of M1 mRNA, M2 mRNA, mRNA3, and mRNA4 of
G11C-CM12 and G12C-CM13 mutants in comparison with those of wild type as detected by real-time RT-PCR. The lower panels show the results
by RT-PCR. They were 123 (M1 mRNA), 276 (M2 mRNA), 204 (mRNA3), and 230 (mRNA4) bp long. (D) Expression of M1 and M2 proteins
from G11C-CM12 and G12C-CM13 mutants at 72 h after transfection of 293T cells with 12 plasmids via reverse genetics.
10878VOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10879
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FIG. 5. (A) Mutations introduced into M2 mRNA 5⬘ ss at positions 51 and 52 and the rescue of these mutants. Underlined letters are the
nucleotides changed in the plasmid used to generate these mutant viruses. Viruses rescued by reverse genetics, those not rescued, and those
rescued yet with a reduced growth rate are described by ⫹, ⫺, and ⫹*, respectively. The gray rectangle marks the nucleotides that encode the ninth
amino acid in M1 and M2 proteins. (B) Detection of M1 mRNA splicing from M2 mRNA 5⬘ ss mutants at 72 h after transfection of 293T cells
with 12 plasmids via reverse genetics. The upper panels show the relative proportions of M1 mRNA, M2 mRNA, mRNA3, and mRNA4 of C51G
and G52C mutants in comparison with those of the wild type as detected by real-time RT-PCR. The lower panels shows the result by RT-PCR
(sizes listed in bp). They were 123 (M1 mRNA), 276 (M2 mRNA), 204 (mRNA3), and 230 (mRNA4) bp long. (C) Expression of M1 and M2
proteins from M2 mRNA 5⬘ ss mutants at 72 h after transfection of 293T cells with 12 plasmids via reverse genetics. The M1 and M2 proteins
were each detected by mouse anti-M1 Ab and 1D6 mouse anti-M2 cytoplasmic tail Ab, respectively. Anti-M2 1D6 Ab (against C terminus
of M2 protein) was used for Western blotting to reduce the interference of Ab affinity caused by amino acid substitution near the N terminus
of the M2 protein.
These results indicate that conserved genomic signatures for influenza A virus was studied. The C-to-G substitution at the 3⬘
mRNA3 5⬘ ss among all influenza A viruses are required for end of the 5⬘ exon (position 51) of M2 mRNA (C51G) was
the formation of the structure of promoter region, which is made to increase the strength of the 5⬘ ss. The resulting mutant
essential for M gene synthesis. (C51G mutant) could not be rescued (Fig. 5A). The M1
A weak splice site for M2 mRNA is essential for efficient mRNA level was reduced moderately in 12-plasmid-trans-
influenza A virus growth. After the importance of mRNA3 5⬘ fected cells in which the C51G mutation was made in the M
ss was determined, the impact of the M2 mRNA 5⬘ ss on the gene (Fig. 5B, lane 3). The amount of M2 mRNA in C51G was10880 CHIANG ET AL. J. VIROL.
twofold higher than that in the wild type (Fig. 5B, lane 7). TABLE 2. Influenza A viruses containing an mRNA4 alternative 5⬘ ss
However, mRNA3 and mRNA4 splicing levels were signifi- Sequence from
Virus strain Accession no.
