Conserved cysteine and histidine residues in the putative zinc finger motif of the influenza A virus M1 protein are not critical for influenza virus replication

The influenza virus matrix protein (M1), the most abundant protein in virus particles, plays a critical role in many aspects of virus replication, from virus entry and uncoating to assembly and budding of the virus particle (reviewed by Lamb & Krug, 2001; Nayak & Hui, 2002). The structure of the N terminus (aa 2164) of M1 has been determined by crystallization, X-ray diffraction, circular dichroism and tritium bombardment (Sha & Luo, 1997; Shishkov et al., 1999; Arzt et al., 2001; Harris et al., 2001). It contains two spatially distinct domains, N and M (Fig. 1B), consisting of helices and loops. Several specific domains, including the lipid-binding domain, the RNA-binding domain, the transcription inhibition domain, the nuclear localization signal (NLS) and a putative zinc finger-binding motif (Wakefield & Brownlee, 1989), have been identified in M1 (Fig. 1A). The putative zinc finger motif of influenza virus M1 (¹⁴⁸CATCEQIADSQHRSH¹⁶²) (Wakefield & Brownlee, 1989) is present at the C terminus of helix 9 (H9) and extends into loop 9 (L9) (Fig. 1A, B) (Sha & Luo, 1997; Arzt et al., 2001; Harris et al., 2001). The sequence represents a typical CCHH zinc finger motif (CX₂₄CX₂₁₅HX₂₆H, where X is any amino acid residue), which is known to be involved in zinc binding (reviewed by Iuchi, 2001). CCHH zinc finger proteins comprise a diverse family of DNA- or RNA-binding proteins and function in regulating transcription (reviewed by Takatsuji, 1998; Iuchi, 2001).

(40K): Fig. 1. (A) Schematic diagram of the zinc finger motif in the H9 (aa 140158) and L9 (aa 159163) domains of the influenza A virus M1 protein (aa 1252). The lipid-binding domain (LBD), RNA-binding domain (RBD), transcription inhibition domain (TID), nuclear localization signal (NLS) and phosphorylation sites are shown. The sequence corresponding to the H9 domain has been boxed. The zinc finger motif (Zn) is located between aa 148 and 162, which overlap H9 and L9. Cysteine and histidine residues in the zinc finger motif are shaded in grey. The putative protein kinase C phosphorylation site Ser161 is indicated (*). (B) Schematic diagram of the helix (H1H9) and loops (aa 2164) of the CCHH motif. The α-helix regions are indicated as cylinders and the putative amino (N) domain, the middle (M) domain and the N and C termini are marked. (C) Helical wheel plot of the C-terminal H9 domain (aa 144158). Cys148 and Cys151 (grey shading) are located at the same side of the helix (bold curve).

Zinc finger-like motifs have been found in a number of viral structural proteins and have been shown to play critical roles in the life cycles of these viruses (Fernandez-Pol et al., 2001). Several lines of evidence have suggested that the CCHH motif in M1 may play an important role in influenza virus replication. Firstly, amino acid sequence alignment analysis shows that the CCHH motif is evolutionarily conserved among the M1 proteins of influenza A and B viruses (Table 1) (Wakefield & Brownlee, 1989). Secondly, zinc ions have been found to be associated with a fraction of influenza virus M1 protein molecules (Elster et al., 1994). Thirdly, Okada et al. (2003) have reported recently a pH-dependent conformational change in the structure of a 28-mer peptide encompassing this region in the presence of zinc and proposed a model for zinc binding. Furthermore, they predicted that a small fraction of M1 molecules which bind zinc are likely to play an important role in virus uncoating at acidic pH. Fourthly, peptide 4, a synthetic zinc finger peptide based on the M1 sequence ¹⁵²EQIADSQHRSHRQMV¹⁶⁶, and peptide 6, corresponding to the M1 sequence ¹⁴⁸CATCEQIADSQHRSHRQMV¹⁶⁶, have been shown to be potent inhibitors of influenza virus transcription (Judd et al., 1992). Peptide 6 was shown also to inhibit CPE in virus-infected MDCK cells in culture (Nasser et al., 1996) and pathogenicity in influenza virus-infected mice (Judd et al., 1997). These studies imply that the CCHH motif at aa 148162 and its zinc-binding function play a critical role in virus replication.

