Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 93 (6)

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling

Affiliations

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling

Laura K Springgay et al. J Virol.

Abstract

Interferon (IFN) production and the subsequent induction of IFN-stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are promyelocytic leukemia nuclear bodies (PML-NBs), which are subnuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologs of IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN, while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3; however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our present study, we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription.IMPORTANCE KSHV and RRV encode a unique set of homologs of cellular IFN regulatory factors, termed vIRFs, which are hypothesized to help these viruses evade the innate immune response and establish infections in their respective hosts. Our work elucidates the role of one RRV vIRF, R12, and demonstrates that RRV can dampen the type I IFN response downstream of IFN signaling, which would be important for establishing a successful infection in vivo.

Keywords: PML; R12; gammaherpesvirus; innate immunity; interferon; rhadinovirus; vIRF.

Figures

FIG 1
FIG 1
RRV is sensitive to IFN, and the vIRFs are necessary for PML-NB disruption. (A) Primary RhFs were infected with WT RRV or RRVvIRF-KO at an MOI of 2 in the presence or absence of 100 U/ml RhIFN-α2. Viral titers were measured at the indicated times postinfection by a plaque assay and are presented as PFU per milliliter. Assays were performed in duplicate, replicates were averaged, and data were analyzed by repeated-measures ANOVA with a post hoc Tukey-Kramer test. Adjusted P values of less than 0.05 were considered significant, and asterisks denote significant P values (*, P ≤ 0.05; **, P ≤ 0.01). (B) tRhFs were infected with WT RRV or RRVvIRF-KO at an MOI of 2 in the presence or absence of 100 U/ml RhIFN-α2. At the indicated times postinfection, cells were fixed with methanol and stained with PML (red)- and RRV-gB (green)-specific antibodies, while nuclei were stained with DAPI (blue). Arrows indicate RRV-gB-positive (gB+) cells that lack PML-NB staining within nuclei. The experiment was performed twice in duplicate, and representative images at a ×40 magnification are shown. (C) PML/RRV-gB-double-positive cells as well as PML-negative/RRV-gB-positive cells were counted at 36 hpi from cells treated as described above for panel B. Data were analyzed by two-tailed Fisher’s exact test, and P values of less than 0.05 were considered significant. (D) For the RRV-gB-positive cells that were positive for PML-NBs in panel C, the number of PML-NBs was counted within the nuclei of each cell. Data were analyzed by unpaired Student’s t test, and P values of less than 0.05 were considered significant. (C and D) Experiments were performed twice, and data from one representative experiment are shown. (E) tRhFs were infected with WT RRV-GFP or RRVvIRF-KO-GFP at an MOI of 5 for 24 h, before sorting the GFP-positive cells by flow cytometry for fluorescein isothiocyanate (FITC). Cytoplasmic and nuclear fractions were prepared from sorted cells, untreated cells, and cells treated with 100 U/ml RhIFN-α2 for 18 h. Cytoplasmic and nuclear proteins were separated before analysis by SDS-PAGE and probed with PML-, lamin A/C-, RRV-ORF52-, and GAPDH-specific antibodies. Densitometry on the PML isoform I/II band was performed and normalized to the lamin A/C loading control. The experiment was performed three times, and data from one representative experiment are shown. IB, immunoblotting.
FIG 2
FIG 2