cantly decreased in the C51G mutant (Fig. 5B, lanes 11 and 15) nt 143 to 151
in comparison with those of the wild type (lanes 10 and 14). A/chicken/Pennsylvania/1/1983 CY015074 GAG/GUACUU
Production levels of M2 protein in the wild type and the C51G (H5N2)
mutant were comparable (Fig. 5C, lanes 1 and 2). However, A/chicken/Pennsylvania/1370/1983 CY015109 GAG/GUGCUU
(H5N2)
the expression level of the M1 protein was markedly reduced in A/fowl/Rostock/45/1934 (H7N1) CY015047 GAG/GUUCUC
the C51G mutant (Fig. 5C, lane 2). A/chicken/FPV/Weybridge M23917 GAG/GUUCUC
(H7N7)
The mutation at position 51 caused the ninth amino acid to A/FPV/Weybridge (H7N7) L37797 GAG/GUUCUC
change from Thr to Arg in both M1 and M2 proteins (Fig. 5A). A/FPV/Rostock/1934 (H7N1) M55474 and M55475 GAG/GUUCUC
A/turkey/North Carolina/12344/03 AY779257 GAG/GUUCUC
Whether or not this change would jeopardize the viability of (H3N2)
recombinant virus was also examined. One A50C C51G mu- A/turkey/Minnesota/764-2/03 AY779258 GAG/GUUCUC
tant, which corresponds to a T9R mutation, and an A50G (H3N2)
A/WSN/33 (H1N1) L25818, M19374, GAG/GUUCUC
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mutant, which corresponds to a T9A mutation, were made and M23920, and
tested. While the former recombinant virus was lethal, the X08088
A/Puerto Rico/8/34 (H1N1) CY009445, V01099, GAG/GUUCUC
latter was rescued, yet it had a reduced growth rate (Fig. 5A). AF389121, and
It seems that Thr at position 9 is essential for virus survival with EF467824
an adequate growth rate. This may explain why the M2 mRNA A/NWS/1933 (H1N1) L25814 GAG/GUUCUC
A/Wilson-Smith/1933 (H1N1) DQ508908 GAG/GUUCUC
5⬘ ss exists in such a sequence combination (AAC/G, a weak 5⬘ A/Alaska/1935 (H1N1) CY019956 GAG/GUUCUC
ss) so that it can encode Thr9. A/Victoria/1968 (H3N2) CY015509 GAG/GUUCUC
A/Philippines/2/82 (H3N2) AF348913 GAG/GUUCUC
On the other hand, the G-to-C substitution was made at A/Port Chalmers/1/1973-mouse X08090 GAG/GUUCUC
position 52 (the first nucleotide of the intron) to knock out the adapted (H3N2)
A/Taiwan/1769/96 (H1N1) AF138710 GAG/GUUCUC
splicing for the production of M2 mRNA. The resulting re- A/swine/Iowa/15/1930 (H1N1) M33045 GAG/GUUCUC
combinant virus was rescued but had a growth rate that was A/swine/Korea/S10/2004 (H1N1) AY790268 GAG/GUUCUC
A/swine/Korea/S81/2004 (H9N2) AY790276 GAG/GUUCUC
lower (by approximately 2 logs) than that of wild type (Fig. 5A; A/swine/Korea/S109/2004 (H9N2) AY790321 GAG/GUUCUC
see Fig. 7A). As expected, M2 mRNA splicing was impaired by A/swine/Korea/S190/2004 (H9N2) AY790298 GAG/GUUCUC
the mutation of G at the donor site of the M2 mRNA intron
(Fig. 5B, lane 8). The G52C mutant did not express the M2
protein (Fig. 5C, lane 3) in the 12-plasmid-transfected cells.
These experimental results have also been obtained by Cheung tuted for G at position 145 and C was substituted for G at
et al. (10). position 146 (Fig. 6B, lanes 15 and 16). The M1 and M2
mRNA4 5ⴕ ss is important for the growth of A/WSN/33. protein expression levels in G145A and G146C mutant virus-
After investigating mRNA3 and M2 mRNA 5⬘ ss, we further transfected cells were lower than those in wild-type-transfected
turned our attention to the downstream mRNA4 5⬘ ss. Unlike cells (Fig. 6C). In order to know whether other mutations at
the highly conserved sequences in mRNA3 and M2 mRNA 5⬘ position 146 would lead to lethal phenotypes, we made two
ss, the mRNA4 5⬘ ss sequences differed among influenza A additional mutants of A/WSN/33 M1 mRNA. The G146A mu-
viruses (38). All of the influenza A virus M gene sequences (up tant (corresponding to an amino acid change from V to I) was
to 19 January 2008) were analyzed. Twenty-two strains out of able to be rescued, yet it had a low growth rate. G146U (re-
6,192 strains contained the potential mRNA4 5⬘ ss (Table 2). sulting in an amino acid change from V to F), on the other
These strains belong to different subtypes and were isolated hand, was a lethal mutation (Fig. 6A). The results indicate that
from various hosts, including humans, birds, and swine. Val at position 41 of M1 protein is important for virus survival
Many of these strains listed in Table 2, like A/chicken/FPV/ and efficient growth.