Table 1. Comparison of influenza A and B virus M1 zinc finger sequences

Therefore, it becomes important to determine the role of the putative zinc finger motif and the adjacent region of the M1 protein in the influenza virus life cycle. In this report, we have used site-directed mutagenesis and rescue of mutant viruses by reverse genetics to define the function of this region in the virus life cycle. Viruses and cell lines.
Influenza virus strain A/WSN/33 (H1N1) was used in these experiments. Stock viruses were prepared by infecting MDCK cells at an m.o.i. of 0·001 and incubating at 33 °C in virus growth medium (VGM) (MEM supplemented with 0·2 % BSA, 4 % BME vitamin, 10 mM HEPES, pH 7·2, 0·155 % NaHCO₃, 0·0015 % DEAEdextran, 100 U penicillin-G ml^-1 and 100 µg streptomycin ml^-1) (Hui & Nayak, 2001, 2002). Culture supernatants at 60 h post-infection (p.i.) were harvested, clarified by centrifugation and stored in aliquots of 0·5 ml at -80 °C. WSN stock virus contained 1·45x10⁹ p.f.u. ml^-1. MDCK cells were cultured in DMEM (Invitrogen) supplemented with 10 % heat-inactivated FBS (Atlanta Biologicals) and antibiotics (100 U penicillin-G ml^-1 and 100 µg streptomycin ml^-1). 293T cells were grown in Opti-MEM (Invitrogen) supplemented with 5 % FBS and antibiotics.

Plaque assay.
Plaque assays were performed and titres (p.f.u. ml^-1) determined as described previously (Hui & Nayak, 2001, 2002). MDCK cell monolayers (1·5x10⁶ cells at a confluency of 100 % in 35 mm dishes) were washed with PBS⁺ (PBS supplemented with 0·5 mM MgCl₂ and 1 mM CaCl₂) and infected with different dilutions of virus for 1 h at 37 °C. The virus inoculum was removed by washing with VGM. Cell monolayers were then overlaid with agar overlay medium (VGM supplemented with 0·6 % low-melting-point agarose and 0·5 µg TPCK-treated trypsin ml^-1) and incubated at 33 °C. Visible plaques were counted at 3 days p.i. and titres were determined. All data are expressed as the mean of three or four independent experiments with SD values less than 10 %. The significance of the difference between values was compared using the Student t-test and P<0·001 was considered significant.

Generation of transfectant viruses using reverse genetics and preparation of mutant stock viruses.
cDNA from the WSN M gene in the Pol IPol II plasmid (Hoffmann et al., 2000) was used as template for site-directed mutagenesis. Mutagenesis reactions were carried out using the QuickChange Site-Directed PCR Mutagenesis kit (Stratagene). Primers used in these reactions will be provided upon request. All mutations were confirmed by sequencing the entire M gene cDNAs. Mutant viruses were rescued from transfected 293T cells using an eight-plasmid Pol IPol II transfection system (Hoffmann et al., 2000), with modifications (Hui et al., 2003). All eight Pol IPol II plasmids, which encode the influenza virus genes, were kindly provided by R. Webster (St. Jude Children's Research Hospital, Memphis, TN, USA). Briefly, 1 µg of each of the eight plasmids (seven Pol IPol II constructs expressing the wt proteins HA, NA, NP, NS, PA, PB1 and PB2 and one Pol IPol II construct expressing either the wt or the mutated M gene) was mixed with transfection reagent [2 µl TransIT LT-1 (Panvera) µg^-1 DNA]. 293T cells (1x10⁶ cells at a confluency of 5070 % in 35 mm dishes) were transfected and incubated at 37 °C for 8 h. Later, the DNA/transfection mixture was replaced with virus rescue medium (Opti-MEM containing 0·3 % BSA, 0·01 % FBS, 100 U penicillin-G ml^-1 and 100 µg streptomycin ml^-1). After 22 h, transfected cells were shifted to 33 °C and incubated for 16 h. Then, TPCK-treated trypsin (Sigma) at a final concentration of 0·5 µg ml^-1 was added and cells were incubated for a further 32 h, at which time supernatants were collected and titrated for infectious virus by plaque assay. Routinely, rescued virus with 10⁷ p.f.u. ml^-1 was obtained directly from the supernatant of transfected cells. Individual plaques were isolated, resuspended in virus dilution buffer (VDB) (PBS⁺ supplemented with 0·2 % BSA, 0·005 % DEAEdextran, 100 U penicillin-G ml^-1 and 100 µg streptomycin ml^-1) and used for stock virus preparation in MDCK cells (1·2x10⁷ cells at a confluency of 100 % in 25 cm² flasks) at an m.o.i. of 0·001. Infected cells were incubated at 33 °C in VGM with trypsin (final concentration 0·5 µg ml^-1) for 48 h. Supernatants were harvested, titrated by plaque assay and used as virus stocks.

