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The adenovirus (Ad) serotype 5 genome encodes two non-coding small RNAs (virus-associated RNAs: VA-RNAI and II), which are approximately 160nt-long RNAs transcribed by RNA polymerase III. It is well-known that VA-RNAI supports Ad infection via the inhibition of double-stranded RNA-dependent protein kinase (PKR), which recognizes double-stranded RNA and acts as an antiviral system. Recent studies revealed that VA-RNAs are processed into VA-RNA-derived microRNAs (miRNAs) (mivaRNAI, II); however, we and another group recently demonstrated that mivaRNAI does not promote Ad replication. On the other hand, the roles of VA-RNAII and VA-RNAII-derived miRNA (mivaRNAII) in Ad replication have remained to be clarified. In this study, we demonstrate mivaRNAII-mediated promotion of Ad replication. Transfection with chemically synthesized 3'-mivaRNAII-138, one of the most abundant mivaRNAII, significantly enhanced Ad replication, while the other species of mivaRNAII did not. We identified 8 putative target genes of 3'-mivaRNAII-138 by microarray analysis and in silico analysis. Among the 8 candidates, knockdown of the cullin4A (CUL4A) gene, which encodes a component of the ubiquitin ligase complex, most significantly enhanced Ad replication. CUL4A expression was significantly suppressed by 3'-mivaRNAII-138 via post-transcriptional gene silencing, indicating that CUL4A is a target gene of 3'-mivaRNAII-138 and mivaRNAII functions as a viral miRNA promoting Ad infection. It has been reported that CUL4A is involved in degradation of c-Jun, which acts as a transcription factor in the Jun-N-terminal kinase (JNK) signaling cascade. Treatment with JNK inhibitors dramatically suppressed Ad replication, suggesting that mivaRNAII-mediated down-regulation of CUL4A enhanced JNK signaling, and thereby promoted Ad infection. [ABSTRACT FROM AUTHOR]

Highlights: [•] Higher amounts of shRNA were incorporated into RISC using HD-Ad-shRNA. [•] Higher levels of knockdown efficiencies were found using HD-Ad-shRNA. [•] Higher amounts of shRNA were incorporated into RISC using AdΔVR-shRNA. [•] Higher levels of knockdown efficiencies were found using AdΔVR-shRNA. [Copyright &y& Elsevier]