Open AccessA Self-Excisable Infectious Bacterial Artificial Chromosome Clone of Varicella-Zoster Virus Allows Analysis of the Essential Tegument Protein Encoded by ORF9
Author(s) -
B. Karsten Tischer,
Benedikt B. Kaufer,
Marvin Sommer,
Felix Wussow,
Ann M. Arvin,
Nikolaus Osterrieder
Publication year - 2007
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.01148-07
Subject(s) - biology , bacterial artificial chromosome , virology , cosmid , virus , replicon , varicella zoster virus , recombinant virus , viral replication , complementation , mutant , microbiology and biotechnology , genetics , gene , recombinant dna , plasmid , genome
In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established inEscherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded byORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogatedORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature ofORF9 for VZV replication was ultimately confirmed by restoration of the growth of theORF9 -deficient mutant virus usingtrans -complementation via baculovirus-mediated gene transfer.