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.
Highlights
Human cytomegalovirus (HCMV) is a ubiquitous pathogen infecting 50% to 90% of the population worldwide, with an extremely high prevalence (.90%) in China
For kinetic studies of miRNA expression, Human embryonic lung fibroblasts (HELs) were infected with HCMV at a multiplicity of infection (MOI) of 5
After confirmation of miRNA expression by RT-PCR at 48 h after transduction, transduced HELs were infected with HCMV at MOIs of 0.01, 0.1, 1, or 5
Summary
Human cytomegalovirus (HCMV) is a ubiquitous pathogen infecting 50% to 90% of the population worldwide, with an extremely high prevalence (.90%) in China. HCMV infection is not believed to be deleterious to immunocompetent individuals It can cause serious, often life-threatening complications in immunocompromised individuals, including solid organ and cell transplant recipients, AIDS patients, and patients suffering from late stage cancers (reviewed by Mercorelli [1]). More than 10,000 miRNAs have been identified in a variety of organisms [5] They participate in developmental processes (hematopoiesis, organogenesis, cell proliferation, differentiation and apoptosis), regulation of virus infection and anti-viral immune responses [4]. Human cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. 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
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