RRV vIRFs do not affect localization of SP100 or Daxx proteins. (A) tRhFs were mock infected or infected with WT RRVBAC or RRVvIRF-KO at an MOI of 2 for 24 h. Cells were fixed with methanol and stained with PML (red)-, SP100 (red)-, Daxx (red)-, and RRV-gB (green)-specific antibodies. Nuclei were stained with DAPI (blue). Arrows indicate gB-positive cells that lack nuclear staining for PML, SP100, or Daxx. Images at a ×40 magnification are presented. (B and C) IFAs were performed as described in the legends of Fig. 1C and D for RRV-gB-positive cells with or without SP100 (B) or Daxx (C). Data were analyzed by two-tailed Fisher’s exact test, and P values of less than 0.05 were considered significant. NS, not significant. (D) SP100 and Daxx dots within uninfected cells were quantified from IFA images and are displayed as the number of punctate dots per nucleus.
FIG 3
FIG 3
RRV vIRF R12 colocalizes with PML-NBs. (A) Doxycycline (Dox)-inducible tRhFs expressing R12-FLAG (tRhF-R12) were grown in the presence or absence of 100 U/ml RhIFN-α2 with or without 2 μg/ml Dox for the indicated hours before fixation with methanol. Fixed cells were stained with FLAG (green)- and PML (red)-specific antibodies, while nuclei were stained with DAPI (blue). White arrows indicate cells with enlarged PML-NBs. Images at a ×40 magnification are presented. The experiment was performed three times, and representative images are shown. (B) Numbers of PML-NBs within nuclei of cells under each culture condition were counted and are presented in a graph. Data were analyzed by unpaired Student’s t test, and P values of less than 0.05 were considered significant. Experiments were performed twice, and data from a representative experiment are shown. (C) tRhF-R12 cells were grown as described above for panel A for the indicated times. Nuclear fractions were isolated, and protein lysates were analyzed by SDS-PAGE and probed with PML-, FLAG-, lamin A/C-, and GAPDH-specific antibodies. Experiments were performed three times, and data from representative experiments are presented. (D) tRhF-R12 cells were treated with Dox for 18 h and then fixed with methanol and stained with FLAG (green)- and PML (red)-specific antibodies. Images at a ×40 magnification are presented, along with a ×63 magnification and z-axis analysis of the cell, highlighted by the white box.
FIG 4
FIG 4
R12 protein coimmunoprecipitates with PML protein. (A) tRhF-R12 cells were grown and nuclear fractions were isolated as described in the legend of Fig. 3C. Nuclear protein lysates were immunoprecipitated (IP) with a PML-specific antibody before resolving proteins on SDS-PAGE gels. Western blots were probed with PML- and FLAG-specific antibodies. (B) Nuclear protein lysates from tRhF-R12 cells cultured as described above for panel A were immunoprecipitated with a FLAG-specific antibody before resolving the proteins on SDS-PAGE gels. Western blots were probed with PML- and FLAG-specific antibodies. < denotes high-molecular-weight R12-FLAG protein, and * denotes the predicted size of the R12-FLAG protein. (C) Input control for panels A and B. Total nuclear lysates were subjected to SDS-PAGE, and Western blots were probed with PML-, FLAG-, lamin A/C-, and GAPDH-specific antibodies. Experiments were performed at least twice, and representative Western blots are shown.
FIG 5
FIG 5
R12 protein is SUMO-1 modified. (A) tRhF-R12 cells were either mock transfected or transfected with a His6–SUMO-1 plasmid and grown with or without 2 μg/ml Dox. His6–SUMO-1 proteins were purified from nuclear lysates using nickel columns and resolved on SDS-PAGE gels, followed by Western blot analysis with FLAG-, PML-, and SUMO-1-specific antibodies. (B) Input controls for panel A. Total nuclear lysates treated as described above for panel A were resolved on SDS-PAGE gels, and Western blots were probed with PML-, SUMO-1-, FLAG-, lamin A/C-, and GAPDH-specific antibodies. < denotes high-molecular-weight R12-FLAG protein, and * denotes the predicted size of the R12-FLAG protein.
FIG 6
FIG 6
