Fig 1.
KSHV ORF20 encodes three isoforms.
(A) KSHV ORF20WT, a member of the UL24 family, can potentially express three isoforms: ORF20FL, starting at methionine 1 (M1) (aa 1–320), ORF20A, starting at leucine 24 (L24) (aa 1–297), and ORF20B, starting at M64 (aa 1–257). (B) Plasmid constructs express the three isoforms singly or in combination with each other as indicated. For analysis of ORF20A, either the genomic L24 start codon (ORF20WT and ORF20FLgA) or genomic L24 with an upstream methionine, indicated with M (ORF20A, ORF20AB) was used. * indicates genomic ORF20A leucine start codon; ORF20A starting with leucine was not detectable by immunoblotting. (C) Expression vectors encoding ORF20WT, individual ORF20 isoforms, or ORF20 isoforms in combination with each other were transfected into 293T cells. Lysates were prepared 24 h later, separated by Bis-Tris PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Empty vector (EV) was included as a control. The immunoblot is representative of four independent experiments.
Fig 2.
KSHV ORF20 isoforms localize predominantly to the nuclei and nucleoli.
(A-B) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, (A) coverslips were fixed directly in 4% paraformaldehyde in PBS (PFA) for whole cell immunofluorescence. (B) For nuclear immunofluorescence, coverslips were incubated in cold 1% NP-40 extraction buffer for 5 min to remove the cytoplasm before PFA fixation. (A-B) All samples were then processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm.
Fig 3.
Quantitative affinity purification coupled to mass spectrometry identifies OASL as an ORF20 interaction partner.
(A) Scheme of the quantitative affinity purification coupled to mass spectrometry (q-AP-MS) workflow used to identify cellular interaction partners of ORF20. In the forward experiment, HeLa S3 cells were labeled with heavy or light amino acids, then transfected with ORF20WT-myc or LacZ-myc, respectively. In the crossover experiment, ORF20WT-myc was labeled with light amino acids and LacZ-myc with heavy amino acids. Lysates were immunoprecipitated with anti-myc magnetic beads, combined for affinity purification, processed, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ORF20 interaction partners were identified based on the increased abundance of heavy labeled proteins compared to light labeled proteins. In the parallel crossover experiment, specific interaction partners were identified based on increased abundance of light labeled proteins. Proteins with a 1:1 ratio of light and heavy amino acids are non-specific binding partners or contaminants. (B) Quantitative proteomics results displayed graphically. The abundances of proteins identified in the forward and crossover experiments are graphed, with ORF20 interacting partners located in the lower right quadrant. Each point represents one protein. 40S and 60S ribosomal proteins are shown in blue and OASL is indicated in red. (C) 293T, 293T OASL-/-, and HeLa S3 cells were transfected with ORF20WT-myc (+) or empty vector (-) and/or RIG-I N (+) or empty vector (-). An anti-myc immunoprecipitation of NP-40 lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-OASL and anti-myc antibodies. *: Nonspecific background band. Immunoblots are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of three independent experiments. (E) HeLa S3 cells were transfected individually with ORF20WT-myc or OASL-V5, then labeled with antibodies against myc or V5 as appropriate (green) and fibrillarin (red). (F) HeLa S3 cells were co-transfected with ORF20WT-myc and OASL-V5, and then labeled with anti-V5 (green) and anti-myc (red) antibodies. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (E) and 20 μm (F).
Table 1.
ORF20 interacting partners identified by q-AP-MS analysis.
Fig 4.
The subcellular localization of OASL-V5 and ORF20-myc isoforms is unaltered in latent and reactivated HuARLT2-rKSHV.219 cells.
