Abstract

The poxvirus, vaccinia, is a large DNA virus which replicates in the cytoplasm of the host cell. The virus is believed to encode most or all of the functions required for the temporally regulated transcription and replication of its 186 kilobase genome. Physical and genetic autonomy from the host make vaccinia a useful eukaryotic organism in which to study replication genes and proteins, using a combination of biochemical and genetic techniques. Essential viral functions for replication are identified by conditional lethal mutants that fail to synthesize DNA at the non-permissive temperature. One such group contains the non-complementing alleles ts17, ts24, ts69 (WR strain). Studies were undertaken to define the phenotype of ts mutants, and to identify and characterize the affected gene and protein. Mutant infection was essentially normal at 32$\sp\circ$C, but at 39$\sp\circ$C the mutants did not incorporate $\sp3$H-thymidine into nascent viral DNA or synthesize late viral proteins. If mutant cultures were shifted to non-permissive conditions at the height of replication, DNA synthesis was halted rapidly, implying that the mutants are defective in DNA elongation. The gene affected in the WR mutants and in ts6389, a DNA-minus mutant of the IHD strain, was mapped by marker rescue and corresponds to open reading frame 5 (orfD5) of the viral HindIII D fragment. The single nucleotide change responsible for the ts defect in each of the WR mutants was determined by DNA sequencing. The mRNAs transcribed from orfD5 early in infection were defined by RNA filter hybridization and S1 nuclease analysis. Hybrid selection and in vitro translation identified the encoded protein of 90 kDa molecular weight. A polyclonal antiserum was raised, and immunoprecipitation of metabolically labeled infected cell extracts defined the pattern of D5 protein synthesis during wild type and mutant infections. Pulse-chase analysis revealed the greater lability of the mutant encoded protein at 39$\sp\circ$C compared to wild type. The D5 protein was overexpressed from a vaccinia recombinant via T7 RNA polymerase to facilitate purification for biochemical and functional analysis.