1 Department of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA
2 SynVax Inc., North Logan, UT 84341, USA
Correspondence
Debi P. Nayak
dnayak{at}ucla.edu
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ABSTRACT |
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INTRODUCTION |
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METHODS |
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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.
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RESULTS |
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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
).
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DISCUSSION |
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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
-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 -strands at the N-terminal sequence followed by one
-helix at the C-terminal half of the X12HXnH motif, forming a
fold (reviewed by Pieler & Bellefroid, 1994
; Iuchi, 2001
). Two cysteine residues in the zinc-binding domain are present in the
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
-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 (Kd=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
1, 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.
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Elster, C., Fourest, E., Baudin, F., Larsen, K., Cusack, S. & Ruigrok, W. H. (1994). A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1RNA interaction. J Gen Virol 75, 3742.[Abstract]
Elster, C., Larsen, K., Gagnon, J., Ruigrok, R. W. H. & Baudin, F. (1997). Influenza virus M1 protein binds to RNA through its nuclear localization signal. J Gen Virol 78, 15891596.[Abstract]
Fernandez-Pol, J. A., Hamilton, P. D. & Klos, D. J. (2001). Essential viral and cellular zinc and iron containing metalloproteins as targets for novel antiviral and anticancer agents: implications for prevention and therapy of viral diseases and cancer. Anticancer Res 21, 931957.[Medline]
Harris, A., Sha, B. & Luo, M. (1999). Structural similarities between influenza virus matrix protein M1 and human immunodeficiency virus matrix and capsid proteins: an evolutionary link between negative-stranded RNA viruses and retroviruses. J Gen Virol 80, 863869.[Abstract]
Harris, A., Forouhar, F., Qiu, S., Sha, B. & Luo, M. (2001). The crystal structure of the influenza matrix protein M1 at neutral pH: M1M1 protein interfaces can rotate in the oligomeric structures of M1. Virology 289, 3444.[CrossRef][Medline]
Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000). A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 61086113.
Hui, E. K.-W. & Nayak, D. P. (2001). Role of ATP in influenza virus budding. Virology 290, 329341.[CrossRef][Medline]
Hui, E. K.-W. & Nayak, D. P. (2002). Role of G protein and protein kinase signalling in influenza virus budding in MDCK cells. J Gen Virol 83, 30553066.
Hui, E. K.-W., Yang, Y. H. & Yung, B. Y.-M. (1992). Schedule-dependent sphinganine potentiation of retinoic acid-induced differentiation, cell growth inhibition, and nucleophosmin translocation in a human leukemia cell line (HL-60). Exp Hematol 20, 454461.[Medline]
Hui, E. K.-W., Barman, S., Yang, T. Y. & Nayak, D. P. (2003). Basic residues of the helix six domain of influenza virus M1 involved in nuclear translocation of M1 can be replaced by PTAP and YPDL late assembly domain motifs. J Virol 77, 70787092.
Iuchi, S. (2001). Three classes of C2H2 zinc finger proteins. Cell Mol Life Sci 58, 625635.[Medline]
Judd, A. K., Sanchez, A., Kharitonenkov, I., Moscona, A., Nasser, E. & Bucher, D. J. (1992). M-protein peptides of influenza virus: application as antiviral agents. In Peptides, pp. 694696. Edited by J. A. Smith & J. E. Rivier. Leiden: ESCOM.
Judd, A. K., Sanchez, A., Bucher, D. J., Huffman, J. H., Bailey, K. & Sidwell, R. W. (1997). In vivo anti-influenza virus activity of a zinc finger peptide. Antimicrob Agents Chemother 41, 687692.[Abstract]
Lamb, R. A. & Krug, R. M. (2001). Orthomyxoviridae: the viruses and their replication. In Fields Virology, pp. 725769. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Lemay, G. & Danis, C. (1994). Reovirus 1 protein: affinity for double-stranded nucleic acids by a small amino-terminal region of the protein independent from the zinc finger motif. J Gen Virol 75, 32613266.[Abstract]
Liu, T. & Ye, Z. (2002). Restriction of viral replication by mutation of the influenza virus matrix protein. J Virol 76, 1305513061.
Matthews, J. M., Kowalski, K., Liew, C. K., Sharpe, B. K., Fox, A. H., Crossley, M. & MacKay, J. P. (2000). A class of zinc finger involved in proteinprotein interactions: biophysical characterization of CCHC fingers from Fog and U-shaped. Eur J Biochem 267, 10301038.
Nasser, E. H., Judd, A. K., Sanchez, A., Anastasiou, D. & Bucher, D. J. (1996). Antiviral activity of influenza virus M1 zinc finger peptides. J Virol 70, 86398644.[Abstract]
Nayak, D. P. & Hui, E. K.-W. (2002). Assembly and morphogenesis of influenza viruses. Recent Res Dev Virol 4, 3554.
Neumann, G., Whitt, M. A. & Kawaoka, Y. (2002). A decade after the generation of a negative-sense RNA virus from cloned cDNA what have we learned? J Gen Virol 83, 26352662.
Okada, A., Miura, T. & Takeuchi, H. (2003). Zinc- and pH-dependent conformational transition in a putative interdomain linker region of the influenza virus matrix protein M1. Biochemistry 42, 19781984.[CrossRef][Medline]
Perez, D. R. & Donis, R. O. (1998). The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo. Virology 249, 5261.[CrossRef][Medline]
Pieler, T. & Bellefroid, E. (1994). Perspectives on zinc finger protein function and evolution: an update. Mol Biol Rep 20, 18.[Medline]
Rey, O. & Nayak, D. P. (1992). Nuclear retention of M1 protein in a temperature-sensitive mutant of influenza (A/WSN/33) virus does not affect nuclear export of viral ribonucleoproteins. J Virol 66, 58155824.[Abstract]
Sha, B. & Luo, M. (1997). Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1. Nat Struct Biol 4, 239244.[Medline]
Shishkov, A. V., Goldanskii, V. I., Baratova, L. A., Fedorova, N. V., Ksenofontov, A. L., Zhirnov, O. P. & Galkin, A. V. (1999). The in situ spatial arrangement of the influenza A virus matrix protein M1 assessed by tritium bombardment. Proc Natl Acad Sci U S A 96, 78277830.
Takatsuji, H. (1998). Zinc finger transcription factors in plants. Cell Mol Life Sci 54, 582596.[CrossRef][Medline]
Wakefield, L. & Brownlee, G. G. (1989). RNA-binding properties of influenza A virus matrix protein M1. Nucleic Acids Res 17, 85698580.[Abstract]
Watanabe, K., Handa, H., Mizumoto, K. & Nagata, K. (1996). Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. J Virol 70, 241247.[Abstract]
Whittaker, G., Kemler, I. & Helenius, A. (1995). Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus. J Virol 69, 439445.[Abstract]
Received 3 June 2003;
accepted 29 July 2003.