Protocols for expression and functional analysis of viral microRNAs

MicroRNAs (miRNAs) are small RNAs of generally 21 to 23 nt that down-regulate target gene expression at the posttranscriptional level. Although miRNAs are endogenously expressed by all animals and plants, numerous DNA viruses have now been identified that also encode miRNAs, presumably to down-modulate protein expression from viral and host transcripts. Although this has been shown in some cases, the function of the majority of viral miRNAs remains unclear. The herpesviruses stand out by making extensive use of miRNA expression during long-term latent infection or lytic replication. Because viral miRNAs are only present in the context of viral infection and can therefore be considered "exogenous," their expression in uninfected cells of the appropriate cell type is a valuable tool to assess their function. Techniques to achieve and validate the expression of viral miRNAs are described in this review.