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. 2005 Mar 15;102(11):4140-5.
doi: 10.1073/pnas.0408994102. Epub 2005 Feb 28.

Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers

M B Reeves  1 , P A MacAry, P J Lehner, J G P Sissons, J H Sinclair
Affiliations

Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers

M B Reeves et al. Proc Natl Acad Sci U S A. .

Abstract

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.

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Figures

Fig. 1.
Fig. 1.
Naturally acquired HCMV genome is carried throughout myeloid differentiation to DC. (a) DNA from monocytes (lane 1), immature monocyte-derived DC (lane 2), and mature monocyte-derived DC (lane 3) from a seronegative donor and monocytes (lane 4), immature monocyte-derived DC (lane 5), and mature monocyte-derived DC (lane 6) from a seropositive donor were amplified in an IE-specific PCR. Negative water controls (lanes 7–9) and positive HCMV DNA controls (lanes 10 and 11) are shown. (b) DNA from monocytes (lane 1), immature monocyte-derived DC (lane 2), and mature monocyte-derived DC (lane 3) from a seronegative donor shown in a and monocytes (lane 4), immature monocyte-derived DC (lane 5), and mature monocyte-derived DC (lane 6) from a seropositive donor shown in a were amplified in a β-globin PCR. (c) DNA from CD34+ cells (lane 2) and mature CD34+ cell-derived DC (lane 3) from a seronegative donor and CD34+ cells (lane 1) and mature CD34+ cell-derived DC (lane 4) from a seropositive donor were amplified in an IE-specific PCR. Negative water controls (lanes 5 and 6) and a positive HCMV DNA control are shown (lane 7). (d) DNA from CD34+ cells (lane 1) and mature CD34+ cell-derived DC (lane 2) from a seronegative donor shown in c and CD34+ cells (lane 3) and mature CD34+ cell-derived DC (lane 4) from a seropositive donor shown in c were amplified by β-globin PCR.
Fig. 2.
Fig. 2.
Differentiation to mature DC induces reactivation of IE gene expression from naturally acquired latent HCMV. (a) RNA from monocytes (lane 1), immature DC (lane 2), and mature monocyte-derived DC (lane 3) from a seronegative donor and monocytes (lane 5), immature DC (lane 6), and mature monocyte-derived DC (lane 7) from a seropositive donor were amplified in an IE-specific RT-PCR. Negative water controls (lanes 4 and 14–16) and a positive HCMV DNA control (lane 17) are shown. RNA samples 1–3 and 5–7 but with no prior RT are shown (in lanes 8–10 and 11–13, respectively) (b) RNA from monocytes (lane 1), immature monocyte-derived DC (lane 2), and mature monocyte-derived DC (lane 3) from the seronegative donor shown in A and monocytes (lane 4), immature monocyte-derived DC (lane 5), and mature monocyte-derived DC (lane 6) from the seropositive donor also shown in A were amplified in a histidyl tRNA synthetase RT-PCR. (c) Duplicate RNA samples from CD34+ cells (lanes 1–4) and mature CD34+ cell-derived DC (lanes 5–8) from two seropositive donors were amplified in an IE-specific RT-PCR. RNA samples 1, 3, 5, and 7 but with no prior RT were amplified by an IE-specific RT-PCR (lanes 9–12, respectively). HCMV DNA (lane 13) and RNA from infected HF (lanes 14 and 15) are shown. (d) RNA from the CD34+ cells (lanes 1 and 2) and mature CD34+ cell-derived DC (lane 3 and 4) from the two seropositive donors shown in c were amplified in a histidyl tRNA synthetase RT-PCR.
Fig. 3.
Fig. 3.
Chromatin remodeling of the latent viral MIEP occurs upon differentiation of monocytes or CD34+ cells to mature DC with reactivation of IE gene expression. DNA associated with histones was immunoprecipitated and used in PCR assays with primers complementary to the MIEP (a. b, and d) or HS4 region of the β-globin gene (c). (a) Seropositive CD34+ cells (lanes 1–3) and seronegative CD34+ cells (lanes 4–6) were incubated with control serum (lanes 1 and 4), anti-HP1 (lanes 2 and 5) or antiacetylated histone H4 (lanes 3 and 6) antibodies. (b) Seropositive mature CD34+ cell-derived DC (lanes 1–3) and seronegative mature CD34+ cell-derived DC (lanes 4–6) were incubated with control serum (lanes 1 and 4), anti-HP1 (lanes 2 and 5) or antiacetylated histone H4 (lanes 3 and 6) antibodies. (c) Seropositive CD34+ cells (lanes 2–4) and seropositive mature CD34+ cell-derived DC (lanes 5–8) were incubated with control serum (lanes 2 and 6), anti-HP1 (lanes 3 and 7) or antiacetylated histone H4 (lanes 4 and 8) antibodies. Ten percent of input controls with no ChIP are shown (lanes 1 and 5) (d) Seropositive monocytes (lanes 1–3) and seropositive mature monocyte-derived DC (lanes 4–6) were incubated with control serum (lanes 1 and 4), anti-HP1 (lanes 2 and 5), or antiacetylated histone H4 (lanes 3 and 6) antibodies.
Fig. 4.
Fig. 4.
HDAC1 expression decreases upon differentiation of monocytes to mature monocyte-derived DC. A total of 105 monocytes (M) or mature monocyte-derived DC (DC) were analyzed by Western blot analysis for expression of the cellular transcription factors YY1 (a), ERF (b), and HDAC1 (c), which are known to be associated with the regulation of the viral MIEP. HDAC1 levels decreased upon differentiation but there was no significant change in expression of YY1 or ERF. A coomassie stain (d) shows that equivalent levels of protein from monocytes (M) and mature DC were loaded.
Fig. 5.
Fig. 5.
Reactivation of naturally acquired HCMV from mature CD34+ cell-derived DC. Mature CD34+ cell-derived DC were cocultured with primary HFs for 13 days, and then the supernatant was transferred to new HF monolayers that were subsequently stained for IE gene expression. Supernatants collected from seronegative mature CD34+ cell-derived DC cocultures (a) and three seropositive mature CD34+-derived DC cocultures (c, e, and g) were tested. The nuclei of infected HF were counterstained with Hoechst 33342 (b, d, f, and h).
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