A haploid HSV-1 genome platform for vector development: testing of the tetracycline-responsive switch shows interference by infected cell protein 0.
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.
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Hena
H
Khalique
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
Jorge
J
López Marco
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain. filip.lim@uam.es.
Filip
http://orcid.org/0000-0001-6570-1017
F
Lim
D045744:Cell Line, Tumor; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D006238:Haploidy; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D009154:Mutation; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000235:genetics; D013754:Tetracyclines / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
D017874:Immediate-Early Proteins; D013754:Tetracyclines; D014764:Viral Proteins; C066469:gamma 34.5 protein, Human herpesvirus 1; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1
DNA technology; Tet system; gene transfer; herpes simplex virus (HSV); viral vector