RT-PCR.
Viral RNA was extracted from culture supernatants using the QIAamp Viral RNA Mini kit (Qiagen). RNA was reverse-transcribed using the OneStep RT-PCR kit (QIAGEN) with oligonucleotides 5'-AGCAAAAGCAGGTAGATATTGAAG-3' and 5'-CATTTGCTCCATAGCCTTAGCTG-3' and used for sequencing.

Labelling and immunoprecipitation of M1 mutant proteins.
At 18 h post-transfection, 293T cells were incubated in methionine- and cysteine-free DMEM (Invitrogen) for 30 min and pulse-labelled with 60 µCi ³⁵S-labelled protein (ICN) for 2 h. Cells were then lysed in 1 ml RIPA buffer [50 mM Tris/HCl, pH 7·5, 150 mM NaCl, 1 % Triton X-100, 0·5 % sodium deoxycholate, 0·1 % SDS and 1x proteinase inhibitor cocktail (Sigma)], immunoprecipitated with anti-M1 antibody (Biodesign International) and analysed by SDS-PAGE (12 %).

Indirect immunofluorescence.
For indirect immunofluorescence, MDCK cells (4x10⁵) grown on tissue culture chamber slides (Nunc) were infected with wt or mutant virus (m.o.i. of 3). At 5 and 12 h p.i., infected cells were washed with PBS⁺ and fixed in 100 % acetone for 10 min at -20 °C. Fixed cells were washed with PBS (three times for 5 min each) and incubated with goat anti-M1 antibodies (diluted 1 : 30 in PBS containing 3 % BSA) for 1 h at room temperature. Cells were then washed three times for 15 min each in PBS and incubated with FITC-conjugated anti-goat IgG (Santa Cruz Biotechnology) diluted 1 : 120 in PBS containing 3 % BSA for 35 min at room temperature. Cells were washed again with PBS (four times for 15 min each), once with water (1 min) and blow-dried. Cells were then mounted in 50 % glycerol in PBS (pH 9) (Hui et al., 1992). Slides were viewed under an Axioskop-2 fluorescence microscope (Carl Zeiss).

Peptide treatment of infected MDCK cells.
24-well plates were seeded with 2x10⁵ MDCK cells per well. Monolayers were washed with PBS⁺ and infected with wt influenza virus at an m.o.i. of 0·05 in 200 µl VDB in the presence of 1 µM peptides (SynVax) (Table 3). After 1 h at 37 °C, cells were incubated with 1 ml VGM in the presence of 1 µM peptide. Then, 200 µl supernatent was collected at 48 and 72 h p.i. and assayed for virus titre.