Weybridge (H7N7) and A/FPV/Weybridge (H7N7), A/fowl/ Attenuated growth characteristics of influenza A viruses
Rostock/45/1934 (H7N1) and A/FPV/Rostock/1934 (H7N1), with mutations at alternative 5ⴕ ss of M1 mRNA. Table 1
and A/WSN/33 (H1N1), A/NWS/1933 (H1N1) and A/Wilson- summarizes the effects of mutations at the alternative 5⬘ ss for
Smith/1933 (H1N1), represent different isolates yet are closely mRNA3, M2 mRNA, and mRNA4 on production of progeny
related to each other. To determine whether the mRNA4 5⬘ ss influenza A viruses. Notably, G12C, C51G, and G146C are
plays a role in viral growth, we introduced two mutations at this lethal mutations, whereas mutants with G11C, G52C, and
5⬘ ss. The G-to-A substitution at position 145 (the 3⬘ end of the G145A can be rescued. The stabilities of the introduced mu-
5⬘ exon) was made to weaken the 5⬘ ss. The resulting mutant tations were examined by sequencing of the full length of the
(the G145A mutant) was rescued, although it had a low growth M gene segment. These recombinant viruses were found to be
rate (Fig. 6A; see Fig. 7A). When G was replaced by C at stable for at least 10 passages and were not reverted to the wild
position 146 (intron donor site) to knock out the production of type. We also did not observe any nucleotide substitution.
mRNA4, the recombinant virus (G146C) was not rescued (Fig. Sequences of other gene segments, including NP and NS
6A). The same results were also obtained for A/Puerto Rico/ genes, that may associate with M gene were also examined and
8/34 (data not shown). found no mutations (data not shown). This study further ex-
Together these data suggest that the existence of mRNA4 5⬘ amined the growth properties of rescued recombinant viruses
ss in certain strains of influenza A viruses, such as A/WSN/33 in MDCK cells. Cells were infected with recombinant viruses
and A/Puerto Rico/8/34, may affect viral growth. In addition, at a multiplicity of infection of 0.001; the viruses yielded in the
the mRNA4 levels were strongly reduced when A was substi- culture supernatant at various times were determined byVOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10881
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FIG. 6. (A) Mutations introduced into mRNA4 5⬘ ss at positions 145 and 146 and rescue of these mutants. Underlined letters are nucleotides
changed in the plasmid used to generate these mutant viruses. Viruses rescued by reverse genetics, those not rescued, and those rescued yet with
a reduced growth rate are described by ⫹, ⫺, and ⫹*, respectively. The gray rectangle marks the nucleotides that encode amino acid 41 in M1
protein. (B) Detection of M1 mRNA splicing from mRNA4 5⬘ ss mutants at 72 h after transfection of 293T cells with 12 plasmids via reverse
genetics. The upper panels show the relative proportions of M1 mRNA, M2 mRNA, mRNA3, and mRNA4 of G145A and G146C mutants in
comparison with those of the wild type as detected by real-time RT-PCR. The lower panels show the results by RT-PCR (sizes listed in bp). They
were 123 (M1 mRNA), 276 (M2 mRNA), 204 (mRNA3), and 230 (mRNA4) bp long. (C) Expression of M1 and M2 proteins from mRNA4 5⬘ ss
mutants at 72 h after transfection of 293T cells with 12 plasmids via reverse genetics. The M1 and M2 proteins were detected by mouse anti-M1
Ab and 14C2 mouse anti-M2 Ab, respectively.
plaque assay. The results demonstrate that G11C, G52C, and wild-type recombinant virus (Fig. 7A). These results demonstrate
G145A mutant viruses grew significantly more slowly than the wild- that variations in M1 mRNA 5⬘ ss attenuated influenza A virus
type virus did (Fig. 7A). The maximum titers of G11C, G145A, and growth. Notably, these mutations did not change any amino acid
G52C viruses were approximately 1 to 2 logs lower than that of the sequence in the encoded virus proteins (Table 1).10882 CHIANG ET AL. J. VIROL.
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FIG. 7. Growth curves and plaque morphology of mutant and wild-type viruses. (A) MDCK cells were infected at a multiplicity of infection of
0.001 with mutant and wild-type viruses. At indicated time points, titers of infectious particles present in the culture medium were determined by
plaque assay. Three independent experiments were performed. (B) Plaque morphology of mutant and wild-type viruses in MDCK cells at 72 h
postinfection.
