Genetic analysis of varicella-zoster virus ORF0 to ORF4 by use of a novel luciferase bacterial artificial chromosome system.
To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(Luc)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(Luc) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.
Zhang
Zhen
Z
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, NJ 07101-1709, USA.
Rowe
Jenny
J
Wang
Weijia
W
Sommer
Marvin
M
Arvin
Ann
A
Moffat
Jennifer
J
Zhu
Hua
H
D000818:Animals; D045744:Cell Line, Tumor; D022202:Chromosomes, Artificial, Bacterial; D004195:Disease Models, Animal; D017353:Gene Deletion; D020043:Genes, Essential; D005822:Genetic Vectors; D016679:Genome, Viral; D006562:Herpes Zoster / Q000821:virology; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D013194:Staining and Labeling / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics*
D014764:Viral Proteins; D008156:Luciferases