Stable expression of R12 protein during RRVvIRF-KO infection results in a loss of PML-NB structures. (A) tRhF-R12 cells were grown in the presence or absence of 2 μg/ml Dox for 18 h, before mock infection or infection with RRVvIRF-KO at an MOI of 2 for 24 h. Cells were fixed with methanol and stained with antibodies specific for PML (red), FLAG (green), and RRV-gB (purple), and nuclei were stained with DAPI (blue). Images at a ×63 magnification were obtained by confocal microscopy. White boxes indicate RRV-gB-positive cells, and yellow boxes indicate R12-expressing cells within uninfected cultures. The experiment was performed four times, and representative images are shown. (B) IFAs were performed as described in the legend of Fig. 1C. Data were analyzed by two-tailed Fisher’s exact test, and P values of less than 0.05 were considered significant. (C) IFAs were performed as described in the legend of Fig. 1D. Data were analyzed by unpaired Student’s t test, and P values of less than 0.05 were considered significant. (D) R12-FLAG-inducible cells were grown and treated as described above for panel A and infected with RRVvIRF-KO at an MOI of 2 for 24 h. Nuclear and cytoplasmic fractions were isolated, and proteins were separated and resolved on SDS-PAGE gels, followed by Western blot analysis. Western blots were probed with PML-, FLAG-, lamin A/C-, RRV-ORF52-, and GAPDH-specific antibodies.
FIG 7
FIG 7
R12 protein expression reduces ISG transcription in the presence of type I IFN. (A) tRF-R12 cells were mock treated or treated with 100 U/ml RhIFN-α2 for 6 h, 2 μg/ml Dox for 12 h, or Dox for 6 h before RhIFN-α2 was added for an additional 6 h. The same experiment was repeated in a tRhF empty vector cell line. Transcript levels were normalized to GAPDH transcript levels and are presented as fold changes over values with RhIFN-α2 treatment. Data were analyzed by unpaired Student’s t test. P values of less than 0.05 were considered significant, and asterisks denote significant P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). Experiments were performed in duplicate, and the averaged results are displayed.
FIG 8
FIG 8
RRVR12FLAG and RRVR12ns construction and characterization. (A) Schematic of the mutations introduced into the RRV genome to generate RRVR12ns and RRVR12FLAG. (B) BamHI restriction digests of WT RRVBAC, RRV-R12-GalK-KO BAC, RRVR12ns BAC, and RRVR12FLAG BAC DNA clones. * denotes the location of the digest band containing the R12 sequence, and < denotes the increased size of the digest band that contains the GalK cassette. (C) Southern blot analysis of BamHI-digested BAC clones probed with GalK- and R12-specific probes. (D) Nuclear and cytoplasmic protein lysates from mock-infected and WT RRVBAC-, RRVvIRF-KO-, RRVR12ns-, and RRVR12FLAG-infected cells (MOI of 2) were collected at 24 h; resolved on SDS-PAGE gels; and probed with FLAG-, lamin A/C-, RRV-ORF52-, and GAPDH-specific antibodies. (E) RT-PCR analysis of total cellular RNA isolated from WT RRVBAC-, RRVR12ns-, or RRVR12FLAG-infected cells with or without reverse transcriptase enzyme using R11-, R13-, and GAPDH-specific primers. PCR products were subjected to agarose gel electrophoresis. (F) One-step (MOI = 2) or multistep (MOI = 0.1) growth analysis of WT RRVBAC, RRVvIRF-KO, RRVR12ns, or RRVR12FLAG. Viral titers at each time point were determined by a plaque assay and are presented as PFU per milliliter. Experiments were performed twice, and data from representative experiments are shown, except for panel F, where the average titer from both experiments is graphed.
FIG 9
FIG 9
Virus-expressed R12 coimmunoprecipitates with PML protein and is required for RRV disruption of PML-NBs. (A) RNA was purified from mock-infected, WT RRVBAC-infected (24 hpi), or RRVR12FLAG-infected cells at the indicated hours postinfection. RT-PCR was performed with or without reverse transcriptase enzyme using R12- and GAPDH-specific primers, and PCR products were subjected to agarose gel electrophoresis. (B) Nuclear lysates from mock-infected, RRVR12FLAG-infected, and RRVR12ns-infected cells (MOI of 2) were collected at the indicated times postinfection; resolved on SDS-PAGE gels; and probed with FLAG-, lamin