The plaque diameter of the G52C recombinant virus after the efficiency of wild-type virus. The M2 mRNA was not
72 h postinfection was approximately 0.5 mm, four times produced in the G52C mutant (the M2 mRNA 5⬘ ss knock-
smaller than that of the wild-type, G11C, and G145A recom- out mutation). On the other hand, when the mRNA4 5⬘ ss
binant viruses (approximately 2 mm in diameter at 72 h postin- was weakened, the production of M2 mRNA also decreased
fection) (Fig. 7B). These data are consistent with the findings approximately 2.3-fold at 24 h (17.49% versus 7.66%; Stu-
that M2 protein expression is not required for the growth of dent’s t test, P ⫽ 0.016) and approximately 1.7-fold at 36 h
the influenza A virus in cell cultures and that influenza A virus (25.5% versus 15.27%; Student’s t test, P ⫽ 0.036) postin-
can undergo multiple cycles of replication without M2 ion fection in comparison to wild-type M2 mRNA production.
channel activity (10, 40). Expression levels of the M1 and M2 proteins at 12, 24, and
Disturbance of M splicing and protein expression in the 36 h after infection of MDCK cells were determined. As time
recombinant mutant virus-infected cells. After demonstrat- increased, the levels of M1 and M2 in wild-type virus- and
ing that growth of G11C, G52C, and G145A mutant recom- mutant-infected cells increased (Fig. 8B). The expression lev-
binant viruses was attenuated by mutations at alternative 5⬘ els of M1 protein in G52C and G145A mutants (Fig. 8B, lanes
ss of M1 mRNA, this study then examined the splicing of M1 8 and 9 and lanes 11 and 12, respectively) were lower than
mRNA and expression levels of M1 and M2 proteins in those in wild-type virus (lanes 2 and 3) at 24 and 36 h after
G11C, G52C, and G145A mutant virus-infected cells. Quan- infection of MDCK cells. Although the G11C mutant did
titative real-time RT-PCR using specific primers for each not change the expression of M1 protein levels, the expres-
spliced mRNA was performed. The amount of mRNA3 in sion of M2 protein was reduced slightly (Fig. 8B, lanes 5 and
wild-type infected cells at 12 h postinfection was approxi- 6). As expected, no M2 protein was detected in the G52
mately 10-fold higher than that at 24 h (7.73% versus 0.77%; mutant virus-infected cells (Fig. 8B, lanes 8 and 9). Further-
Student’s t test, P ⫽ 0.0003) and approximately 8-fold higher more, expression of M2 proteins was significantly decreased
than that at 36 h (7.73% versus 1%; Student’s t test, P ⫽ in the G145A mutant (lanes 11 and 12). Taken together, the
0.0004) postinfection (Fig. 8A). In contrast, as time in- results suggest that mutations in the alternative 5⬘ ss of M1
creased, the amount of M2 mRNA in wild-type virus in- mRNA affect the production of M2 mRNA and the protein
creased. When the mRNA3 5⬘ ss was weakened (via the it encodes.
G-to-C mutation at position 11), splicing efficiency of the
producing M2 mRNA increased approximately 1.3-fold at DISCUSSION
24 h (23.09% versus 17.49%; Student’s t test, P ⫽ 0.0011)
and approximately 1.3-fold at 36 h (32.99% versus 25.5%; The influenza virus harbors an enormous genomic diversity
Student’s t test, P ⫽ 0.0029) postinfection in comparison to because of gene reassortment or accumulation of point muta-VOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10883
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FIG. 8. (A) Relative proportions of M1 mRNA, M2 mRNA, mRNA3, and mRNA4 derived from G11C, G52C, and G145A mutants and
wild-type virus at 12, 24, and 36 h postinfection (hr). The amounts of RNA were quantified by real-time RT-PCR. Relative proportions of M2
mRNAs of recombinant viruses at 12, 24, and 36 h postinfection are shown. The relative proportions of mRNA3 for the wild type at 12, 24, and
36 h postinfection are also shown. (B) Detection of M1 and M2 proteins in MDCK cells infected by G11C, G52C, and G145A mutants and
wild-type virus at 12, 24, and 36 h postinfection (hpi).
