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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.