Table 3. Amino acid sequences of synthetic peptides

To investigate the function of the CCHH motif of the M1 protein in influenza virus production, we mutated initially the residues of the CCHH motif individually, changing cysteine at positions 148 and 151 and histidine at positions 159 and 162 to alanine to generate mutants with the motifs ACHH, CAHH, CCAH and CCHA (Table 2). While CCHH fingers are, in general, identified with DNA- or RNA-binding properties, the CCHC motif has been shown to mediate proteinprotein interactions with other proteins (Matthews et al., 2000). Therefore, we also mutated His¹⁶²→Cys to create the mutant CCHC (Table 2). cDNA transfection showed that all mutant proteins were expressed efficiently in transfected 293T cells (Fig. 2C). We then used the Pol IPol II transfection system (Hoffmann et al., 2000; reviewed by Neumann et al., 2002) to rescue mutant influenza virus from each of the mutated cDNAs in 293T cells (Hui et al., 2003). Mutant viruses were rescued successfully from each of the mutated cDNAs. Similarly, we made a series of double (AAHH, ACAH, ACHA, CAAH, CAHA and CCAA), triple (AAHA, ACAA and CAAA) and quadruple (AAAA) mutations of the CCHH motif by alanine replacement in different combinations (Table 2). Again, each of these mutant proteins was expressed efficiently in 293T cells after cDNA transfection (Fig. 2C). We also rescued infectious virus from each of these mutated M1 cDNAs successfully. We isolated viral RNA from rescued virus particles and used RT-PCR to confirm the mutations in the viral genome (data not shown). To determine if these mutations affected the M1 phenotype, we examined the intracellular location of M1 early and late in the infectious cycle, since defective nuclear export of M1 has been shown to affect virus growth and replication (Rey & Nayak, 1992). Accordingly, MDCK cells were infected with virus, fixed at 5 and 12 h p.i. and analysed by indirect immunofluorescence by staining for M1. Previously, we and others (Rey & Nayak, 1992; Whittaker et al., 1995; Hui et al., 2003) have shown that in influenza virus-infected cells, M1 is localized predominantly in the nucleus early in the infectious cycle (5 h p.i.) but becomes cytoplasmic later (12 h p.i.) in the infectious cycle, indicating the exit of the M1vRNP (viral ribonucleoprotein) complex from the nucleus to the cytoplasm. Infection of MDCK cells in all 15 mutant viruses (Table 2), including the three triple A and quadruple A mutants, showed similar cytoplasmic and nuclear distribution of M1 as the wt virus at both 5 and 12 h p.i. Like wt virus-infected cells, M1 was both nuclear and cytoplasmic at 5 h p.i., whereas M1 was predominantly cytoplasmic late in the infectious cycle at 12 h p.i. (data not shown), suggesting that the mutation in the CCHH motif did not affect M1 transport and localization.

Table 2. Alanine replacements in the CCHH motif of M1

(36K): Fig. 2. (A) Titre of transfectant viruses. Transfectant viruses were rescued using the eight-plasmid transfection system in 293T cells. MDCK cells were then infected with transfectant virus at an m.o.i. of 0·001 and maintained in VGM containing 0·5 µg trypsin ml-1, as described in Methods. Supernatants were collected at 48 h p.i. and assayed for virus titre by plaque assay. Data represent mean titres (n=3). (B) Plaque sizes for different mutant viruses on MDCK cells. Plaques were visualized at day 3 and diameters were measured. Data represent mean diameters (n=4). No significant differences in plaque titre or plaque diameter were observed among wt and mutant viruses. (C) Immunoprecipitation of M1 mutant proteins. At 18 h post-transfection, 293T cells were pulse-labelled for 2 h. Cells were then lysed in RIPA buffer, immunoprecipitated with anti-M1 antibody and resolved by SDS-PAGE (12 %).