tions. Many studies have revealed that sequence variations in weaker signal than mRNA3 is for splicing? (iii) As mRNA4 5⬘
the coding region change virus phenotypes. However, the im- ss exists only in certain strains of influenza A viruses, how does
pact of sequence variation in noncoding regions or splice sites it affect the viability of these strains? By introducing mutations
has not been extensively studied. This work clearly demon- into the splice site junctions and examining M RNA synthesis,
strated that the mutations at alternative 5⬘ ss of M1 mRNA splicing, and protein expression in detail, we revealed clues to
were lethal or attenuated the growth rate for influenza A virus. answer these questions. We firstly observed that the mRNA3
These findings may explain why these splicing signatures must 5⬘ ss has been preserved because the splice site signature G/G
be preserved throughout viral evolution. at positions 11 and 12 of M1 mRNA corresponds to CC at the
Why do additional splice sites in the M gene of influenza A 3⬘ end of vRNA, which is important to form a promoter struc-
viruses exist despite the fact that only M1 mRNA and M2 ture needed for efficient viral RNA synthesis. Secondly, our
mRNA encode two functional viral M1 and M2 proteins? Spe- mutation analysis shows that changing the M2 mRNA 5⬘ ss into
cifically, we asked three questions in the beginning of this a stronger one would inevitably prevent the M2 protein residue
study. (i) Why does the mRNA3 5⬘ ss exist and why has it been at position 9 from being coding as Thr, which would adversely
preserved during evolution? (ii) Why is M2 mRNA 5⬘ ss a affect the virus viability and growth rate. This explains why the10884 CHIANG ET AL. J. VIROL.
M2 mRNA 5⬘ ss has to stay in a weaker form than mRNA3 (10) and Watanabe et al. (40), who showed that M2 protein
does. Lastly, for mRNA4 5⬘ ss, we noticed that the signature G expression in cell culture was not required for the growth of
as the first intron nucleotide for mRNA4 is responsible for influenza A virus. Interestingly, the mutant with the C51G
encoding Val at position 41 of M1 protein, and this might be mutation (which enhances the weak M2 mRNA 5⬘ ss) with an
important for virus survival and efficient growth. This could increased amount of produced M2 protein was not active in
explain why the mRNA4 5⬘ ss is there. There is, however, viral growth (Fig, 5A). Unlike the abundant M2 protein in the
another novel reason for the existence of the mRNA4 5⬘ ss. We C51G mutant, M1 protein was expressed only in a fairly small
found that the mutation at position 145 (the last nucleotide of amount (Fig. 5C). Strong M2 mRNA 5⬘ ss may end up with
the 5⬘ exon for mRNA4) did not change any amino acid in M1 extremely efficient splicing of M1 mRNA, resulting in only a
or M2 protein but did attenuate the viral growth rate (Fig. 7A). tiny amount of precursor left (Fig. 5B). Without sufficient M1
This attenuation might have been a result from the disturbance protein, the virus may not assemble very effectively. Addition-
of M mRNA splicing (Fig. 8). ally, the C51G mutation changed the amino acid sequence
It has been proposed that the mRNA3 5⬘ ss is spliced early from Thr to Arg at the ninth position of M1 and M2 proteins
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in infection to ensure that the M2 protein is expressed when (Table 1). Previous reports indicate failure to generate a series
needed (36, 37). Splicing of the M2 mRNA 5⬘ ss occurs late in of recombinant viruses with mutations at the dinucleotide GU
infection, when alternative splicing occurring at mRNA3 5⬘ ss of the 5⬘ exon of M2 mRNA 5⬘ ss, because the 9th or 10th
is blocked by the binding of the viral polymerase complex at amino acid of the M1 protein involved in lipid membrane
the first 11 or 12 nucleotides of the 5⬘ end of M1 mRNA (37). binding in the process of virus replication were altered by those
Two mRNA3 5⬘ ss mutants, with mutation G11C (which weak- mutations (10, 35). The ninth amino acid substitution in the
ens the mRNA3 5⬘ ss) or G12C (which knocks out the mRNA3 M1 protein affected rescue of the C51G mutant, a conclusion
5⬘ ss), were generated to affect the production of mRNA3. A in agreement with the result obtained by Cheung et al. (10),
decrease in mRNA3 and an increase in M2 mRNA in these suggesting that the ninth amino acid Thr is essential for pro-
mutants were expected. Increased M2 mRNA levels in G11C duction of influenza A virus.
