Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong SAR
Correspondence
L. L. M. Poon
llmpoon{at}hkucc.hku.hk
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ABSTRACT |
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INTRODUCTION |
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The genome of influenza A virus contains eight RNA segments (vRNA) of negative polarity. Each segment associates with the nucleoprotein (NP) and polymerase subunits (i.e. PB2, PB1 and PA) to form a viral ribonucleoprotein (vRNP) complex. Influenza A virus uses a different strategy from eukaryotes for mRNA synthesis. The viral mRNA is generated exclusively by the vRNP (Huang et al., 1990; Fodor et al., 1994
; Poon et al., 1999
; Leahy et al., 2001
). With a genome of less than 14 kb, the virus has developed several strategies to expand its genome-coding capacity (Lamb & Takeda, 2001
). These strategies include translation of unspliced, spliced (NS segment) and alternatively spliced (M segment) mRNAs and bicistronic mRNAs (PB1 segment). Of these eight vRNA segments, the M1 mRNA generated from the M vRNP can further undergo alternative splicing to generate two different spliced viral mRNA molecules (Inglis & Brown, 1981
). The influenza A virus M gene has two alternative 5' splice sites: a proximal 5' splice site (corresponding to nt 5253 of M cRNA), producing M2 mRNA, and a distal 5' splice site (corresponding to nt 1213 of M cRNA), producing M3 mRNA (Shih et al., 1995
). It has been noted that M3 mRNA has almost no coding capacity, whereas M1 and M2 mRNAs encode structural and ion-channel proteins, respectively (Valcarcel et al., 1991
). The choice of alternative splicing sites is suggested to be controlled by the viral polymerase (Shih et al., 1995
) and related to a host splicing factor (Shih & Krug, 1996
).
The splicing of influenza A virus mRNA has been studied via both in vitro and in vivo assays (Lamb & Lai, 1984; Plotch & Krug, 1986
; Agris et al., 1989
; Alonso-Caplen & Krug, 1991
; Valcarcel et al., 1991
, 1993
; Nemeroff et al., 1992
; Shih et al., 1995
; Shih & Krug, 1996
). However, the majority of these studies were based on transcripts that were not synthesized by vRNPs. Sequence analyses of the influenza M and NS genes have shown that the viral introns possess typical features of U2-type splicing sequences, suggesting that the viral introns can be excised by U2-type spliceosomes. In particular, the introns of the M and NS genes contain invariant GU and AG dinucleotides at the 5' and 3' ends, respectively (Lamb & Lai, 1980
). In vitro experiments have shown that small nuclear RNPs can bind to viral-like mRNA transcripts to form U2-type spliceosomes (Agris et al., 1989
). In addition, M mRNA has also been found to contain an enhancer sequence to facilitate the binding of cellular splicing factors (Shih & Krug, 1996
).
In this study, we tried to inhibit M2 protein expression by introducing mutations at the 5'-proximal splicing site of the M gene. In addition, we investigated the effect of mutations on the invariant GU dinucleotide at the 5'-proximal splicing site of the M gene. Rather than using classical in vitro splicing systems or viral-like RNA transcripts in our study, the viral transcripts we studied were generated entirely from recombinant vRNPs by using in vivo systems. We found that mutation of the dinucleotide sequence abolished splicing for M2 mRNA production. Furthermore, recombinant viruses with mutations at this unique sequence were attenuated in cell culture and failed to express M2 ion-channel protein.
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METHODS |
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Construction of M vRNA expression plasmids with mutations at the invariant 5' GU at the 5'-proximal splicing site.
Plasmids pPOLI-M-RT (Fodor et al., 1999, 2002
) and pHW187-M (Hoffmann et al., 2000
) were used to express M vRNA of influenza A/WSN/33 virus. Mutations at the 5'-proximal GU splicing sequence were introduced into these plasmids by inverse PCR techniques and all mutated sequences were confirmed by DNA sequencing.
Expression of M vRNP in transfected cells.
