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

Abstract

The influenza virus matrix protein (M1) possesses a cysteine and histidine (CCHH) motif in the helix 9 (H9) and adjacent region ( 148 CATCEQIADSQHRSH 162 ). The CCHH motif has been proposed as a putative zinc finger motif and zinc-binding activity has been implicated in virus uncoating as well as transcription inhibition and mRNA regulation. The function of the CCHH motif in the influenza virus life cycle was investigated by site-directed mutagenesis (alanine replacement) and by rescuing mutant viruses by reverse genetics. Mutant viruses containing an alanine replacement of the cysteine and histidine residues, either individually or in combination, were seen to exhibit wt phenotype in multiple virus growth cycles and plaque morphology. In addition, synthetic peptides containing the putative zinc finger motif did not inhibit virus replication in MDCK cells. However, mutation of Ala 155 in H9 was lethal for rescuing infectious virus.These data show that the CCHH motif does not provide a critical function in the influenza virus life cycle in cell culture and that the zinc-binding function may not be involved in virus biology. However, the lethal phenotype of the Ala 155 mutation shows that the H9 region of M1 provides some other critical function(s) in virus replication.

Mutations in Influenza Virus M1 CCHH, the Putative Zinc Finger Motif, Cause Attenuation in Mice and Protect Mice against Lethal Influenza Virus Infection

“…By using reverse genetics, we could rescue mutant viruses from each of mutated CCHH cDNAs. Each of these CCHH mutant (25). The new triple mutant (AAAH), which was not reported previously, also exhibited the WT phenotype in MDCK cells (Table 1, set I).…”

“…The CCHH motif in helix 9 (H9) of the M1 protein has been presumed to be the zinc finger motif (71). To determine the role of the CCHH motif and the adjacent region of H9, we made single, double, triple, and quadruple mutations in the CCHH residues by alanine replacement as report previously (25). A new triple mutant (AAAH), not reported previously, was also created ( Table 1, set I).…”

“…In addition, we also made single and multiple mutations of the entire H9 region by alanine replacement. We then used a Pol I-Pol II transfection system to rescue mutant influenza virus from each of the mutated cDNAs in human embryonic kidney 293T cells (22,25). By using reverse genetics, we could rescue mutant viruses from each of mutated CCHH cDNAs.…”

“…Therefore, we have examined the role of the M1 protein in virulence in mice. In this report, we show that CCHH, the putative zinc finger motif, which does not play an important role in virus growth in MDCK cells in culture (25), plays a critical role in virulence in mice. We found that some CCHH mutant viruses were highly attenuated in mice.…”

“…In our previous study (25), we observed that the mutations in the conserved CCHH motif (the putative zinc finger motif) present in helix nine (H9) and the adjacent region of M1 did not affect virus replication in Madin-Darby canine kidney (MDCK) cells in culture (25). However, the significance of the CCHH motif in influenza virus biology is an enigma, since the zinc finger peptide (peptide 6 [CATCEQIADSQHRSHRQ MV]) had been shown to be effective against type A influenza virus infection in BALB/c mice (28).…”