Combinatorial targeting of 2 different steps in adenoviral DNA replication by herpes simplex virus thymidine kinase and artificial microRNA expression for the inhibition of virus multiplication in the presence of ganciclovir
نویسندگان
چکیده
BACKGROUND Human adenoviruses are a frequent threat to immunocompromised patients, and disseminated disease is associated with severe morbidity and mortality. Current drugs are not capable of preventing all fatalities, thus indicating the need for alternative treatment strategies. Adenoviruses can be rendered susceptible to antiherpetic prodrugs such as ganciclovir (GCV), upon expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in adenovirus-infected cells. Furthermore, adenoviruses are amenable to post-transcriptional gene silencing via small interfering RNAs (siRNAs) or artificial micro RNAs (amiRNAs). RESULTS In this study, we combined these 2 approaches by constructing a combinatorial gene expression cassette that comprises the HSV-TK gene and multiple copies of an amiRNA directed against the mRNA encoding the adenoviral preterminal protein (pTP). HSV-TK gene expression was controlled by the adenoviral E4 promoter, which is activated in the presence of the adenoviral E1 gene products (i.e., when adenovirus is present in the cell). When inserted into a replication-deficient (E1-, E3-deleted) adenoviral vector, this cassette effectively inhibited the replication of wild-type adenovirus in vitro. The reduction rate mediated by the combinatorial approach was higher compared to that achieved by either of the 2 approaches alone, and these obvious additive effects became most pronounced when the GCV concentration was low. CONCLUSIONS The concept presented here has the potential to aid in the inhibition of wild-type adenovirus replication. Furthermore, the combinatorial expression cassette may constitute a safeguard to potentially control unintended replication of adenoviral vectors and to prevent immune responses provoked by them.
منابع مشابه
PCR detection of thymidine kinase gen of latent herpes simplex Virus type 1 in mice trigeminal ganglia
Herpes simplex virus type 1 establishes a latent infection in the peripheral nervous system following primary infection. During latent infection, virus genome exhibit limited transcription, with the HSV LATs consistently detected in latency infected ganaglia. Following ocular infection viral latency develops in the trigeminal ganglia. In this study PCR has been used for detection of HSV-1 nuc...
متن کاملThe Effects and Pharmacokinetics of Acyclovir in Neonates
Acyclovir (9-[2-hydroxyethoxymethyl] guanine) is an acyclic nucleoside analogue of guanosine which is a potent and selective antiviral agent. Acycloviris converted to the monophosphate by thymidine kinase the virus-specific form of this enzyme and is subsequently converted to the triphosphate by the host cell kinase. Acyclovir triphosphate inhibits viral DNA-polymerase terminating the chain...
متن کاملAn Enterovirus-Like RNA Construct for Colon Cancer Suicide Gene Therapy
Background: In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy. Methods: Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3’ non-translated region of Poliovirus to design an en...
متن کاملRestriction Enzyme Analysis of Thymidine Kinase Gene in Four Iranian Isolates of Herpes Simplex Virus Type 1
متن کامل
Replication Characteristics of Herpes Simplex Virus Type-1 (HSV-1) Recombinants in 3 Types of Tissue Cultures
A complication in the analysis of the role of ICP34.5 gene in the herpes simplex virus type-1 (HSV-1) lifecycle is the presence of overlapping antisense gene, open reading frame P (ORF P), which is also deleted in HSV-1 ICP34.5 negative mutants. A HSV-1 wild type strain (17+) ICP34.5/ORF P deletion mutant (1716) is totally avirulent in animal models and impaired in a number of in vitro function...
متن کامل