HuARLT2-rKSHV.219 cells were transiently transduced with lentiviruses encoding the indicated construct. 4 days post transduction, cells were seeded onto glass coverslips. The following day, cells were untreated (A, latent infection) or reactivated with sodium butyrate and RTA-expressing baculovirus (B, reactivated). Infected cells express GFP, reactivated cells express RFP, and transduced proteins were detected with anti-myc or anti-V5 antibodies and an anti-mouse Alexa Fluor 647 coupled antibody (pink). Nuclei were counterstained with Hoechst (blue). Images are representative of at least 2 independent experiments. Scale bar = 10 μm.
Fig 5.
The interaction of ORF20 with OASL is conserved among the members of the UL24 family.
(A) HeLa cells were transfected with the indicated plasmid and seeded onto coverslips. 48 h post transfection, coverslips were fixed in PFA and processed for anti-myc (green) and anti-fibrillarin (red) immunofluorescence. Nuclei were counterstained with Hoechst (blue). Images are representative of three independent experiments. Scale bar = 10 μm (B) 293T cells were transfected with myc-tagged UL24, UL76, M76, or ORF20WT. Lysates were prepared 24 h later, separated by SDS-PAGE, and anti-myc and anti-tubulin immunoblotting was performed. Data are representative of four independent experiments. (C) 293T cells were transfected with the indicated myc-tagged KSHV ORF20 form or MHV68 ORF20. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with an anti-myc antibody. Data are representative of two independent experiments. (D) 293T cells were transfected with myc-tagged UL24 homologs MCMV M76, HCMV UL76, or HSV-1 UL24, LacZ-myc, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Data are representative of two independent experiments.
Fig 6.
OASL RNA binding and ubiquitin-like domains are not required for interaction with ORF20.
(A) Multiple OASL constructs were utilized, including WT OASL and OASL mutants, including ΔUBL lacking the ubiquitin-like domain, the P-loop mutants V67G and N72K, the RNA binding mutants R45E/K66E/R196E/K200E (RKRK), K63E, and K66E, and the catalytic triad mutants, E81A, E83A, and T152A. (B) 293T cells were transfected with myc-tagged ORF20WT or LacZ, V5-tagged OAS1, WT or mutant OASL, and/or empty vector (EV) as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed and input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of at least three independent experiments. Similar results were obtained with simultaneous and sequential antibody exposures. (C) The amino acid sequence for ORF20B is shown, with the predicted nuclear localization sequence indicated in red, the predicted nucleolar localization sequence indicated in green, and predicted disordered regions underlined. The predictions formed the basis for three ORF20B C-terminal truncation mutants: ORF20B 1–186, 1–220, and 1–235. (D) 293T cells were transfected with myc-tagged ORF20 isoforms, ORF20B mutants, or LacZ, OASL-V5, and/or EV as indicated. An anti-myc immunoprecipitation of RIPA lysates was performed. Input lysates and immunoprecipitates were immunoblotted with anti-V5 and anti-myc antibodies. Immunoblots are representative of three independent experiments.
Fig 7.
Quantitative proteomics identified nucleolar and ribosomal interaction partners of OASL.
(A) q-AP-MS was performed as shown in Fig 3A. For the forward experiment, heavy-labeled HeLa S3 cells were transfected with OASL-myc and light-labeled cells were transfected with LacZ-myc. In the crossover experiment, OASL-myc was labeled with light amino acids and LacZ-myc with heavy amino acids. Cell lysates were immunoprecipitated with anti-myc magnetic beads and analyzed by LC-MS/MS. Specific interacting partners of OASL identified in both the forward and crossover experiments are located in the lower right quadrant. 40S and 60S ribosomal proteins are shown in blue and nucleolar proteins are indicated in green. (B) The interacting partners shown in Tables 1 and 2 were compared using VennDis to determine the number of specific and shared interacting partners for ORF20 and OASL (related to S1 Supporting Information).
Table 2.
OASL interacting partners identified by q-AP-MS.
Fig 8.
ORF20 and OASL copurify with ribosomal subunits and polysomes.
