Functional role of HCMV miRNAs

miRNAs are a species of small-regulatory RNA that post-transcriptionally regulate gene expression via the RNA induced silencing complex (RISC). They are encoded ubiquitously among animals and plants, and have recently been shown to be encoded by the majority of herpesviruses. It seems likely that herpesvirus encoded miRNAs have evolved as a tool for the manipulation of host-cellular and viral-gene expression during infection. Human cytomegalovirus (HCMV) is a clinically important herpesvirus that represents a significant cause of morbidity and mortality in the immune-compromised. HCMV encodes as many as 25 miRNAs during infection, but the function of the majority of these is not known. Identifying the targets of HCMV miRNAs will not only establish a basis for understanding the role of miRNAs within the context of HCMV infection, but also provide a means for discovering novel host-virus interactions. Using RISC immunoprecipitation and siRNA screening, host-cellular targets of viral miRNAs that play important roles in the biology of HCMV were identified. ATP6VOC, a key component of the vacuolar-ATPase, was shown to be a target of miR-US25-1 and subsequent siRNA knockdown of ATP6VOC resulted in the almost complete inhibition of infectious virion production. Despite this, ATP6VOC knock-down did not inhibit viral entry, DNA synthesis, or gene expression, highlighting a possible role for ATP6VOC in the assembly and egress of HCMV. A critical step in HCMV assembly and egress is the formation of the juxta-nuclear virion assembly compartment (VAC). The HCMV VAC is derived from host-cellular endocytic and secretory vacuoles, and is crucial for the efficient nuclear egress of nucleocapsids, cyotplasmic tegumentation, final envelopment, and the egress of mature virions. Using siRNA knock-down, immunofluorescence-microscopy, and western-blot analysis, a crucial role for ATP6VOC and v-ATPase function in the formation of the VAC was demonstrated. siRNA knock-down of ATP6VOC resulted in a failure in the reorganisation of trans-golgi and early-endosomal compartments during infection, resulting in a failure in VAC formation. These findings demonstrate a crucial role for ATP6VOC during infection, and in so doing identify a novel host factor that is required for HCMV assembly.