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Journal of Virology, January 2000, p. 74-82, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Respiratory Syncytial Virus That Lacks Open Reading Frame 2 of the M2 Gene (M2-2) Has Altered Growth Characteristics and Is Attenuated in Rodents

Hong Jin,* Xing Cheng, Helen Z. Y. Zhou, Shengqiang Li, and Adam Seddiqui

Aviron, Mountain View, California 94043

Received 28 May 1999/Accepted 20 September 1999

The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2Delta M2-2). Recombinant virus rA2Delta M2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2Delta M2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2Delta M2-2-infected cells. By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2Delta M2-2. rA2Delta M2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2Delta M2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV. rA2Delta M2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRC5 cells but replicated efficiently in Vero and LLC-MK2 cells. Replication of rA2Delta M2-2 in the upper and lower respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2Delta M2-2 was able to provide protection against challenge with wild-type RSV A2. The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2Delta M2-2 suggested that rA2Delta M2-2 may be a potential candidate for use as a live attenuated vaccine.

* Corresponding author. Mailing address: Aviron, 297 North Bernardo Ave., Mountain View, CA 94043. Phone: (650) 919-6587. Fax: (650) 919-6611. E-mail: hjin{at}aviron.com.

Journal of Virology, January 2000, p. 74-82, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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