Molecular genetic analysis of vaccinia virus genes which confer resistance to alpha-amanitin

Graduation date: 1989Figures in original document are black and white photocopies. Best scan available.Several laboratories have shown that the VV\ud replication cycle is dependent on the host cell nucleus.\ud The basic biological observation is that if the host cell\ud nucleus is physically removed by cytochalasin B-mediated\ud enucleation, functionally inactivated by UV-irradiation, or\ud selectively blocked with a-amanitin, the results are the\ud same: W is able to initiate infection and carry out\ud macromolecular syntheses, but no infectious progeny virions\ud are assembled.\ud To elucidate the molecular mechanism of this virus-host\ud cell interaction, one approach is to identify the\ud viral gene(s) responsible for interaction with the host\ud nucleus and subject them to a detailed molecular analyses. The genomic location of the gene(s) which provide an\ud α-amanitin resistant phenotype to viral mutants have been\ud mapped to Hindlll N/M region of the genome by the use of\ud marker rescue techniques [E.C. Villarreal and D.E. Hruby\ud (1986) Journal of Virology 57: 65-70] . In an attempt to\ud understand the genetic organization of this part of the\ud genome, the entire Hindlll N and part of the neighboring\ud Hindlll M DNA fragments have been sequenced. The sequencing\ud data revealed two complete leftward reading open reading\ud frames (ORFs, N2 and Ml). In order to determine which of\ud the two ORFs is responsible for confering resistance to\ud a-amanitin, marker rescue analyses on a VV dual mutant\ud (arts7) that has both temperature sensitive and resistant\ud to α-amanitin was carried out. This showed that N2 ORF is\ud the gene responsible for both phenotypes. To analyze\ud regulatory sequences responsible for expression of the N2\ud and Ml genes, putative cis-acting regulatory sequences\ud upstream from the N2 and Ml genes were isolated and abutted\ud it to a bacterial reporter gene [chloramphenicol acetyl\ud transferase (CAT)]. To assay for regulatory sequences,\ud these promoter:CAT plasmids were initially used in\ud transient expression protocols and subsequently these\ud plasmids were used to make recombinant viruses