A haploid HSV‐1 genome platform for vector development: testing of the tetracycline‐responsive switch shows interference by infected cell protein 0. (October 2016)

• Record Type: Journal Article
• Authors: Khalique, Hena; López Marco, Jorge; Lim, Filip
• Is Part Of: Journal of gene medicine. Volume 18:Number 10(2016:Oct.)
• Page Start: 302
• Page End: 311
• Publication Date: 2016-10
• Subjects: DNA technology -- gene transfer -- herpes simplex virus (HSV) -- Tet system -- viral vector; Genetic transformation -- Periodicals; Gene Transfer -- Periodicals; Gene Therapy -- Periodicals; 616.042
• DOI: 10.1002/jgm.2929
• Languages: English
• ISSNs: 1099-498X

Abstract

Abstract: Background: Although herpes simplex virus type 1 (HSV‐1) has outstanding properties for gene delivery vectors and its genome is available in bacterial artificial chromosomes (BACs) for mutagenesis studies, one impediment is the presence of approximately 15.4 kb of DNA sequences that are duplicated in the HSV‐1 genome, complicating vector construction and stability. Methods: As a useful platform for building HSV‐1 vectors, we have constructed a fully haploid HSV‐1 genome BAC by deletion of one of these repeats, confirming that viral propagation in culture is not impaired. We used this ΔIR mutant to subsequently investigate whether the insertion of tetracycline‐responsive tetO elements into the ICP34.5 – ICP0 gene region can be used to control HSV‐1 lytic replication. Results: The results of the present study show that ΔIR mutants deleted for ICP34.5 are viable for replication but not when the ICP0 promoter is also disrupted, thus indicating that regulation of infected cell protein 0 (ICP0) levels in the absence of ICP34.5 could be a viable means for controlling growth of HSV‐1 vectors. Surprisingly, however, the tetO elements inserted into the ICP0 promoter did not confer ligand responsiveness to growth or ICP0 expression. Further analysis by transfection experiments revealed that ICP0 itself interferes with the tetracycline switch and reduces the the inducibility of this system. Conclusions: Our new haploid HSV‐1 BAC is a useful platform for building multiply deleted HSV‐1 vectors. Deletion of the gene for ICP34.5 in this backbone renders viral growth dependent on ICP0, although ICP0 expression could not be regulated by tet‐responsive transcriptional regulators.