Since we have shown recently that the NLS function of M1 was not critical for M1 function and virus replication (Hui et al., 2003), M1 transport and localization may not indicate virus phenotype. Therefore, we wanted to determine the effect of these mutations on the infectious cycle, including virus replication and plaque morphology. Since multiple cycles of growth were the most sensitive indicator of infectious virus production, we infected MDCK cells with each of the 15 mutant viruses at a very low m.o.i. (0·001 p.f.u. per cell) in the presence of trypsin (0·5 µg ml^-1). Supernatants at 48 h p.i. were titrated by plaque assay, as described in Methods. Results show that each of the mutant viruses, whether possessing single, double, triple or even quadruple mutations in the CCHH motif, exhibited the wt phenotype in multiple growth cycles in MDCK cells (Fig. 2). Also, since we have noted that plaque morphology and plaque diameter are highly sensitive indicators for multiple cycles of virus growth (Hui et al., 2003), we also examined plaque morphology and plaque characteristics of each mutant virus in MDCK cells in the presence of trypsin, as described in Methods. Each mutant virus produced clear plaques and there was no significant variation in plaque diameter among the virus mutants compared to those of wt virus (Fig. 2B). Taken together, these results demonstrate that mutations in the CCHH motif, either alone or in combination, did not have a significant effect on virus replication, either in multiple growth cycles or in plaque size and morphology.

As indicated earlier, synthetic peptides corresponding to M1 zinc finger sequences were shown to inhibit virus transcription in vitro and CPE in MDCK cells as well as virus pathogenesis in mice (Judd et al., 1992, 1997; Nasser et al., 1996). Therefore, synthetic peptides (Table 3) were used to determine their effect on virus growth in MDCK cells. Initially, we used peptides at 1 µM concentration, since this concentration was shown to inhibit transcription and prevent virus-induced CPE (Nasser et al., 1996; Judd et al., 1997). Results show that neither the putative zinc finger peptides nor the control peptides had any effect on virus growth at either 48 (Fig. 3) or 72 h p.i. (data not shown). All virus-infected cells with or without peptide exhibited similar CPE, suggesting that they did not have any effect on CPE in infected cells (data not shown). Even the peptides at higher concentrations (5 and 25 µM) did not show any significant inhibitory effect on virus growth or CPE (data not shown). Therefore, no measurable antiviral activity was seen in the presence of these CCHH motif peptides, supporting the phenotype and growth characteristics of mutant viruses observed (Fig. 2).

(18K): Fig. 3. Effect of synthetic peptide corresponding to the CCHH motif of M1 on virus production. 24-well plates were seeded with 2x105 MDCK cells per well. Monolayers were infected with wt influenza virus at an m.o.i. of 0·05 in the presence of 1 µM peptide, as indicated. After 1 h at 37 °C, cells were incubated with VGM in the presence of 1 µM peptide. Then, 200 µl supernatant was collected at 48 and 72 h and plaque assayed. Results show virus titres at 48 h p.i. No significant inhibition of virus titre was observed in CCHH peptide-treated cells.

During site-directed PCR mutagenesis and M gene cDNA cloning, we obtained a number of double and triple mutants involving adjacent sequences, albeit infrequently (Table 4). Attempts to rescue these double or triple mutants into infectious virus were always negative, even after repeated attempts (five or more) (Table 4). These mutant M gene cDNAs were sequenced completely to ensure that there was no other mutation and that they expressed mutant M1 proteins upon transfection of 293T cells (data not shown). Sequence analysis showed that Ala¹⁵⁵ was always mutated in these mutant M gene cDNAs (Table 4). Therefore, we wanted to determine if combinations of these mutations or mutation of Ala¹⁵⁵ alone was responsible for the lethal phenotype in virus rescue. Since the majority of these mutant cDNAs contained the Ala¹⁵⁵→Gly mutation, we made a single mutation Ala¹⁵⁵→Gly and attempted to rescue infectious virus. We observed that even a single mutation of Ala¹⁵⁵→Gly was lethal and infectious virus could not be rescued (Table 4). These results show that Ala¹⁵⁵ is critically required for M1 protein function.