A/C-, and GAPDH-specific antibodies. (C) WT RRVBAC-, RRVR12FLAG-, RRVR12ns-, or RRVvIRF-KO-infected cells were fixed after 24 hpi and stained with RRV-gB- and PML-specific antibodies, while nuclei were stained with DAPI. IFAs were performed as described in the legend of Fig. 1C, and data were analyzed by two-tailed Fisher’s exact test, with P values of less than 0.05 being considered significant. (D) IFAs were further performed as described in the legend of Fig. 1D, and data were analyzed by unpaired Student’s t test, with P values of less than 0.05 being considered significant. (E) Nuclear and cytoplasmic fractions were isolated from mock-infected or RRVR12FLAG-infected cells at 6, 10, 14, 16, 18, and 24 hpi. Nuclear lysates were immunoprecipitated with a FLAG-specific antibody, resolved by SDS-PAGE, and analyzed by Western blotting using a PML-specific antibody. Nuclear and cytoplasmic proteins were analyzed by SDS-PAGE and Western blotting using FLAG-, PML-, lamin A/C-, RRV-ORF52-, and GAPDH-specific antibodies as controls for protein analysis. The experiment in panel A was performed twice, and experiments in panels B to E were performed at least three times. Data from representative experiments and representative images are shown.
FIG 10
FIG 10
RRV R12 inhibits ISG induction downstream of IFN signaling and aids RRV replication in the presence of IFN. (A) tRhFs were infected with WT RRVBAC, RRVvIRF-KO, RRVR12FLAG, or RRVR12ns at an MOI of 2 for 18 h. Afterwards, 100 U/ml RhIFN-α2 was added to the infected cell culture media for an additional 6 h. RNA was purified, and cDNA was synthesized before transcript levels were measured by quantitative PCR. Transcript levels were normalized to GAPDH transcript levels and are presented as fold changes relative to the values for the RhIFN-α2 sample. Data were analyzed by unpaired Student’s t test. P values of less than 0.05 were considered significant, and asterisks denote significant P values (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). (B) PML protein levels were knocked down using shRNA specific for PML. Nuclear lysates from tRhF cells, shPML-tRhF cells, and shControl-tRhF cells treated with 100 U/ml RhIFN-α2 for 18 h were resolved on SDS-PAGE gels, and Western blots were probed with PML- and lamin A/C-specific antibodies. PML protein bands were quantified by densitometry. (C) The shPML cell line was infected with WT RRVBAC, RRVvIRF-KO, RRVR12FLAG, or RRVR12ns identically as described above for panel A and treated with or without 100 U/ml RhIFN-α2 for an additional 6 h. As controls, tRhF or shControl tRhF cells were treated with 100 U/ml RhIFN-α2 for 6 h. RNA was purified, and cDNA was synthesized before transcript levels were measured by quantitative PCR. Transcript levels were normalized to GAPDH transcript levels and are presented as fold changes over values for shControl cells treated with RhIFN-α2 for 6 h. Data were analyzed by unpaired Student’s t test. P values of less than 0.05 were considered significant, and asterisks denote significant P values. Experiments were performed in duplicate, and averaged results are shown. (D) Primary RhFs were infected with WT RRV, RRVvIRF-KO, RRVR12FLAG, or RRVR12ns at an MOI of 2.5 in the presence of 100 U/ml RhIFN-α2. Viral titers were measured at the indicated times postinfection by a plaque assay and are presented as PFU per milliliter. Data were analyzed by repeated-measures ANOVA with a post hoc Tukey-Kramer test. Adjusted P values of less than 0.05 were considered significant, and asterisks denote significant P values. 1, WT RRV versus RRVvIRF-KO and RRVR12FLAG versus RRVvIRF-KO; 2, WT RRV versus RRVR12ns and RRVR12ns versus RRVR12FLAG; 3, RRVR12ns versus WT RRV; 4, WT RRV versus RRVvIRF-KO and RRVR12ns versus RRVR12FLAG; 5, RRVR12FLAG versus RRVvIRF-KO. The experiment was performed twice, and the averages from both experiments are graphed.

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

Publication types

MeSH terms

Feedback