Plasmids pcDNA-PB2, pcDNA-PB1, pcDNA-PA, pcDNA-NP and pPOLI-M-RT were used to express M vRNP of influenza A/WSN/33 virus in transfected cells (Fodor et al., 1999, 2002
). Briefly, 1 µg of each of the plasmids was transfected into 293T cells by using Lipofectamine 2000 (Invitrogen) as instructed by the manufacturer. The medium of transfected cells was replaced by MEM with 10 % FCS and 1 % P/S at 24 h post-transfection.
Generation of recombinant influenza viruses.
To generate recombinant influenza A/WSN/33 virus, eight plasmids (pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M and pHW188-NS) were used in this study (Hoffmann et al., 2000). Transfection experiments were performed as described by Hoffmann et al. (2000)
.
RNA extraction and cDNA synthesis.
Total RNA from transfected or infected cells was harvested by using an RNeasy Mini kit (Qiagen). In a typical reverse-transcription reaction, 3 µg total RNA was reverse-transcribed into cDNA by SuperScript II reverse transcriptase (Invitrogen). For the detection of vRNA, 400 ng vRNA-specific primer complementary to the 3' end of vRNA (Hoffmann et al., 2001) was used in reverse-transcription reactions. By contrast, for the detection of M mRNA molecules, 20 pmol M mRNA-specific primer (5'-TTTTTTTTTTTTTTTACTC-3'; underlined sequence corresponds to nt 2326 of the M vRNA) was used in reverse-transcription reactions.
Quantitative RT-PCR assays for vRNA and mRNA.
For quantification of M vRNA and mRNA, a SYBR green-based real-time PCR method was employed. Real-time PCR experiments were performed by using a LightCycler (Roche). Plasmids containing the target sequence were used as positive controls. Primers for the M vRNA and M1 mRNA were 5'-GACCAATCCTGTCACCTC-3' (corresponding to nt 171188 of the M cRNA) and 5'-GATCTCCGTTCCCATTAAGAG-3' (corresponding to nt 735755 of the M vRNA). For M2 and M3 mRNA detection, intron-spanning primers were used. Primers for the M2 RNA were 5'-GAGGTCGAAACGCCTAT-3' (underlined sequence corresponds to nt 4151 of the M cRNA, italic sequence corresponds to nt 740745 of the M cRNA) and 5'-CTCCAGCTCTATGTTGACAAA-3' (corresponding to nt 2444 of the M vRNA). Primers for the M3 mRNA were 5'-CATAGCAAAAGCAGGCCTA-3' (underlined sequence corresponds to the first 11 nt of the M cRNA, italic sequence corresponds to nt 740744 of the M cRNA) and 5'-CATAGACTCTGGCACTCC-3' (underlined sequence corresponds to nt 102116 of the M vRNA). The amplification program started with one cycle at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 5 s (56 °C for M3 mRNA detection) and 72 °C for 15 s. The specificity of the assay was confirmed by melting-curve analysis at the end of each run (6595 °C, 0·1 °C s1). Amplified products were further analysed by agarose-gel electrophoresis and sequencing.
Indirect immunostaining of M2 protein.
Confluent monolayers of MDCK cells grown on glass coverslips were washed with PBS and infected at an m.o.i. of 1 with influenza A viruses. Infected cells were harvested at 8 h post-infection, washed twice in PBS and fixed in 4 % paraformaldehyde in PBS. Fixed cells were permeabilized with 0·5 % Triton X-100 in PBS for 30 min, followed by two washes in PBS. Cells were then incubated with 14C2 mouse anti-M2 IgG monoclonal antibody (mAb) (diluted 1 : 1000; Affinity Bioreagents) overnight at 4 °C. After two washes in PBS with 0·01 % Triton X-100, cells were incubated with rabbit anti-mouse antibody conjugated with fluorescein isothiocyanate (diluted 1 : 100; Zymed Laboratories) for 1 h at room temperature. Samples were washed twice in PBS containing 0·01 % Triton X-100 and counterstained for 1 min with propidium iodide solution (BD Biosciences Pharmingen) for DNA staining. After two more washes with PBS, coverslips were mounted by using ProLong anti-fading reagent (Molecular Probes). The surface fluorescence of cells was observed with a Zeiss Axiophot Microscope and images were taken by using a Leica CCD camera.
Virus purification and Western blot analysis.
