Suppressors of a host range mutation in the rabbitpox virus serpin SPI-1 map to proteins essential for viral DNA replication.
The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, P.O. Box 100266, Gainesville, FL 32610. rmoyer@ufl.edu.
Benjamin G
BG
Luttge
Richard W
RW
Moyer
D000595:Amino Acid Sequence; D000818:Animals; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D016153:Genes, Suppressor; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D014616:Vaccinia virus / Q000235:genetics*; D014779:Virus Replication
D015843:Serpins; D004259:DNA-Directed DNA Polymerase