Research ArticleVIROLOGY

Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein

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Science Advances  27 Nov 2015:
Vol. 1, no. 10, e1501164
DOI: 10.1126/sciadv.1501164
  • Fig. 1 UL138 inhibits VPA-responsive IE1 expression during HCMV latency.

    (A) Schematic of recombinant AD169 viruses encoding UL138 (see also fig. S2A). (B) RNA from THP-1 and primary CD34+ cells infected at a multiplicity of infection (MOI) of 1 with the indicated viruses for 18 or 24 hours, respectively, was analyzed for transcript expression of the indicated gene by reverse transcription polymerase chain reaction (RT-PCR). RNA from normal human dermal fibroblasts (NHDFs) infected with AD-138HA at an MOI of 1 for 24 hours serves as a positive control. (C) RNA and DNA from samples in (B) were analyzed by quantitative RT-PCR (qRT-PCR) or qPCR, respectively, and normalized to cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Viral transcripts were normalized to viral DNA and are shown relative to that of NHDFs (HF). (D and E) CD34+ cells infected at an MOI of 1 for 24 hours in the absence (−) or presence (+) of VPA (1 mM) analyzed for transcript expression by RT-PCR (D) or qRT-PCR (E). (F to H) THP-1 cells infected at an MOI of 3 for 18 hours in the absence (−) or presence (+) of VPA analyzed for transcript expression by RT-PCR (F) or qRT-PCR (G) and protein expression by Western blot (H). (I) ESCs infected with the indicated virus at an MOI of 3 for 24 hours analyzed for transcript expression of the indicated gene by RT-PCR. (J) ESCs infected as in (I) were analyzed for IE1 transcripts per viral genome as in (C). (K) ESCs infected with the indicated virus at an MOI of 3 for 24 hours in the absence (−) or presence (+) of VPA analyzed for transcript expression of the indicated gene by RT-PCR. (L) Samples from (K) were analyzed by qRT-PCR. Data are means ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, or ***P < 0.001 by Student’s t test.

  • Fig. 2 UL138 is not required for HDAC-independent repression of IE gene expression in clinical strains and is not sufficient to maintain latency.

    (A to D) CD34+ (A and B) or THP-1 (C and D) cells infected with AD169, TB40/E, or TB40/E-ΔUL138 (TB-Δ138) at an MOI of 1 in the absence (−) or presence (+) of VPA were analyzed for the indicated transcripts by RT-PCR (A and C) or qRT-PCR (B and D). (E) Lysates from THP-1 cells infected as in (C) analyzed by Western blot. (F) Infectious virions produced by ESCs infected with the indicated virus at an MOI of 3 for 10 days were quantitated by plaque assay. (G to J) CD34+ (G and H) or THP-1 (I and J) cells infected with AD169, TB40/E with ULb′ replaced with that of AD169 (TB w/AD-ULb′), or TB40/E at an MOI of 1 in the absence (−) or presence (+) of VPA were analyzed by RT-PCR (G and I) or qRT-PCR (H and J). (K) Lysates from THP-1 cells infected as in (I) analyzed by Western blot. Data are means ± SEM from three independent experiments. *P < 0.05 or **P < 0.01 by Student’s t test. n.s., not significant (P = 0.29).

  • Fig. 3 UL138 inhibits demethylation of histones at the MIEP to facilitate silencing of IE1 expression during latency.

    (A to D) ChIP assays for repressive histone methylation marks H3K9me2 (A and C) and H3K27me3 (B and D) at the MIEP in VPA-treated THP-1 (A and B) or CD34+ (C and D) cells infected with the indicated virus at an MOI of 3 or 1 for 18 or 24 hours, respectively. (E) ChIP assays for the presence of the histone demethylase JMJD3 at the MIEP in VPA-treated THP-1 cells infected with live (−) or UV-inactivated (+) AD169 at an MOI of 1 for 18 hours. (F) THP-1 cells infected as in (E) analyzed for IE1 transcripts by qRT-PCR. (G) ChIP assay for JMJD3 at the MIEP in VPA-treated THP-1 cells infected with AD169 at an MOI of 1 for 6 hours in the absence (−) or presence (+) of actinomycin D (ActD). (H) THP-1 cells infected as in (G) analyzed for IE1 transcripts by qRT-PCR. (I to L) ChIP assays for the presence of JMJD3 (I and J) or LSD1 (K and L) at the MIEP in VPA-treated THP-1 (I and K) or CD34+ (J and L) cells infected with the indicated virus at an MOI of 3 or 1 for 18 or 24 hours, respectively. (M and N) THP-1 cells were infected with AD169 at an MOI of 1 in the absence (−) or presence (+) of VPA and the histone demethylase inhibitor OG-L002, GSK-J4, or ML324 for 18 hours and analyzed by qRT-PCR (M) or Western blot (N). (O) CD34+ cells were infected with AD169 at an MOI of 1 in the absence (−) or presence (+) of VPA, OG-L002, GSK-J4, or ML324 for 24 hours and analyzed by RT-PCR. In (A) to (E), (G), and (I) to (L), gray bars represent immunoglobulin G (IgG) controls. Data are means ± SEM from at least three independent experiments. *P ≤ 0.05, **P < 0.01, or ***P < 0.001 by Student’s t test. n.s., not significant (P > 0.1). For all ChIP assays, the signal from a specific antibody was significantly enriched over the IgG signal (P < 0.05) with the exception of LSD1 ChIP in AD-138HA–infected CD34+ cells (L), which is not significantly different from IgG (P = 0.18).