MDCK cells were infected with wild-type or mutant virus at an m.o.i. of 0·01. Virus was harvested at 3 days post-infection and purified by using sucrose gradients as described previously (Poon et al., 2000). Purified virus was lysed in lysis buffer [0·6 M KCl, 50 mM Tris/HCl (pH 7·5), 0·5 % Triton X-100]. Viral lysates were analysed by 12 % SDS-PAGE and transferred to a PVDF membrane (Amersham Biosciences). Transferred membrane was first blocked for 1 h at room temperature with PBS containing 5 % skimmed milk and 0·1 % Tween 20 (Sigma, ICI Americas) followed by overnight incubation with 14C2 mouse anti-M2 IgG mAb (Affinity Bioreagents). The membrane was washed three times with PBS containing 0·1 % Tween 20 and then incubated for 1 h at room temperature with a 1 : 5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Zymed Laboratories). Signals were detected by using the Western Immunoblot ECL Plus detection system, following the manufacturer's instructions (Amersham Biosciences).
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RESULTS |
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From our transfection experiments (Fig. 1), we demonstrated that mutations at the 5'-proximal splicing site inhibited M2 mRNA synthesis. It was therefore of interest to test whether these recombinant viruses also failed to produce M2 mRNA. Two of the mutated viruses (AU and CU) were selected for further analysis. MDCK cells were infected by wild-type or mutants at an m.o.i. of 2. Total RNA from infected cells was harvested at various time points post-infection. The amounts of M1, M2 and M3 mRNA in these samples were quantified by real-time RT-PCR. Consistent with the observations deduced from the transfection experiments, M vRNA (not shown), M1 mRNA and M3 mRNA were detectable in all samples (Fig. 2a
), indicating that these mutated vRNA segments were templates for viral transcription and replication in infected MDCK cells. By contrast, M2 mRNA could only be detected in MDCK cells infected with wild-type virus, but not with the mutants.
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Recombinant viruses with mutations at the 5'-proximal splicing site are attenuated
Viruses with mutations at the 5'-proximal splicing site failed to synthesize spliced M2 mRNA. Although these mutants were able to replicate in cell culture, they could only produce pinpoint plaques when grown on MDCK cells. The plaque sizes of the mutants (0·5 mm diameter) were three to four times smaller than those of the wild-type (Fig. 3a
). To characterize the growth properties of these mutants in detail, MDCK cells were infected with the mutant viruses at an m.o.i. of 0·01. At various time points post-infection, the number of infectious progeny viral particles released into the medium was determined by plaque assay. The maximum viral titres of these mutants were about 2 log units lower than that of the wild-type (Fig. 3b
). In addition, we determined the stability of these mutations by passaging the mutants in MDCK cells 10 times. The desired mutations were all retained, indicating that the introduced mutations were stable in routine cell culture. These results suggested that, at least under cell-culture conditions, M2 protein might not be absolutely essential for virus replication. However, our results also indicated that lack of M2 protein expression severely affected growth of the virus.
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Virus morphology
M2 protein has been reported to be associated with virus morphology (Roberts et al., 1998). We examined the morphology of the splicing mutants by electron microscopy. Both splicing mutants were spherical in shape and morphologically indistinguishable from the wild-type (data not shown). The sizes of the AU (138·83±15·70 nm, n=100) and CU (134·98±14·42 nm, n=100) splicing mutants were not statistically significantly different from that of the wild-type (139·60±20·20 nm, n=100) (P>0·05, Student's t-test).
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DISCUSSION |
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None of the mutants was capable of synthesizing M2 ion-channel protein (Fig. 4a, b). The lack of M2 protein expression in these mutants was validated by transfection experiments (Fig. 4c
). In addition, using a highly sensitive quantitative RT-PCR assay with a detection limit of 10 copies per reaction (data not shown), we failed to detect M2 mRNA from these mutants. Thus, it is highly unlikely that these mutants expressed a low level of M2 protein in infected cells. Although these mutants might have been expected to revert to the wild-type by altering a single nucleotide, they were found to be stable for at least 10 passages.