recombinant virus-infected cells were observed at 24 h and 36 h A/Puerto Rico/8/34 (H1N1) is another influenza A virus that
postinfection (Fig. 8A). Splicing of the mRNA3 knockout mu-
also contains mRNA4 5⬘ ss. We have introduced G145A and
tation (G12C) could not be validated, because precursor M1
G146C mutations into A/Puerto Rico/8/34 through an eight-
mRNA was barely detectable (Fig. 2B, lane 4). The undetect-
plasmid-based reverse genetics system (18). In a result similar
able level of M1 mRNA was possibly due to the impaired M
to the results obtained from WSN, the G145A mutant was
gene synthesis (Fig. 3B and C). Impaired synthesis was likely
rescued but had a low growth rate, and G146C was a lethal
because the sequence signatures for mRNA3 5⬘ ss are also
mutation. This study also demonstrated that a mutation cor-
important signatures for the M vRNA promoter. The terminal
responding to a change of Val to Leu (G146C) at position 41
13 and 12 nucleotides of the 5⬘ and 3⬘ ends, respectively, are
of M1 mRNA was a lethal mutation for the A/WSN/33 influ-
highly conserved in eight influenza A virus RNA segments.
enza A virus (Table 1). Amino acid changes at positions 41, 95,
The first 12 to 14 nucleotides at the 3⬘ end of the vRNAs and
and 218 in the M1 protein of A/WSN/33 would result in the
the first 11 to 13 nucleotides at the 3⬘ end of the cRNA
comprise the core promoter region (25, 29–31, 33, 34, 42). A filamentous phenotype (12). Assembly of the G146C mutant is
conventional chloramphenicol acetyltransferase reporter assay affected by the amino acid change at position 41. The knockout
system demonstrated that disruption of base pairs at position mutation in mRNA4 5⬘ ss (G146C) is a nonsynonymous mu-
11 or 12 in the 5⬘ strand of cRNA significantly reduced the tation. Therefore, it is difficult to assess the importance of
vRNA level, indicating that base-pairing between the 5⬘ and 3⬘ mRNA4 5⬘ ss with knockout mutation experiments. However,
ends of the cRNA promoter is essential for viral replication when the last nucleotide of the 5⬘ exon at position 145 was
(11, 21). The mRNA3 5⬘ ss is located at nt 11 and 12 of M1 changed from G to A, this synonymous mutation caused the
mRNA, corresponding to nt 11 and 12 of the M cRNA pro- recombinant virus to have a growth rate lower than that of
moter region (Fig. 3A). Therefore, the G11C and G12C mu- wild-type virus (Fig. 7A), suggesting that the splicing site sig-
tants were expected to disrupt the base-pairing of the cRNA nature is important for the A/WSN/33 virus. The functions of
promoter. Experimental findings demonstrated that the G11C the resulting amino acids in the M1 protein remain unclear.
and G12C mutants reduced the M vRNA level in 12-plasmid- Table 2 lists some key prototype viruses that have been pas-
transfected cells (Fig. 3B and C). These results are consistent saged multiple times and are high-yield viruses, and the exis-
with findings obtained by Crow et al. (11) and Kim et al. (21). tence of mRNA4 5⬘ ss could be one of the reasons promoting
In addition to the role of the mRNA3 5⬘ ss in modulating the such high-yield characteristics. Therefore, it is necessary to
use of the alternative 5⬘ ss of M1 mRNA in infected cells (37), check whether mRNA4 can be translated into protein in virus-
the fact that G at position 12 is within the vRNA promoter infected cells. If it was translated, it would yield a 54-residue
region definitely accounts for why the mRNA3 5⬘ ss sequence polypeptide that has 40 and 14 amino acids that are identical to
is highly conserved in all influenza A viruses. those of the N and C termini of the M1 protein, respectively
The M2 mRNA 5⬘ ss exists for M2 protein production. The (Fig. 1). We have indeed performed the test and did not detect
M2 protein is essential for vRNP uncoating during viral entry this 54-residue peptide in infected cells by using M1 Ab. We
(17). This study demonstrates that the mutant with the G52C have generated polyclonal Abs against those 20 residues at the
mutation (which knocks out the M2 mRNA 5⬘ ss) and lacking C terminus of the putative M4 protein; however, those Abs
the M2 protein grew slowly in MDCK cells (Fig. 7A), a result could not clearly detect any bands potentially representing M4
that is in agreement with the findings obtained by Cheung et al. (by molecular weight) either. Therefore, it remains unclearVOL. 82, 2008 M1 mRNA 5⬘ SPLICE SITES AND VIRUS VIABILITY 10885
whether mRNA4 can be translated to a functional protein in 11. Crow, M., T. Deng, M. Addley, and G. G. Brownlee. 2004. Mutational anal-
ysis of the influenza virus cRNA promoter and identification of nucleotides
infected cells. critical for replication. J. Virol. 78:6263–6270.
