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. 2020 Aug 4;117(31):18764-18770.
doi: 10.1073/pnas.2002651117. Epub 2020 Jul 21.

FOXO transcription factors activate alternative major immediate early promoters to induce human cytomegalovirus reactivation

Andrew E Hale  1   2 , Donna Collins-McMillen  3 , Erik M Lenarcic  1   2 , Suzu Igarashi  3 , Jeremy P Kamil  4 , Felicia Goodrum  3   5 , Nathaniel J Moorman  6   2
Affiliations

FOXO transcription factors activate alternative major immediate early promoters to induce human cytomegalovirus reactivation

Andrew E Hale et al. Proc Natl Acad Sci U S A. .

Abstract

Human progenitor cells (HPCs) support human cytomegalovirus (HCMV) latency, and their differentiation along the myeloid lineage triggers cellular cues that drive reactivation. A key step during HCMV reactivation in latently infected HPCs is reexpression of viral major immediate early (MIE) genes. We recently determined that the major immediate early promoter (MIEP), which is primarily responsible for MIE gene expression during lytic replication, remains silent during reactivation. Instead, alternative promoters in the MIE locus are induced by reactivation stimuli. Here, we find that forkhead family (FOXO) transcription factors are critical for activation of alternative MIE promoters during HCMV reactivation, as mutating FOXO binding sites in alternative MIE promoters decreased HCMV IE gene expression upon reactivation and significantly decreased the production of infectious virus from latently infected primary CD34+ HPCs. These findings establish a mechanistic link by which infected cells sense environmental cues to regulate latency and reactivation, and emphasize the role of contextual activation of alternative MIE promoters as the primary drivers of reactivation.

Keywords: FOXO; HCMV; herpesvirus; latency; reactivation.

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Conflict of interest statement

Competing interest statement: J.P.K., F.G., and N.J.M. have filed a patent for manipulation of intronic promoters to reduce reactivation of viral vaccines.

