A conserved gammaherpesvirus protein kinase targets histone deacetylases 1 and 2 to facilitate viral replication in primary macrophages

Abstract

Gammaherpesviruses are ubiquitious pathogens that establish lifelong infection and are associated with several malignancies. All gammaherpesviruses encode a conserved protein kinase that facilitates viral replication and chronic infection and thus represents an attractive therapeutic target. In this study, we identify a novel function of gammaherpesvirus protein kinase as a regulator of class I histone deacetylases (HDAC). Mouse gammaherpesvirus 68 (MHV68)-encoded protein kinase orf36 interacted with HDAC1 and 2 and prevented association of these HDACs with the viral promoter driving expression of RTA, a critical immediate early transcriptional activator. Furthermore, the ability to interact with HDAC1 and 2 was not limited to the MHV68 orf36, as BGLF4, a related viral protein kinase encoded by Epstein-Barr virus, interacted with HDAC1 in vitro. Importantly, targeting of HDAC1 and 2 by orf36 was independent of the kinase's enzymatic activity. Additionally, orf36 expression, but not its enzymatic activity, induced changes in the global deacetylase activity observed in infected primary macrophages. Combined deficiency of HDAC1 and 2 rescued attenuated replication and viral DNA synthesis of the orf36 null MHV68 mutant, indicating that the regulation of HDAC1 and 2 by orf36 was relevant for viral replication. Understanding the mechanism by which orf36 facilitates viral replication, including through HDAC targeting, will facilitate the development of improved therapeutics against gammaherpesvirus kinases.

Fig 1

Global inhibition of HDACs rescues RTA transcription in the absence of functional orf36. (A) Primary bone marrow-derived BL6 macrophages were infected at a multiplicity of infection (MOI) of 1 PFU/cell with wild-type (WT) or indicated orf36 mutant MHV68 viruses and treated with 40 nM TSA immediately after virus absorption. Total RNA was collected at 16 hpi, and RTA mRNA levels were measured by qRT-PCR. Data were pooled from three independent experiments. (B) Primary macrophages were mock infected or infected at an MOI of 1 with the MHV68 virus expressing Flag-tagged orf36 (36F). Orf36 and β-actin protein levels were measured at the indicated times postinfection.

Fig 2

Orf36 inhibits association of HDAC1 and 2 with the distal RTA promoter. (A, B) Primary macrophages were infected as indicated, and chromatin was collected at 16 hpi and subjected to ChIP using HDAC1-specific (A) or HDAC2-specific (B) antibodies or a nonspecific IgG control. Enrichment of RTA core and distal promoter sequences was calculated using the ΔΔC_T method. Results are presented as fold enrichment over IgG. (C) Schematic of the core and distal RTA promoters. Arrows above the sequence indicate the locations of PCR primers used in experiments depicted in panels A and B. (D, E) Primary macrophages were infected, and chromatin was harvested as described for panels A and B. ChIP enrichment of orilyt and orf57 promoter sequences immunoprecipitated with anti-HDAC1 were calculated as above. (F) ChIP was performed using chromatin from primary macrophages infected as indicated for 5 h at an MOI of 1. (G) Primary macrophages were infected with wild-type (WT) MHV68 or the N36S mutant at an MOI of 10. Chromatin was harvested at 16 h postinfection and subjected to ChIP as described above. All ChIP data in this figure were pooled from 3 to 5 independent experiments; error bars represent the standard errors of the means. *, P < 0.05.

Fig 3

orf36 and BGLF4 interact with HDAC1 and -2. (A) Primary macrophages were infected at an MOI of 10 with wild-type or 36F MHV68 virus for 30 h, and lysates were immunoprecipitated with anti-HDAC1 or anti-Flag antibodies. Immunoprecipitates were probed for HDAC1. (B to D) Radioactively labeled proteins were generated using the in vitro transcription/translation system (TNT). TNT-generated proteins were combined and immunoprecipitated using indicated antibodies (against HDAC1, HDAC2, Flag, or HA tag). Immunoprecipitated complexes were analyzed by SDS-PAGE and autoradiography. Images are representative of at least three independent experiments. Signal in lanes 1 to 5 represents input.