Table 4. Mutations in the putative zinc finger region that could not be rescued

The influenza virus M1 protein plays a critical role in many aspects of virus replication and possesses specific domains for specific functions in the infectious cycle (Fig. 1A). Over the last few years, we have attempted to define the role of these putative functional domains of M1 in the life cycle of infectious influenza virus. Recently, we have shown that the NLS of M1 is not critical for the virus life cycle but this region is likely to possess a late (L) assembly domain motif and that NLS mutations can be rescued by L domain motifs, such as PTAP and YPDL (Hui et al., 2003). M1 also possesses a CCHH motif in H9 and in the adjacent loop, L9, which has been proposed to function as a putative zinc-binding motif. Both cysteine residues are present on the same side of the helix (Fig. 1C) and are accessible from the surface (Okada et al., 2003). The function and role of this CCHH motif in virus biology has been controversial. On the one hand, this motif has been proposed to bind zinc (Elster et al., 1994) and play an important role in the virus life cycle, including transcription inhibition by M1 as well as uncoating and virus pathogenesis (Judd et al., 1992, 1997; Nasser et al., 1996; Okada et al., 2003). On the other hand, structural consideration of this region has implied that the CCHH motif may not bind zinc and that neither this region nor its zinc-binding property may be critical in vRNP-binding or in M1-mediated transcription inhibition of the influenza virus vRNP (Watanabe et al., 1996; Elster et al., 1997; Perez & Donis, 1998). Therefore, it became important to define the functional significance of the CCHH motif in virus replication. Our results with mutant viruses demonstrate clearly that the CCHH motif of M1 is not critical for the influenza virus life cycle and, therefore, that the zinc-binding function may not be involved in the infectious cycle of the virus.

Results on the mutation of the CCHH motif presented here are contradictory to reports published earlier, in that peptides corresponding to the putative zinc finger motif inhibited virus pathogenesis in mice and CPE in cell culture (Judd et al., 1992, 1997; Nasser et al., 1996). Since virus pathogenesis studies were done in mice, it is possible that the CCHH motif may have an important role in virus growth and pathogenesis in mice that is not observed in cell culture. However, it is difficult to reconcile our result with the inhibitory effect of peptides, observed as virus-induced CPE in MDCK cells (Nasser et al., 1996), except that in these studies the effect of peptides on virus growth was not determined and different virus strains were used. We used influenza virus strain A/WSN/33 in this study, whereas strains A/PR/8/34 and A/Victoria/3/75 were used in earlier studies (Nasser et al., 1996).

Recently, Okada et al. (2003) have determined the effect of zinc on the structural conformation of an M1 peptide and also postulated the role of zinc-bound M1 molecules in virus uncoating. They used a 28-mer peptide (Ac-¹³⁹TTEVAFGLVCATCEQIADSQHRSHRQMV¹⁶⁶-amide) encompassing the CCHH motif to study the effect of zinc ions on the conformation of this peptide. They observed that Cys¹⁴⁸ and Cys¹⁵¹ are located on the same side of the H9 domain (Fig. 1C) and are not buried within the domain but are accessible from the surface (Okada et al., 2003). The peptide takes a partially unfolded conformation at neutral pH, with tetrahedral coordination of two cysteine and two histidine residues to a zinc ion in the centre. His¹⁵⁹ and His¹⁶¹ present on the C-terminal side of H9 are also accessible to zinc at a neutral pH. However, upon acidification, the peptide releases zinc molecules and assumes an α-helix-rich conformation. They proposed that the pH-dependent conformation transition of M1 and release of zinc ions from a small number of zinc-bound M1 molecules at low pH may play an important role in releasing M1 from the vRNP and facilitating influenza virus uncoating. However, data presented in our report do not support any role of zinc-bound M1 in uncoating the vRNP or in transcription inhibition of vRNP, since mutant viruses exhibited the same wt virus replication in MDCK cells.

