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. 2013 Feb 12:10:51.
doi: 10.1186/1743-422X-10-51.

The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1

Yujing Huang  1 , Ying Qi, Yanping Ma, Rong He, Yaohua Ji, Zhengrong Sun, Qiang Ruan
Affiliations

The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1

Yujing Huang et al. Virol J. .

Abstract

Background: Interleukin-32 (IL-32) is an important factor in innate and adaptive immune responses, which activates the p38MAPK, NF-kappa B and AP-1 signaling pathways. Recent reports have highlighted that IL-32 is regulated during viral infection in humans.

Methods: Enzyme-linked immunosorbent assays (ELISA) were carried out to detect IL-32 levels in serum samples. Detailed kinetics of the transcription of IL-32 mRNA and expression of IL-32 protein during human cytomegalovirus (HCMV) infection were determined by semi-quantitative RT-PCR and western blot, respectively. The expression levels of hcmv-miR-UL112-1 were detected using TaqMan® miRNA assays during a time course of 96 hours. The effects of hcmv-miR-UL112-1 on IL-32 expression were demonstrated by luciferase assay and western blot, respectively.

Results: Serum levels of IL-32 in HCMV-IgM positive patients (indicating an active HCMV infection) were significantly higher than those in HCMV-IgM negative controls. HCMV infection activated cellular IL-32 transcription mainly in the immediately early (IE) phase and elevated IL-32 protein levels between 6 and 72 hours post infection (hpi) in the human embryonic lung fibroblast cell line, MRC-5. The expression of hcmv-miR-UL112-1 was detected at 24 hpi and increased gradually as the HCMV-infection process was prolonged. In addition, it was demonstrated that hcmv-miR-UL112-1 targets a sequence in the IL-32 3'-UTR. The protein level of IL-32 in HEK293 cells could be functionally down-regulated by transfected hcmv-miR-UL112-1.

Conclusions: IL-32 expression was induced by active HCMV infection and could be functionally down-regulated by ectopically expressed hcmv-miR-UL112-1. Our data may indicate a new strategy of immune evasion by HCMV through post-transcriptional regulation.

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Figures

Figure 1
Figure 1
An illustration showing IL-32 levels in serum samples detected by ELISA. Serum samples were from actively HCMV-infected patients (n=40), HCMV previously infected individuals (n=17) and healthy individuals (n=15), respectively.
Figure 2
Figure 2
Expression of IL-32 induced by HCMV infection in MRC-5 cells. (A) IL-32 mRNA levels in HCMV infected cells were compared to that in uninfected MRC-5 cells. β-actin was used as an internal control. The universal primers for all IL-32 transcripts were used. U is for uninfected MRC-5; IE is for immediate early stage; E is for early stage; L is for late stage. (B) IL-32 mRNA levels were represented relative to that in IE stage. (C) IL-32 protein levels in HCMV infected MRC-5 cells were detected at different time points. IL-32 densitometer values were normalized by β-actin values. (D) The IL-32 protein levels were represented relative to that in infected cells collected at 6 hpi. (E) The kinetics of hcmv-miR-UL112-1 expression were measured using TaqMan® miRNA assays. The expression of hcmv-miR-UL112-1 could be detected at 24 hpi and increased gradually as the HCMV-infection process was prolonged.
Figure 3
Figure 3
Down-regulation of IL-32 expression by hcmv-miR-UL112-1. (A) The diagram shows the predicted sequences of hcmv-miR-UL112-1 binding to IL-32 mRNA. (B) As a candidate target, IL-32 was validated for its ability to inhibit expression of a luciferase reporter construct in the presence of hcmv-miR-UL112-1 (pS-miR-UL112-1). Results are shown as percentage expression of negative control sample (pS-neg) following correction for transfection levels according to the control of renilla luciferase expression. Values are shown as means ± standard deviations for triplicate samples. (C) HEK 293 cells were transfected with pS-miR-UL112-1 or control vector respectively. Cells were collected 48 hpi and were subjected to western blot analysis using the indicated antibodies. IL-32 densitometer values were normalized to that of the β-actin values. (D) The amounts of proteins presented in panel C were quantified by densitometry. Results are shown as percentage expression of the negative control sample (Empty).
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