Importantly, this study demonstrated growth rates for G11C, 12. Elleman, C. J., and W. S. Barclay. 2004. The M1 matrix protein controls the
G52C, and G145A mutant viruses that were lower than that of filamentous phenotype of influenza A virus. Virology 321:144–153.
13. Flick, R., and G. Hobom. 1999. Interaction of influenza virus polymerase
the wild-type virus (Fig. 7A). Notably, these nucleotide muta- with viral RNA in the ‘corkscrew’ conformation. J. Gen. Virol. 80:2565–2572.
tions did not alter any amino acid sequence in the M1 and M2 14. Flick, R., G. Neumann, E. Hoffmann, E. Neumeier, and G. Hobom. 1996.
proteins. A disturbance in M1 mRNA splicing in those mutant Promoter elements in the influenza vRNA terminal structure. RNA 2:1046–
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recombinant virus-infected cells was observed (Fig. 8A). When 15. Fodor, E., L. Devenish, O. G. Engelhardt, P. Palese, G. G. Brownlee, and A.
the upstream mRNA3 5⬘ ss is weak, M2 mRNA 5⬘ ss can be Garcia-Sastre. 1999. Rescue of influenza A virus from recombinant DNA.
used more efficiently to produce more M2 mRNA; however, J. Virol. 73:9679–9682.
16. Gómez-Puertas, P., C. Albo, E. Perez-Pastrana, A. Vivo, and A. Portela.
when the downstream mRNA4 5⬘ ss is weakened, the utiliza- 2000. Influenza virus matrix protein is the major driving force in virus
tion of M2 mRNA 5⬘ ss also decreased, resulting in a level of budding. J. Virol. 74:11538–11547.
M2 mRNA lower than that of wild-type virus (Fig. 8A). The 17. Helenius, A. 1992. Unpacking the incoming influenza virus. Cell 69:577–578.
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region. The protein expression levels for both M1 and M2 in 19. Hui, E. K., S. Barman, D. H. Tang, B. France, and D. P. Nayak. 2006. YRKL
sequence of influenza virus M1 functions as the L domain motif and interacts
those virus-infected cells were decreased (Fig. 8B). It has been with VPS28 and Cdc42. J. Virol. 80:2291–2308.
reported that low expression levels of M1 and M2 proteins in 20. Hui, E. K., S. Barman, T. Y. Yang, and D. P. Nayak. 2003. Basic residues of
the helix six domain of influenza virus M1 involved in nuclear translocation
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In summary, several mutations were introduced into recom- 21. Kim, H. J., E. Fodor, G. G. Brownlee, and B. L. Seong. 1997. Mutational
analysis of the RNA-fork model of the influenza A virus vRNA promoter in
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ACKNOWLEDGMENTS derived from genome RNA segment 7 of influenza virus: colinear and in-
terrupted mRNAs code for overlapping proteins. Proc. Natl. Acad. Sci. USA
We thank the National Science Council of Taiwan, the Republic of 78:4170–4174.
China (NSC-96-2320-B-182-006) and Chang Gung Memorial Hospital 25. Li, X., and P. Palese. 1992. Mutational analysis of the promoter required for
(CMRPD150162) for financially supporting this research. influenza virus virion RNA synthesis. J. Virol. 66:4331–4338.
George G. Brownlee and Ervin Fodor are appreciated for providing 26. Liang, Y., Y. Hong, and T. G. Parslow. 2005. cis-Acting packaging signals in
the reverse genetics plasmids used in this study, and Robert A. Lamb the influenza virus PB1, PB2, and PA genomic RNA segments. J. Virol.
is appreciated for providing M2 cytoplasmic tail Ab 1D6. 79:10348–10355.
27. Mullin, A. E., R. M. Dalton, M. J. Amorim, D. Elton, and P. Digard. 2004.
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