The vaccinia virus H5R gene encodes late gene transcription factor 4: purification, cloning, and overexpression

The vaccinia virus late stage-specific transcription factor P3 was purified to homogeneity from HeLa cells that were infected in the presence of an inhibitor of viral DNA replication. The purified 36-kDa protein was digested with trypsin, and the peptides were analyzed by mass spectroscopy and amino-terminal sequencing. The purified factor was identified as the product of the vaccinia virus H5R open reading frame by both methods. A recombinant baculovirus was engineered to express the H5R open reading frame. The partially purified recombinant protein could replace the vaccinia virus P3 factor in transcription assays. On the basis of these findings, we assigned the H5R gene product the name viral late gene transcription factor 4 (VLTF-4). Unlike VLTF-1, -2, and -3, which are synthesized exclusively after viral DNA replication, VLTF-4 is synthesized before and after viral DNA synthesis. Indirect immunofluorescence of infected cells with anti-H5R protein antiserum demonstrated that VLTF-4 is diffusely distributed in the cytoplasm when DNA replication is blocked but is localized to discrete viral DNA-containing factories during a productive infection. Its expression pattern and subcellular distribution suggest that the H5R gene product may have multiple roles in the viral life cycle.