title: Comprehensive analysis of human cytomegalovirus microRNA expression during lytic and quiescent infection.
abstract: BACKGROUND
  Human 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.
  
  
  METHODS
  Viral 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.
  
  
  RESULTS
  Abundant 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.
  
  
  CONCLUSIONS
  Viral 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.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Zhang-Zhou
authors.initials: ZZ
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Pan
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Ling-Feng
authors.initials: LF
authors.lastname: Miao
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Ye
authors.affiliation: INSERM-U1043/CNRS-U5282/Paul Sabatier University, Toulouse, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Chavanas
authors.affiliation: INSERM-U1043/CNRS-U5282/Paul Sabatier University, Toulouse, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Davrinche
authors.affiliation: Department of Pediatrics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: McVoy
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
authors.forename: Min-Hua
authors.initials: MH
authors.lastname: Luo
mesh_terms: D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000821:virology; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D035683:MicroRNAs / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral