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. 2018 Apr;3(4):503-513.
doi: 10.1038/s41564-018-0131-9. Epub 2018 Mar 27.

Human cytomegalovirus reprogrammes haematopoietic progenitor cells into immunosuppressive monocytes to achieve latency

Dihan Zhu  1 , Chaoyun Pan  1 , Jingxue Sheng  2 , Hongwei Liang  1   3 , Zhen Bian  1   3 , Yuan Liu  3 , Phong Trang  2   4 , Jianguo Wu  5 , Fenyong Liu  6 , Chen-Yu Zhang  7 , Ke Zen  8   9
Affiliations

Human cytomegalovirus reprogrammes haematopoietic progenitor cells into immunosuppressive monocytes to achieve latency

Dihan Zhu et al. Nat Microbiol. 2018 Apr.

Abstract

The precise cell type hosting latent human cytomegalovirus (HCMV) remains elusive. Here, we report that HCMV reprogrammes human haematopoietic progenitor cells (HPCs) into a unique monocyte subset to achieve latency. Unlike conventional monocytes, this monocyte subset possesses higher levels of B7-H4, IL-10 and inducible nitric oxide synthase (iNOS), a longer lifespan and strong immunosuppressive capacity. Cell sorting of peripheral blood from latently infected human donors confirms that only this monocyte subset, representing less than 0.1% of peripheral mononuclear cells, is HCMV genome-positive but immediate-early-negative. Mechanistic studies demonstrate that HCMV promotes the differentiation of HPCs into this monocyte subset by activating cellular signal transducer and activator of transcription 3 (STAT3). In turn, this monocyte subset generates a high level of nitric oxide (NO) to silence HCMV immediate-early transcription and promote viral latency. By contrast, the US28-knockout HCMV mutant, which is incapable of activating STAT3, fails to reprogramme the HPCs and achieve latency. Our findings reveal that via activating the STAT3-iNOS-NO axis, HCMV differentiates human HPCs into a longevous, immunosuppressive monocyte subset for viral latency.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

