Overexpression of the M2-2 protein of respiratory syncytial virus inhibits viral replication.
The M2-2 protein of respiratory syncytial virus (RSV) is involved in regulation of viral RNA transcription and replication. Encoded by the next-to-last gene of RSV, the M2-2 open reading frame (ORF) overlaps with the upstream M2-1 ORF, suggesting that the production of the M2-2 protein might be tightly regulated during virus replication. To evaluate the effect of M2-2 overexpression on RSV replication, the M2-2 gene was separated from M2-1 by leaving it at the position prior to the M2-1 or moving it to the promoter proximal position as an independent transcriptional unit in the RSV A2 genome. Although recombinant viruses bearing the shuffled M2-2 gene were recovered and expressed higher levels of M2-2, most of these viruses grew poorly in HEp-2 cells. Sequence analysis revealed that various mutations (substitution, insertion, and deletion) occurred in the M2-2 gene, resulting in reduced M2-2 activity as measured by the RSV minigenome system. Further examination of the M2-2 sequence and its function showed that either one of the first two AUG codons located at the 5' end of M2-2 could be used to produce a functional M2-2 protein and that deletion of the first six amino acids from its N terminus or four amino acids from its C terminus greatly reduced its function. The effect of M2-2 protein on RSV replication was also studied by examining RSV replication in cells transiently expressing M2-2. The M2-2 protein expressed at a high level completely inhibited RSV replication. These results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication.
Cheng
Xing
X
MedImmune Vaccines Inc., 297 N. Bernardo Ave., Mountain View, CA 94043, USA.
Park
HyunJung
H
Zhou
Helen
H
Jin
Hong
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology; D015183:Restriction Mapping; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication / Q000502:physiology*
D018076:DNA, Complementary; C507355:M2-2 protein, respiratory syncytial virus; D012367:RNA, Viral; D014764:Viral Proteins