DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER

Gammaherpesvirüsler çeşitli tiplerde kanserlerin gelişimi ile ilişkilidir ve bu virüslerin patogenezi birçok çalışmaya konu olmuştur. Bu virüsler litik ve latent adı verilen iki farklı yaşam döngüleriyle enfekte hücrenin anahtar mekanizmalarını kendi lehlerine düzenlemek için çok sayıda viral kökenli mikroRNA (miRNA) kodlarlar. Bu nedenle, daha iyi terapötik maddeler geliştirmek için miRNA ve bunların mRNA hedef etkileşimlerinin anlaşılması önemlidir. Burada, viral tRNA tarafından üretilen ve Blimp1 transkriptini hedefleyen, bir shRNA’nın viral enfeksiyon aşamasında hedef genin ifadesini istatistiksel açıdan anlamlı oranda azaltabildiğini lusiferaz deneyi ile gösterdik. Bu deney dizaynı önemli olan miRNA-mRNA etkileşimlerini test etmek açısından bir kavram ispatı sunmayı hedeflemektedir. Ayrıca oldukça kısa bir promotör boyutuna sahip olan Murine Gammaherpesvirus 68 viral tRNA4, yaklaşık 180 nukleotid uzunluğunda bir diziden iki adet shRNA üretebilmektedir. İstenilen shRNA’nın anlatımı için yalnızca sınırlı bir alan mevcutsa, viral tRNA promotrü etkin bir shRNA anlatımı sistemi sunmaktadır.

DESIGN AND GENERATION OF A RECOMBINANT GAMMAHERPESVIRUS ENCODING shRNA FROM A NATIVE VIRAL tRNA PROMOTER

Gammaherpesviruses are associated with multiple types of tumor development and understanding the pathogenesis of these viruses has been the subject of various studies. Throughout the lytic and latent life cycle, these viruses utilize numerous virally encoded microRNAs (miRNAs) to regulate the key mechanisms of the infected cell in their favor. Therefore, it is important to understand the miRNA and their mRNA target interactions for developing better therapeutics. In this study, the strategy and design of a recombinant virus expressing a short hairpin RNA (shRNA) element targeting the host B-lymphocyte-induced maturation protein 1 (Blimp1) transcript was evaluated. Here we have shown that viral tRNA-driven expression of anti-Blimp1 shRNA is able to reduce the target gene expression at a statistically significant level as assessed by luciferase assay during virus infection. This proof-of-principle experiment provides a means to study important miRNA-mRNA interactions in vivo. Further, the very short promoter of the murine gammaherpesvirus 68 (MHV68) viral tRNA (vtRNA4) has the ability to generate two shRNAs from a ~180 nucleotide sequence. If there is a size limit for the shRNA construct, viral tRNA promoter provides an effective shRNA expression system.

Keywords:

Blimp1 protein, Gammaherpesvirus, MHV68 Red mediated mutagenesis, shRNA,

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