Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

Abstract

Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate–glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.

Human Cytomegalovirus Delays Neutrophil Apoptosis and Stimulates the Release of a Prosurvival Secretome

“…Of particular interest are UL36, an inhibitor of caspase-induced apoptosis ( 75 ), its antiapoptotic intronic micro-RNA mir-UL36-5p ( 90 ), and the pro-survival non-coding RNA b2.7 ( 78 ). All three are present in or carried on the virion ( 91 , 92 ), carry at least minor mutations in AD169 ( 88 , 89 , 91 ), and are targets for further investigation.…”

The Clinical Application of MicroRNAs in Infectious Disease

“…Manipulating viral miRNAs using miRNA mimics or antagomiRs could hold therapeutic potential. Several studies show viral miRNAs are excreted in exosomes of infected cells, and in some cases this appears to modulate innate immune responses in recipient cells including dendritic cells and macrophages; however, the functional significance of this remains to be seen ( 186 – 190 ).…”

The Diverse Roles of microRNAs at the Host–Virus Interface

“…Evidence suggests that these so-called “virional” miRNAs are selectively packaged inside virions during encapsidation or envelopment, but it is still unclear if they directly contribute to infection or pathogenesis in vivo. However, similar virional miRNAs have been reported in other viral infections, including HCMV and HIV-1, suggesting that this may be a common strategy used to control gene expression early in infection [ 75 , 76 ].…”