On the other hand, a number of observations support our finding in that the putative zinc finger motif of influenza virus M1 may not play a critical role in virus biology. Firstly, the structure of the putative M1 motif is different from that of the common functional CCHH zinc fingers, which possess two to three β-strands at the N-terminal sequence followed by one α-helix at the C-terminal half of the X₁₂HX_nH motif, forming a ββα fold (reviewed by Pieler & Bellefroid, 1994; Iuchi, 2001). Two cysteine residues in the zinc-binding domain are present in the β structure. Although the structure of the C-terminal sequence of the proposed zinc finger motif in L9 of influenza virus M1 has not been defined, two cysteines in the middle of the H9 domain are in the α-helix conformation (Fig. 1) (Sha & Luo, 1997; Shishkov et al., 1999; Arzt et al., 2001; Harris et al., 2001; Okada et al., 2003). Therefore, these residues are unlikely to form a coordinate complex with zinc. This is, however, contrary to the proposed model of zinc-bound M1 peptide with a partially unfolded helix at neutral pH (Okada et al., 2003). Secondly, by atomic absorption spectroscopy, only a small proportion of the M1 protein contained zinc ions (<10 % in influenza A virus and 1520 % in influenza B virus) (Elster et al., 1994), suggesting that zinc binding may not be critical for M1 function in virus biology. However, Okada et al. (2003) proposed that a small number of zinc-bound M1 molecules may play a special role in virus uncoating. Thirdly, circular dichroism analysis showed that the mutant M1 protein (SCHH) bound to RNA with the same affinity (K_d=6x10^-8 M) as wt M1 (CCHH), suggesting that zinc binding does not affect the RNA-binding properties of M1 (Elster et al., 1997). Also, a deletion mutant lacking the C-terminal half of M1 (aa 91242), which includes the CCHH motif, caused transcription inhibition effectively in transfected cells (Perez & Donis, 1998). Similarly, recombinant M1 and a deletion mutant lacking the CCHH motif expressed in Escherichia coli bound RNA and inhibited RNA transcription effectively in vitro in the absence of zinc (Watanabe et al., 1996). Also, in some viral proteins, such as the reovirus inner capsid protein λ1, the CCHH motif (CX₂CX₁₆HX₂H) has been shown to be unrelated to nucleic acid binding (Lemay & Danis, 1994). Fourthly, a mutant M1 protein with a SCHH motif did not have any effect on the influenza virus life cycle (Liu & Ye, 2002). Finally, influenza virus strains containing a mutation of Cys¹⁴⁸ or His¹⁶² have been found in nature (Table 5). Taken together, these findings support our results that the cysteine and histidine residues of the CCHH motif and the zinc-binding function of the CCHH motif are not essential in the influenza virus life cycle in MDCK cells. However, as mentioned above, these studies cannot rule out the role of the CCHH motif and the zinc-binding function in virus pathogenesis in animals.

Table 5. Mutations found in the CCHH motif of influenza A virus strains

We observed that mutations involving Ala¹⁵⁵→Gly in H9 were always lethal in virus rescue, suggesting that H9 may provide other critical function(s) in influenza virus biology. There could be a number of reasons why a single Ala¹⁵⁵→Gly mutation (Table 4) was lethal. Glycine being a helix breaker may have caused alterations in the structural conformation by destabilizing H9 and possibly the rest of the protein. The M domain of M1, including H9, has been implicated as being involved in cooperative interactions among M1 molecules required for both transcription inhibition and mRNA regulation as well as virus assembly and budding (Sha & Luo, 1997; Harris et al., 1999, 2001; Arzt et al., 2001). Since Ala¹⁵⁵ is in a unique position at the end of H9, any replacement with a residue larger than alanine would destabilize H9. This could explain why the replacement of Ala¹⁵⁵ with aspartic acid or threonine in combination with other mutations (Table 4) was also lethal. Therefore, further detailed analysis of this region in defining its functional significance in the virus life cycle will be important and is in progress.

In summary, we have shown that the CCHH motif, the putative zinc-binding motif in H9 of M1, does not provide a critical function in virus replication and that virus mutants in the CCHH motif can replicate as efficiently as the wt virus in MDCK cells. Experiments are in progress to determine if this domain plays an important role in virus pathogenesis.

This work was supported by USPHS grants (AI 16348 and AI 41681) and Stein Oppenheimer Endowment Award. We thank Shankari Somayaji for her help in creating some M1 mutants and plaque assay, and Tae Yong Yang and Ee Ming Yap for their help with DNA preparation.