Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability.
The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.
Tang
R S
RS
Aviron, Mountain View, California 94043, USA.
Nguyen
N
N
Cheng
X
X
Jin
H
H
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000502:physiology*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology
D018076:DNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; D003545:Cysteine