Figures

Fig. 1.
Fig. 1.
HCMV intronic promoters contain FOXO TF consensus sites. (A) Schematic showing the location of the intronic promoters iP1 and iP2 and potential FOXO TF binding sites in the major immediate early intronic promoter locus. (B) The intronic promoters were searched against the two consensus binding motifs for FOXO transcription factors. The sequences of the three potential FOXO binding sites are shown. The bases highlighted in red were changed to CC in the mutants described in Figs. 2–6.
Fig. 2.
Fig. 2.
(A) MIE intronic promoters are activated by FOXO. (A and B) HeLa cells were cotransfected with FOXO overexpression plasmids and previously described luciferase reporter constructs containing the four promoters in the MIE genomic locus 5′ of the luciferase gene; the distal promoter (dP), the major immediate early promoter (MIEP), intronic promoter 1 (iP1), and intronic promoter 2 (iP2). Luciferase activity was measured at 24 h after transfection and normalized to the amount of protein in the sample. A previously characterized positive control ([+] control) was included which contains three consensus FOXO binding sites 5′ of the luciferase gene. The parental luciferase plasmid pGL3 basic, which lacks a promoter upstream of luciferase, served as a control. The graphs show the fold change in activity of each promoter in the presence of FOXO3a (A) or FOXO1 (B) compared to the pGL3 basic control. (C and D) Each potential FOXO binding site in the iP2 promoter was mutated either alone (mut1, mut2, and mut3) or in combination (mut123).The reporters were cotransfected with either FOXO3a (C) or FOXO1 (D), and luciferase assays were performed as above (n = 3; *P < 0.05, **P < 0.05, ***P < 0.005).
Fig. 3.
Fig. 3.
FOXO3a stimulates transcription from iP2 in the context of the MIE genomic locus. (A) A plasmid containing the regulatory and coding regions for the HCMV IE1 and IE2 genes (pSVH) or a variant of pSVH lacking the core MIEP (pSVHΔMIEP) was transfected into HeLa cells together with a FOXO1 or FOXO3a expression plasmid, or an empty expression vector (EV) control. Cells were harvested at 24 h after infection, and cell lysates were analyzed by Western blot using the indicated antibodies. Results are representative of three independent experiments. (B) Each FOXO consensus site in iP2 was mutated in the context of the pSVHΔMIEP plasmid, either alone (mut1, mut2, and mut3) or in combination (mut123). Cells were transfected with the indicated plasmid along with a FOXO3a expression plasmid (FOXO3a) or the EV control and harvested 24 h later. Cell lysates were analyzed by Western blot using the indicated antibodies. The results are representative of three independent experiments. (C) Cells were transfected and harvested as in B. RNA was extracted and analyzed by qRT-PCR using primers specific for UL123 (encoding IE1), UL122 (encoding IE2), iP1, or iP2. The graphs show the fold change in RNA abundance compared to cells transfected with pSVHDMIEP and the empty expression vector control (n = 3; *P < 0.05; NS = not significant).
Fig. 4.
Fig. 4.
FOXO3a directly binds to FOXO consensus sites in iP2. (A) Recombinant purified FOXO3a was incubated with double-stranded biotinylated probes containing the indicated FOXO consensus sites in iP2 (site 1, site 2, and site 3), or mutated sequences containing the changes shown in Fig. 1 (site 1 mut, site 2 mut, and site 3 mut). Protein:DNA complexes were resolved on nondenaturing acrylamide gels, and the complexes were visualized by chemiluminescence. The data are representative of three independent experiments. (B) Graph showing the decrease in FOXO3a binding to the mutant probes compared to the wild-type probes, which are set to 100%. The graph shows the mean of three independent experiments (*P < 0.05; ***P < 0.0005).
Fig. 5.
Fig. 5.
FOXO consensus sites are required for reexpression of MIE genes in THP-1 cells. (A) Schematic of THP-1 model for reexpression of MIE genes after stimulation with TPA. THP-1 cells were infected with TB40/E WT or FOXOmut123. At 5 dpi, cells were treated with TPA to stimulate macrophage differentiation and reexpression of viral genes, or with a DMSO vehicle control. (B) Equal infection (GFP+) of THP-1 cells with each virus was determined by flow cytometric analysis at 24 h postinfection (hpi). Biological replicates are represented as single points around the mean. SE is shown. (C) Total DNA was isolated and viral genomes were quantified using a primer to the β2.7 region of the HCMV genome relative to a cellular gene, RNase-P. Ratio of viral-to-cellular DNA at 3 and 5 dpi are normalized to their respective 1 dpi. Data from five independent biological replicates are shown; SEM is depicted. Two-way analysis of variance (ANOVA) showed no statistically significant change in genome copy for either infection over the time course. (D) Protein lysates were harvested at the indicated time points and immunoblotted to detect viral IE1 and IE2 with tubulin as a loading control. (E) MIE transcripts derived from the MIEP, iP1, or iP2 were quantified using reverse transcriptase quantitative PCR relative to the low-abundance housekeeping gene H6PD. Error bars represent the average of three independent experiments amplified in triplicate and SEM is shown. Statistical significance (*P ≤ 0.05) was determined by multiple paired t tests comparing accumulation of each transcript in WT versus mutant virus infection at each time point.
Fig. 6.
Fig. 6.
FOXO binding sites are necessary for efficient HCMV reactivation in CD34+ HPCs. CD34+ HPCs were infected with TB40/E WT or FOXOmut123 at a MOI of 2. Pure populations of infected (GFP+) CD34+ HPCs were isolated by FACS and seeded into long-term bone marrow cultures. (A) At 10 dpi, viable CD34+ HPCs (reactivation) or an equivalent cell lysate (prereactivation control) were seeded by limiting dilution onto fibroblast monolayers in a cytokine-rich media to promote myeloid differentiation. Infectious centers (GFP+ fibroblasts) were scored 14 d later and are expressed as frequency of infectious centers. Error bars represent SEM for three independent biological replicates. Statistical significance was determined using two-way analysis of variance (ANOVA) with repeated measures by both factors (wild type vs. mutant and prereactivation vs. reactivation where *P ≤ 0.05). (B) Total DNA was isolated at 10 dpi and viral genomes were quantified relative to cellular DNA by qPCR using primers to the β2.7 region of the HCMV genome and the cellular gene RNase-P. Data from two independent biological replicates are shown; mean is depicted.
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