Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells

Abstract

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to oaamanitin concentrations up to 100 p.g/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be oa-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.

Isolation and characterization of monoclonal antibodies directed against two subunits of rabbit poxvirus-associated, DNA-directed RNA polymerase

“…The localization of the RNA polymerase subunits to the nucleus or cytoplasm (or both) of infected cells is of particular interest because there is still a question as to whether a nuclear phase is necessary for successful poxvirus development to occur. Recently, there have been reports not only of viral DNA (1,18) and RNA (3,9) synthesis but also of significant levels of viral DNA-directed RNA polymerase activity within the nucleus of infected cells (38). Immunofluorescence studies of intact infected cells with antibodies directed against the viral RNA polymerase would allow us to directly address the question of the possible nuclear localization of this enzyme.…”

Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization

“…(3) suggested that not only was there viral RNA within the nucleus, but that some of these RNAs might be unique to the nucleus. More recently, it has been reported that full-length vaccinia virus DNA is synthesized in vitro in nuclei purified from infected cells (1) and that a substantial amount of the virus-specified DNA-directed RNA polymerase can be isolated from the nuclei of infected cells (24).…”