Fig 4

HDAC1 colocalizes with viral replication compartments. Primary macrophages were infected at an MOI of 10 with wild-type or orf36 mutant MHV68 viruses. At 16 hpi, cells were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole) and antibodies directed against MHV68 ssDBP and HDAC1. Images were merged, with HDAC1 in green, ssDBP in red, and DAPI in blue. Colocalization was tested using Pearson's correlation coefficient.

Fig 5

orf36 modulates global HDAC activity throughout lytic infection of primary macrophages. (A to C) Primary macrophages were mock infected or infected at an MOI of 1 or 10 with wild-type or orf36 mutant MHV68 viruses. HDAC enzymatic activity, total protein concentration, and HDAC1, HDAC2, and β-actin protein levels were measured in whole-cell lysates collected at the indicated times postinfection. HDAC activity was normalized to total protein content and, subsequently, to mock-infected lysates. In parallel, HDAC activity was measured in cell lysates spiked with TSA. HDAC activity data were pooled from 3 to 6 independent experiments; each condition was assessed in two replicates within each experiment. Densitometric analysis was used to measure HDAC1 and -2 protein content, normalized to β-actin. Normalized values are listed below the respective lanes. (D) Primary macrophages were nucleofected with the indicated plasmids, and HDAC activity and protein levels were measured as above at 6 h postnucleofection. (E) Primary macrophages were mock infected or infected with 36F MHV68 at an MOI of 10. orf36 and β-actin protein levels were measured at the indicated times postinfection. All error bars represent one standard error of the mean. *, P < 0.05; ***, P < 0.001.

Fig 6

Conditional depletion of HDAC1 and -2 in primary bone marrow-derived macrophages. (A) Schematic of the ER-Cre-mediated recombination of the conditional HDAC1 and HDAC2 alleles. Numbered boxes represent exons, and circles represent loxP sites for Cre-mediated recombination. Primers are indicated using arrows above the approximate annealing loci. Recombination products after 4-hydroxytamoxifen treatment are depicted below the genomic loci. (B to E) Bone marrow isolated from ER-Cre-positive or -negative mice with conditional HDAC1 and -2 alleles (F/F) was treated with 1 μM 4-hydroxytamoxifen (OHT) or carrier solution at 4 and 7 days of in vitro macrophage differentiation. (B, C) Total DNA was isolated on day 12 of culture, recombined HDAC1 and -2 alleles were detected by PCR (B), and the intact HDAC1 allele was quantified by real-time PCR using primers F and R1 with data pooled from 2 independent experiments (C). (D to F) Cell lysates were harvested on day 12 of culture, and protein levels of HDAC1, HDAC2, and histone H3 were measured by Western analysis (D and E); HDAC1 and -2 protein content was analyzed by densitometry and normalized to H3 (F). Data were pooled from 3 independent experiments.

Fig 7

Effects of HDAC1 and -2 depletion on MHV68 replication. (A) Primary macrophages generated and treated as described for Fig. 6 were infected at an MOI of 1 (in the absence of OHT) with wild-type MHV68 and orf36 mutant viruses on day 13 of in vitro culture. RNA was collected at 16 h postinfection, and RTA expression was measured via qRT-PCR and normalized to corresponding GAPDH mRNA levels using the ΔC_T method. Data were pooled from 2 to 4 experiments. (B) Primary, OHT-treated macrophages with indicated Cre genotypes were infected at an MOI of 1 with wild-type or N36S MHV68 as described for panel A, and viral yield was measured at the indicated times postinfection. (C) Primary, OHT-treated macrophages with the indicated Cre genotypes were infected at an MOI of 10, and viral DNA accumulation was measured at the indicated time points by real-time PCR. *, P < 0.05. Data are representative of three independent experiments. Error bars represent a standard error of the mean.

Fig 8

Working model. orf36 targets HDAC1 and 2 to facilitate MHV68 replication. At the core RTA promoter, orf36, in an enzymatic activity-dependent manner, induces phosphorylation of H2AX. At the distal promoter, orf36 inhibits HDAC1 and -2 association with this RTA promoter independent of the viral kinase enzymatic activity. However, orf36 enzymatic activity is likely necessary to also target as-yet-unidentified HDAC in order to enhance RTA promoter activity. In addition to directly regulating RTA promoters, orf36 counteracts HDAC1 and -2 to promote viral DNA synthesis and MHV68 replication in primary macrophages.