  • Fig. 4 UL138 prevents recruitment of CtBP1 to the MIEP to silence IE1 transcription during latency.

    (A and B) ChIP assays for CtBP1 at the MIEP in VPA-treated CD34+ (A) or THP-1 (B) cells infected with the indicated virus at an MOI of 1 or 3 for 24 or 18 hours, respectively. (C) ChIP assays for CtBP1 at the MIEP in VPA-treated THP-1 cells infected with AD169 at an MOI of 3 in the absence (−) or presence (+) of the CtBP1 inhibitor MTOB for 18 hours. In (A) to (C), gray bars represent IgG controls. (D and E) THP-1 (D) or CD34+ (E) cells were infected with AD169 at an MOI of 1 in the absence (−) or presence (+) of VPA or MTOB and analyzed for IE1 expression by Western blot (D) or RT-PCR (E). (F and G) NHDFs infected with AD169 (F) or HSV-1 KOS (G) at an MOI of 0.1 in the absence (−) or presence (+) of MTOB or ML324 for 6 hours and analyzed for IE1 (F) or ICP27 (G) expression by qRT-PCR. Data are means ± SEM from at least three independent experiments. *P < 0.05 or ***P < 0.001 by Student’s t test. For all ChIP assays, the signal from the CtBP1 antibody was significantly enriched over the IgG signal (P < 0.05) with the exception of AD-138HA–infected CD34+ cells (A), which is not significantly different from IgG (P = 0.2).

  • Fig. 5 Model for the regulation of HCMV IE gene expression by cellular intrinsic defenses during the establishment of latency.

    The Daxx intrinsic defense is active during latency and restricts IE gene expression by recruiting an HDAC to deacetylate histones associated with the MIEP. Artificial inactivation of this defense with the HDAC inhibitor VPA results in activation of IE gene expression. The antilatent histone KDM and CtBP1-mediated intrinsic defense facilitates activation of IE gene expression by removing repressive histone methylations from the MIEP. Pharmacological inhibition with KDM inhibitors (KDMi) or the CtBP1 inhibitor MTOB or expression of UL138 prevents KDM-mediated activation of IE gene expression and promotes the establishment of latency.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/10/e1501164/DC1

    Materials and Methods

    Fig. S1. UL138 represses an MIEP reporter in THP-1 cells.

    Fig. S2. Phenotypic characterization of AD-UL138HA.

    Fig. S3. UL138HA expressed from recombinant AD169 localizes to the Golgi.

    Fig. S4. AD-138HA enters CD34+ cells and expresses UL138.

    Fig. S5. Expression of viral transcripts during experimental latent infections.

    Fig. S6. UL138 is required for the phenotype of AD-138HA.

    Fig. S7. Phenotypic characterization of TB40/E-Δ138.

    Fig. S8. Model for redundant and nonredundant roles of UL138 in the establishment and maintenance of HCMV latency.

    References (8386)

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. UL138 represses an MIEP reporter in THP-1 cells.
    • Fig. S2. Phenotypic characterization of AD-UL138HA.
    • Fig. S3. UL138HA expressed from recombinant AD169 localizes to the Golgi.
    • Fig. S4. AD-138HA enters CD34+ cells and expresses UL138.
    • Fig. S5. Expression of viral transcripts during experimental latent infections.
    • Fig. S6. UL138 is required for the phenotype of AD-138HA.
    • Fig. S7. Phenotypic characterization of TB40/E-Δ138.
    • Fig. S8. Model for redundant and nonredundant roles of UL138 in the establishment and maintenance of HCMV latency.
    • References (83–86)

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