Fig. 1 |
Fig. 1 |. HCMV NR-1 infection reprogrammes human CD34+ HPCs to achieve latent infection.
a, NR-1 successfully established latency in HPCs. HPCs isolated from bone marrow were infected with NR-1 or deactivated NR-1 (Mock) at a multiplicity of 2 p.f.u per cell. Virus was reactivated by TPA (20 ng ml−1) followed by coculture with HFF-1 cells. Left, levels of HCMV genome and IE1 in HPCs following NR-1 or Mock infection. Middle and right panels represent the quantitative PCR with reverse transcription result of IE1 expression and virus replication of GFP-expressing NR-1 after reactivating virus from latent infection in HPCs (NR-1, 14 dpi), respectively. b, Levels of HCMV IE1, IE2 and lytic infection-associated genes UL54, UL94, UL100 and UL105 in HPCs following the infection with NR-1 or Mock. c, Levels of HCMV latency-associated genes UL138, LUNA and US28 in HPCs following the infection with NR-1 or Mock. d, Alteration of transcription profiling of CD34+ HPCs by NR-1 latent infection (14 dpi). The red-coloured molecules are upregulated (fold change > 2) by NR-1 infection compared to Mock infection, whereas the blue-coloured molecules are downregulated (fold change < 0.5) by NR-1 infection compared to Mock infection. e, The activating (red-coloured) and suppressive (blue-coloured) signal pathways in NR-1 latently infected HPCs compared to those in Mock-infected HPCs. Data are presented as the mean ± s.e.m. of three independent experiments. CTL, control; ND, not detected; dpr, days post reactivation.
Fig. 2 |
Fig. 2 |. HCMV NR-1 infection reprogrammes human CD34+ HPCs into a long-life monocyte subset at the late stage of infection.
a, Expression of surface markers on conventional mature monocytes and HPCs infected with NR-1 or Mock at 14 dpi. BM, bone marrow. b,c, NR-1-infected HPCs at 14 dpi displayed delayed apoptosis (b) and increased cell viability (c) compared to mature monocytes and Mock-infected HPCs. d, Cytokine level in mature monocytes and HPCs infected with NR-1 or Mock at 14 dpi. Data are presented as the mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 as determined by the two-tailed t-test (the P values are detailed in Supplementary Table 1).
Fig. 3 |
Fig. 3 |. NR-1-infected CD34+ HPCs at a late stage of infection (14 dpi or later) posess a strong immunosuppressive capacity to T-cell proliferation in a manner of Mo-MDSCs but not granulocytic MDSCs.
a, NR-1-infected HPCs (14 dpi) suppress CD4 and CD8 T-cell proliferation induced by ConA. The number in the top left corner of each plot represents the percentage of proliferated cells, and the number in the top right corner represents the percentage of non-proliferated cells. b–d, NR-1-infected HPCs expressed high levels of cellular iNOS (b) and NO (c) but not ROS (d) compared to Mock-infected HPCs. Data are presented as the mean ± s.e.m. of three independent experiments. **P < 0.01 as determined by the two-tailed t-test (the P values are detailed in Supplementary Table 1). ND, not detected.
Fig. 4 |
Fig. 4 |. Cellular iNOS/NO induced by STAT3 activity play a critical role in suppressing HCMV IE1 expression and viral replication in HPCs.
a,b, Levels of iNOS (a) and NO (b) in HPCs transfected with iNOS siRNA or scramble oligonucleotide. c, Increase of HCMV IE1 expression and genome replication in NR-1-infected HPCs after iNOS siRNA transfection. d, Depletion of cellular NO by NG-M-L-Arg. e, Increase of HCMV IE1 expression and genome replication in NR-1-infected HPCs after direct depletion of cellular NO by NG-M-L-Arg. f, Lentivirus-mediated iNOS overexpression in HL-60 cells increased intracellular NO level. g, Increase of NO level in HL-60 cells suppressed viral IE1 activity and genome replication. h,i, Inhibition of cellular NO on the activity of HCMV IE1 promoter. A luciferase reporter consisting of IE1/2 promoter region was constructed in pMIR-REPORT plasmid (h) and then transfected into HL-60 and THP-1 cells, respectively. HL-60 and THP-1 cells were treated with LV-iNOS or LV-iNOS siRNA to overexpress or knock down iNOS, respectively, prior to NR-1 infection at MOI of 2. Cellular luciferase activity (i) was assayed. Data are presented as the mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 as determined by the two-tailed t-test (the P values are detailed in Supplementary Table 1). LV, lentivirus vector.
Fig. 5 |
Fig. 5 |. Role of STAT3 signalling pathway in modulating HPC differentiation during HCMV latent infection.
a,b, Western blot images (a) and analysis (b) the levels of STAT3, pSTAT3, STAT1 and pSTAT1 in NR-1-infected HPCs. Note that NR-1 infection strongly induces STAT3 but not STAT1 activity in HPCs. c, Treatment with lentivirus-mediated STAT3 siRNA or control oligonucleotide delivery did not affect the viability of HPCs. d–g, Reduction of STAT3 and pSTAT3 via STAT3 siRNA decreased the cellular levels of iNOS (d) and NO (e), but enhanced IE1 expression (f) and viral replication (g) in NR-1-infected HPCs at 14 dpi. Data are presented as the mean ± s.e.m. of three independent experiments. *P < 0.05, **P < 0.01 as determined by the two-tailed t-test (the P values are detailed in Supplementary Table 1). CTL, control; ND, not detected.
Fig. 6 |
Fig. 6 |. US28-KO cannot establish latency in human CD34+ HPCs because it fails to activate the STAT3–iNOS–NO axis and reprogramme HPCs to an immunosuppressive monocyte subset.
a–c, US28-KO or Mock infection in human HPCs failed to activate STAT3 (a), or increase cellular levels of iNOS (b) and NO (c). d,e, US28-KO infection failed to reprogramme HPCs to the B7-H4+CD16+ immunosuppressive monocyte subset, as indicated by negative or low expression of B7-H4 and CD16 (d), as well as no suppression of CD4 T-cell proliferation (e). f, US28-KO infection failed to achieve latency in HPCs. g, Levels of viral genome and IE1 expression in HPCs infected with US28-KO or NR-1. h, NOS overexpression suppressed viral IE1 and genome expression in the HPCs infected with US28-KO. i, iNOS overexpression decreased the apoptotic rate of US28-KO-infected HPCs. j,k, Depleting cellular NO by NG-M-L-Arg abolished the effect of iNOS overexpression on facilitating US28-KO latency. NG-M-L-Arg depleted NO in US28-KO-infected HPCs that were overexpressed with iNOS (j), leading to significant increase of HCMV IE1 and genome levels (k). Data are presented as the mean ± s.e.m. of three independent experiments. **P < 0.01, ***P < 0.001 as determined by the two-tailed t-test (the P values are detailed in Supplementary Table 1).
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