Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection

Abstract

BackgroundHuman cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.MethodsViral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay.ResultsAbundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33.ConclusionsViral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

“…miR-UL112.1 targets MICB a cellular stress (infection)-induced ligand, which usually functions by binding the homodimeric NK cell activating receptor NKG2D; 54 miR-US4.1 downregulates ERAP-1 which is an aminopeptidase which trims peptides for presentation by MHC Class I and reduced ERAP-1 expression decreasing HCMV-specific CD8 1 CTL recognition of HCMVinfected cells; 55 miR-UL148D targets the chemokine CCL5 (RANTES) which is a T-cell chemoattractant. 56 Interestingly, miR-UL112.1 and miR-US4.1 have been detected in quiescently infected THP1 cells 57,58 and we have also detected all three viral mRNAs in experimentally latent CD34 1 cells and CD14 1 monocytes (IHW Gdansk, Poland, 2012 and CMV meeting San Francisco, USA, 2013; Lau et al, unpubl. data).…”

“…For instance, it could allow an orchestrated manipulation of cellular gene expression without the need to express viral proteins which, otherwise, could be targeted by host immune mechanisms. Reports using a quiescent THP1 model infected with either Towne 57 or Toledo 58 isolates of HCMV have identified a number of expressed viral miRNAs, although their functions during quiescence was not addressed and, at least for Towne, it is unclear how this might inform us as Towne does not usually efficiently infect myeloid cells 59 and is depleted of some important viral coding regions. 60 In our attempts to address this, we have used RT-qPCR to detect viral miRNAs expression in primary CD34 1 myeloid progenitors and CD14 1 monocytes experimentally latently infected with a clinical isolate of HCMV.…”

Human Cytomegalovirus Infection Dysregulates the Localization and Stability of NICD1 and Jag1 in Neural Progenitor Cells

“…The Institutional Review Board approved (WIVH10201202) the isolation of primary human embryonic lung fibroblasts (HELs) and NPCs from postmortem fetal embryo tissue and waived the need for consent. The original source of the postmortem fetal embryo tissue was Zhongnan Hospital (21,29).…”

“…HCMV Towne strain (ATCC VR977) was used for all the experiments and propagated in HELs as described previously (14,29,31). UV-inactivated Towne strain virus was prepared and used as described previously (13,30,32).…”

The Cell Biology of Cytomegalovirus: Implications for Transplantation

“…This tissue specificity suggests that CMV may activate multiple transcriptional programs depending on the The mechanisms underlying tissue specificity of viral gene expression may also relate to recently described and generally conserved microRNAs (miRNAs) of CMV (77)(78)(79)(80)(81). These small, noncoding RNA species have diverse effects including regulation of viral replication and immune evasion, which appear to vary by the cell type infected (77)(78)(79)(80)(81)(82)(83). HCMV-miR-UL112-3p, for example, appears to affect both the stress-induced ligand MICB and the antiviral interferon-response element IRF-1 (80,84).…”