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Journal of Virology, September 2004, p. 9585-9591, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9585-9591.2004

Introduction of a Temperature-Sensitive Phenotype into Influenza A/WSN/33 Virus by Altering the Basic Amino Acid Domain of Influenza Virus Matrix Protein

Teresa Liu and Zhiping Ye^*

Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, Office of Vaccine Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland

Received 1 March 2004/ Accepted 13 May 2004

Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.

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* Corresponding author: Mailing address: CBER/FDA, Bldg. 29A, Rm. 2B17, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 435-5197. Fax: (301) 480-3157. E-mail: yez{at}cber.fda.gov.

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Journal of Virology, September 2004, p. 9585-9591, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9585-9591.2004

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