Shen Zhang-Zhou, Pan Xing, Miao Ling-Feng, Ye Han-Qing, Chavanas Stéphane, Davrinche Christian, McVoy Michael, Luo Min-Hua
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
INSERM-U1043/CNRS-U5282/Paul Sabatier University, Toulouse, France.
PLoS One. 2014 Feb 12;9(2):e88531. doi: 10.1371/journal.pone.0088531. eCollection 2014.
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.
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.
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.
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.
人巨细胞病毒（HCMV）编码微小RNA（miRNA），在允许性细胞的裂解感染期间，这些miRNA作为基因表达的转录后调节因子发挥作用。一些miRNA已被证明可抑制病毒复制，这可能有助于HCMV建立或维持潜伏感染。然而，尚未使用代表允许性（裂解性）、半允许性与非允许性（潜伏样）感染的细胞培养系统，对HCMV miRNA表达进行全面检查和比较。
通过miRNA特异性茎环RT-PCR测定HCMV感染期间病毒miRNA水平和表达动力学。检测了HCMV感染的THP-1细胞（非允许性）、分化的THP-1细胞（d-THP-1，半允许性）和人胚肺成纤维细胞（HEL，完全允许性）。通过蛋白质免疫印迹、RT-PCR、qPCR和蚀斑试验确定所选miRNA对HCMV感染（基因表达、基因组复制和病毒释放）的影响。
在HEL细胞的裂解感染期间观察到15种HCMV miRNA的丰富表达；在感染后48小时出现最高峰诱导（11至1502倍）。在d-THP-1细胞中，检测到14种miRNA有中度诱导（3至288倍），但表达动力学相对于HEL细胞通常延迟24小时。相比之下，在THP-1细胞的静止感染期间，仅三种miRNA被诱导至低水平（3至4倍）。有趣的是，miR-UL70-3p在HEL细胞中诱导较差（1.5倍），在THP-1细胞中中度诱导（4倍），而在d-THP-1细胞中强烈诱导（58倍），表明miR-UL70-3p在THP-1细胞和d-THP-1细胞中可能具有特定作用。进一步评估了miR-US33、-UL22A和-UL70对HEL细胞中HCMV复制的影响。miR-UL22A和miR-UL70的异位表达不影响HEL细胞中的HCMV复制，而miR-US33抑制HCMV复制并降低HCMV US29 mRNA水平，证实US29是miR-US33的靶标。
允许性、半允许性和静止感染之间的病毒miRNA表达动力学不同，并且miR-US33下调HCMV复制。这些结果表明，miR-US33可能起到损害进入裂解复制的作用，从而促进潜伏状态的建立。
