Fig. 1.

Removal of the miR-UL112-1 target site leads to induction of immediate early (IE) gene expression in the absence of virion production. (a) The target site of miR-UL112-1 in the 3′ UTR of IE72 was deleted to generate a recombinant virus in which IE72 is no longer targeted by miR-UL112-1. (b) Monocytes (mono) were either uninfected, infected with wild-type TB40E (WT) or the miR-UL112-1 target site mutant virus (∆112-1) and latency was established for 4 days before harvesting and analysing mRNA levels for viral products UL138, IE and pp28 relative to cellular gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Alternatively, these gene products were analysed following reactivation from latency by differentiation to DCs (reactivation). (c) Both latent monocytes and reactivating DCs, described in (a), were also co-cultured with indicator fibroblasts and supernatants were harvested and seeded onto fresh fibroblasts for 24 h before fixing and immunofluorescence staining for IE protein, which was used to determined the IE foci forming units. Error bars shown in (b) and (c) denote the standard error of two independent experiments, with each containing three technical repeats.