293T cells were cotransfected with myc-tagged ORF20 (A-B), OASL (C-D), or ORF20 and OASL together (E-F), then ribosomes were purified by 5–45% sucrose gradient fractionation in the presence of EDTA (A, C, E) or MgCl2 (B, D, F). Fractionation profiles were determined by absorbance at 254 nm. 40S and 60S indicate the small and large ribosomal subunits, respectively, and 80S indicates the monosome peak. Fractions were collected and analyzed for 18S and 28S rRNA by denaturing gel electrophoresis and for OASL and ORF20 expression by anti-myc immunoblotting. Results are representative of two independent experiments.
Fig 9.
ORF20 does not affect translation rates, but enhances expression of endogenous OASL.
(A) 293T cells were co-transfected with EV, myc-tagged ORF20WT, ORF20FL, or ORF20B, and either EV or RIG-I N. 24 h post transfection, cells were treated for 15 minutes with 5μg/ml puromycin, then lysed and analyzed by immunoblotting. Anti-puromycin, anti-OASL, anti-myc, and anti-actin immunoblotting was performed sequentially. Data are representative of two independent experiments. *: Nonspecific background band. (B) 293T cells were co-transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (C) HEK293 cells were transfected with either EV or ORF20WT-myc. 24h later, cells were transfected with 5’pppRNA complexed with Lipofectamine 2000 for approximately 24h. (D) 293T cells were reverse transfected with control, IRF3, IFNAR, or STAT1 siRNAs as indicated. 48h later, cells were transfected with EV or ORF20WT-myc, and either EV or RIG-I N, for 24h. (B, C, D) Cells were lysed, RNA was isolated, mRNA-specific cDNA was reverse transcribed, and the amount of OASL was determined using relative quantification to GAPDH levels and the 2-ΔΔCT method. Data shown are means + SD of combined duplicates from at least two experiments. ns, not significant, * P<0.05, ** P<0.01, *** P<0.001.
Fig 10.
OASL expression is beneficial for MHV68 and KSHV replication.
(A-F, H) 293T OASL-/- cells were reconstituted with empty vector (EV), mOASL1, or hOASL as indicated. (A-B) 24 h later, cells were infected with VSV-GFP for 16 h. The GFP intensity in fixed cells was determined by flow cytometry. (A) Histograms of the VSV-GFP signal for duplicates from one representative experiment are shown. (B) The number of highly-GFP positive cells was determined and scaled to EV. Means + SD of duplicates from two independent experiments are shown. (C-D) 24 h post transfection, cells were infected with MHV68-GFP at an MOI of 0.05 for 2 h at 37°C. Supernatant was harvested at the indicated times and the viral titer was determined by TCID50 on M2-10B4 cells. Means ± SD of triplicates from two independent experiments are shown. (E, F, H) 24 h post transfection, cells were infected with (E) MHV68-GFP, (F) KSHVLYT wildtype, or (H) KSHVLYT ORF20stop. 20–24 h later, cells were detached, fixed, and the number of GFP-positive cells was determined by flow cytometry. Means + SD of 4–6 replicates total from two independent experiments are shown. (G) In KSHVLYT ORF20stop, the ORF20 GAG codon for E69 is replaced with the stop codon TAG. For all subfigures: ns, not significant, * P<0.05, **P<0.01, *** P<0.001.
Fig 11.
OASL and ORF20 are expressed in reactivated HuARLT2-rKSHV.219 cells.
Uninfected HuARLT2 and latently infected HuARLT2-rKSHV.219 cells were seeded in 6-well plates. HuARLT2-rKSHV.219 cells were untreated (-) or reactivated by addition of sodium butyrate and RTA-expressing baculovirus, then incubated for the indicated times. RNA was prepared and (A) OASL (B) ORF20 (C) ORF16 (D) ORF46 or (E) K8.1 mRNA levels were determined by q-RT-PCR. The averages + SD of duplicates from 1 representative of 3 total experiments are shown. nd, not detected, ns, not significant, ** P<0.01, ***P<0.001.