Article Figures & Data
Figures
- Fig. 1.
(A) Cysteine scanning mutagenesis of the M2-1 gene. The underlined cysteine residues at position 7, 15, 21, and 96 were changed to glycine, and the mutants are denoted C96G, C15G, C21G, and C96G. (B) Three C-terminal-end truncations were engineered by the introduction of tandem stop codons, indicated by asterisks. Mutants with a truncation of 67, 46, or 17 amino acids are designated Tr67, Tr46, or Tr17.
- Fig. 2.
(A) Expression of β-galactosidase is dependent on the M2-1 protein. The indicated amount of pM2-1 expression plasmid was transfected into MVA-T7-infected HEp-2 cells together with 0.2 μg of pN, 0.2 μg of pP, 0.2 μg of pL, and 0.2 μg of pRSVLacZ minigenome. Thirty-six hours after transfection, the β-galactosidase activity was measured by incubating the cell lysates with 5 mM CPRG for 5 min. An average of three transfection experiments is shown. (B) Comparison of β-galactosidase activity from cells transfected with wt and mutant M2-1 expression plasmids. Cells were transfected with 0.3 μg of wt M2-1 plasmid, and the β-galactosidase activity following 30 min of incubation with CPRG is shown. (C) β-Galactosidase activities from cells transfected with wt, C96G, or Tr17 M2-1. The enzymatic activity was monitored for 45 min after incubation of the cell lysates with CPRG. The signal was linearly responsive up to an OD550 of 3.0. (D) Northern blot of total RNA extracted from HEp-2 cells transfected with wt and mutant M2-1 expression plasmids. The blot was hybridized with a riboprobe specific for β-galactosidase mRNA. The LacZ antigenome is also indicated.
- Fig. 3.
Multiple-step replication cycles in Vero and HEp-2 cells. Vero cells (top) or HEp-2 cells (bottom) were infected with rA2, rA2-Tr17, or rA2-C96G at an MOI of 0.1 PFU/cell. Aliquots of culture medium in duplicates were harvested at 24-h intervals, and the virus was quantitated by plaque assay on Vero cells.
- Fig. 4.
Northern blot analyses of viral RNA expression. HEp-2 cells were infected with rA2, rA2-Tr17, or rA2-C96G at an MOI of 0.1 PFU/ml. Total cellular RNA was extracted at 24 or 48 h postinfection (A) or at 48 h postinfection (B). The RNA blots were hybridized with DIG-UTP-labeled riboprobe specific to M2 (A) or F (B) mRNAs. The mRNA and read-through transcripts are indicated.
- Fig. 5.
(A) Western blot of cell lysates infected with rA2, rA2-C96G, or rA2-Tr17. Total cellular proteins from infected cells were separated on 15% polyacrylamide gels containing 0.1% SDS, and the blots were hybridized separately with anti-M2-1 or anti-N antibodies. (B) Immunoprecipitation of labeled RSV polypeptides from infected HEp-2 or Vero cells. HEp-2 or Vero cells were infected with each virus as indicated, and the proteins were labeled with [35S]methionine and [35S]cysteine at 16 h postinfection. The labeled proteins were immunoprecipitated with anti-RSV or anti-M2-2 antibodies. The F1 protein from RSV-infected HEp-2 and Vero cells showed different gel mobilities, and less F1 was produced in rA2-Tr17-infected Vero cells in this particular experiment.
- Fig. 6.
Immunoprecipitation of the M2-1 proteins from infected cells. HEp-2 cells were infected with rA2, rA2T17, or rA2C96G at an MOI of 1 PFU/cell. The labeled M2-1 proteins were immunoprecipitated with anti-M2-1 antibody and denatured in the presence (right panel) or absence (left panel) of β-mercaptoethanol. Immunoprecipitated M2-1 proteins were resolved by SDS–-15% polyacrylamide gel electrophoresis. The different forms of M2-1 on the SDS-polyacrylamide gel are indicated as a, b, c, c*, or d.