The p.f.u./HA ratios of our studied viruses were similar (data not shown), suggesting that the lack of M2 expression did not affect the formation of infectious viral particles. Several previous findings have suggested that M2 protein is not absolutely essential for influenza virus. First, recombinant viruses with defective M2 proteins have been generated by two independent groups (Watanabe et al., 2001; Takeda et al., 2002
). In addition, M2 protein has been found not to be required for viral-like particle formation (Mena et al., 1996
), indicating that M2 protein is not essential for the budding process. Here, we further demonstrated that M2 protein expression is not required for influenza virus to grow in cell culture. Interestingly, unlike the M2 truncated mutants described by Watanabe et al. (2001)
, our M2 splicing mutants showed similar growth kinetics to the wild-type. This discrepancy might be partly due to the different viral strains used in the studies. Nonetheless, the recombinant viruses generated by us and others (Takeda et al., 2002
; Watanabe et al., 2002
) were all attenuated in animals or in cell culture, indicating that the M2 protein plays an important role in efficient virus replication.
In this study, we did not investigate the exact mechanism of attenuation of the mutants. The ion-channel activity of the M2 protein is essential for modulating the pH of the trans-Golgi of virus-infected cells and the virion interior. In the early phase of viral infection, the dissociation of vRNPs from M1 protein is triggered by the proton influx from endosomes to virion interiors via the M2 ion channel (Bui et al., 1996). Using ion-channel inhibitors (e.g. amantadine) to block the ion-channel activity could inhibit the dissociation of M1 protein from vRNPs during viral entry (Kemler et al., 1994
). For some influenza virus subtypes, these inhibitors could also induce conformational changes of cleaved HA in the trans-Golgi network (Ciampor et al., 1992
; Takeuchi & Lamb, 1994
). As the HA of our parental strain (i.e. A/WSN/33) is not cleaved by endogenous proteinase (Goto & Kawaoka, 1998
, 2000
), it would maintain its native structure within the trans-Golgi network. Thus, the absence of M2 expression in our mutants would have little impact on the HA conformation in infected cells. A more plausible hypothesis is that, in the absence of M2 ion-channel protein, vRNPs within virions cannot be acidified by endosomes in the early stage of viral infection. As a result, the dissociation of M1 protein from vRNPs would be inhibited, thereby affecting normal virus transcription and replication. Alternatively, the lack of M2 ion-channel protein might affect other biological processes of the virus. Further work, such as studies of transcription and replication kinetics and the nuclear importing of vRNP of these mutants during early viral infection, is required to elucidate the underlying reason for the attenuation of these mutants.
Of the 15 splicing mutants, only those viruses containing a GU to AU, CU or UU mutation could be rescued. Interestingly, these three mutations did not alter the M1 protein-coding sequence. By contrast, all of the other mutations resulted in changes to aa 9 (Thr) or 10 (Tyr) of the M1 protein. Our results thus suggest that these two amino acid residues are critical for the virus. These two residues are located at the first helical structure of the M1 protein and are involved in lipid-membrane binding (Sha & Luo, 1997). Further investigation is required to elucidate the precise effect of these mutations on the M1 protein.
Our results have demonstrated a novel method for generating attenuated influenza viruses. Sequence analyses of influenza M mRNA indicated that the invariant dinucleotide sequence is absolutely conserved (data not shown). Thus, this approach can be used to generate attenuated viruses with other genetic backgrounds. However, one should be aware that this strategy might not be applicable to strains that encode an intracellularly cleavable HA (Sugrue et al., 1990; Takeuchi & Lamb, 1994
).
Finally, the splicing mutants in this study might be used to study other biological processes of influenza A virus. For example, it has been suggested that the M1 and M2 proteins encoded by the M gene segment contribute to filamentous virus morphology (Hughey et al., 1995; Roberts et al., 1998
; Bourmakina & García-Sastre, 2003
). The virus strain used in this study (A/WSN/33) displayed a spherical morphology and we did not observe any changes in virus morphology in our mutants by electron microscopic examination. However, it would be possible to delineate the roles of the M1 and M2 genes in virus morphology by applying similar mutations to strains that are predominantly filamentous in shape (Bourmakina & García-Sastre, 2003
; Elleman & Barclay, 2004
).
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ACKNOWLEDGEMENTS |
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Received 27 October 2004;
accepted 12 January 2005.
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