Confluent BSC-40 cells were infected at moi = 5 with either wtVV (1st two columns) or Cts9 (last two columns) at 31C (top row) or 40C (lower row). the four cysteine residues which are known in the VV WR A28 protein to participate in two intramolecular disulfide SBI-553 bonds created from the vaccinia-encoded redox pathway (Senkevich et al., 2002; Senkevich et al., 2004a). Our studies having a temperature-sensitive VV mutant in the A28 protein show that deletion of 14 amino acids from your A28 C-terminus, including the fourth conserved cysteine residue, remarkably results in a computer virus that is viable at 31C. However, at 40C the A28 ts SBI-553 mutant synthesizes adult virions that are unable to enter cells, and are defective in mediating cell-cell fusion following low pH treatment. RESULTS Mapping of the mutations Cts6 and Cts9 to the A28 gene region by marker save The heat sensitive vaccinia computer virus mutants Cts6 and Cts9 comprise a complementation group that has a normal phenotype, that is, they display crazy type protein and DNA synthesis in the nonpermissive heat of 40C (Condit & Motyczka, 1981). Recombination between the two mutants was undetectable, suggesting the mutations lay close collectively (Condit & Motyczka, 1981). Using a library of overlapping cosmid clones, Thompson Rabbit Polyclonal to CKLF3 and Condit (1986) showed that Cts6 mapped to an approximately 3 kb region of DNA located in the middle of the entomopoxvirus and entomopoxvirus. Interestingly, the Cts6/Cts9 truncation deletes the fourth conserved cysteine at position 139 in the A28 protein, closest to the C-terminus. Purified mature virions of Cts6 produced at both permissive and non-permissive temperatures contain the truncated A28 protein Analysis of the defect in the Cts9 mutant in infected cells in the nonpermissive heat of 40C by electron microscopy did not reveal any obvious abnormality. All the phases of virion morphogenesis were observed at both 31C and 40C, and apparently normal mature virions were produced following contamination by Cts9 at 40C (data not shown). This obtaining was consistent with earlier observations that protein and DNA synthesis were not grossly perturbed in the mutant contamination (Condit & Motyczka, 1981). The presence of mutant A28 protein in the virions produced at both 31C and 40C was investigated by immunoblotting with a polyclonal antibody against a synthetic peptide spanning residues 56-75 of wild type A28 protein (Materials and Methods). The antibody enabled visualization of the wild-type A28 protein in purified MV of wild type computer virus (Fig. 3A). A similar band was not detected in extracts from uninfected cells (data not shown). A faster-migrating band relative to the wt A28 protein was detected in purified MV of Cts9 produced at either 31C or at 40C (Fig. 3A), indicating that the truncated A28 protein remained associated with virions synthesized at either heat. The mobility difference between wt and Cts9 A28 protein was clearly visible following extended electrophoresis in a 10%-20% gradient Tris-Tricine gel (Fig. 3A) but was less evident following separation for shorter occasions (Fig. 3B). Open in a separate window Physique 3 Immunoblot analysis of purified wild type and mutant virionsA. Different mobilities of A28 protein from WT and Cts9 virions. Virions were incubated in the presence of 50 mM Tris for 30 minutes at 37C, pelleted in a microfuge for 30 minutes, and subjected to electrophoresis on a 10-20% acrylamide Tris-tricine gel before immunoblotting with anti-A28 antibody. The nature of the A28 allele (WT or Cts9) and the heat at which the virions were produced are shown above each SBI-553 lane. B. Western blot analysis of protein distribution in fractionated virions. Wild type (WT) or mutant (Cts9) virions produced at 31C or 40C were separated in the absence (?) or presence (+) of DTT into core (pellet, P) and membrane (supernatant, S) fractions and run on a 10-20% Tris-Tricine gel. Proteins were subjected to Western blot analysis with anti-A28,.