Kaposi's Sarcoma-Associated Herpesvirus Processivity Factor, ORF59, Binds to Canonical and Linker Histones, and Its Carboxy Terminus Is Dispensable for Viral DNA Synthesis

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus and the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. During lytic reactivation, there is a temporal cascade of viral gene expression that results in the production of new virions. One of the viral factors that is expressed during reactivation is open reading frame 59 (ORF59), the viral DNA polymerase processivity factor. ORF59 plays an essential role for DNA synthesis and is required for the nuclear localization of the viral DNA polymerase (ORF9) to the origin of lytic replication (oriLyt). In addition to its functions in viral DNA synthesis, ORF59 has been shown to interact with chromatin complexes, including histones and cellular methyltransferases. In this study, a series of KSHV BACmids containing 50-amino acid (aa) deletions within ORF59 were generated to determine the interaction domains between ORF59 and histones, as well as to assess the effects on replication fitness as a result of these interactions. These studies show that in the context of infection, ORF59 51 to 100 and 151 to 200 amino acids (aa) are required for interaction with histones, and ORF59 301 to 396 aa are not required for DNA synthesis. Since full-length ORF59 is known to localize to the nucleus, we performed an immunofluorescent assay (IFA) with the ORF59 deletion mutants and showed that all deletions are localized to the nucleus; this includes the ORF59 deletion without the previously identified nuclear localization signal (NLS). These studies further characterize ORF59 and demonstrate its essential role during lytic replication.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus and the causative agent of potentially fatal malignancies. Lytic replication of KSHV is an essential part of the viral life cycle, allowing for virus dissemination within the infected host and shedding to infect naive hosts. Viral DNA synthesis is a critical step in the production of new infectious virions. One of the proteins that is vital to this process is open reading frame 59 (ORF59), the viral encoded polymerase processivity factor. Previous work has demonstrated that the function of ORF59 is closely connected to its association with other viral and cellular factors. The studies presented here extend that work to include the interaction between ORF59 and histones. This interaction offers an additional level of regulation of the chromatinized viral genome, ultimately influencing DNA synthesis and transcription dynamics.

Keywords: DNA replication; KSHV; Kaposi's sarcoma-associated herpesvirus; ORF59; histones; processivity factor.

FIG 1

Generation and expression of KSHV BAC16 with in-frame HA tag of ORF59 and ORF59 deletions. (A) Schematic of ORF59 full-length protein showing previously identified interaction domains and sequential ORF59 50-aa deletion mutants described in this report. (B) KSHV BAC16 ORF59-HA and deletions were constructed using homologous recombination with insertion of the kanamycin I-SceI cassette, which contained an in-frame HA-tag to the carboxy terminus of ORF59, followed by seamless removal of the kanamycin cassette. The insertion and removal of the kanamycin cassette was confirmed by Southern blot analysis using a probe specific to ORF58/59. (C) Expression of ORF59 in iSLK cells upon reactivation. iSLK cells were transfected with either BAC16 ORF59-HA or ORF59 deletions and selected with 1 μg/ml puromycin, 250 μg/ml G418, and 1,200 μg/ml hygromycin. BACmid-containing cells were treated with doxycycline and TPA/NaB as indicated to reactivate virus. Protein was harvested at 48 and 72 hpi along with untreated control.

FIG 2

ORF59 301 to 350 aa are not required for interaction with histones during lytic reactivation of KSHV. Co-IP was performed to determine the interaction domain of ORF59 with native histones in the context of infection. Protein lysates were harvested 48 hpi, and protein complexes were isolated using HA-specific antibody and protein G magnetic beads in the presence of EtBr. Immunoprecipitations were analyzed by SDS-PAGE and visualized using histones and HA-specific antibodies.

FIG 3

ORF59 and histone interactions are DNA-independent. 293L transfected with pXI ORF59-HA and induced iSLK BAC16 ORF59-HA cells were treated with micrococcal nuclease and used for co-IP to determine if ORF59-histone interactions are dependent on the presence of DNA. (A) DNA was collected and extracted prior to and following nuclease treatment. DNA was run on 1% agarose gel. (B) Following treatment, protein complexes were isolated using HA-specific antibody and protein G magnetic beads. Immunoprecipitations were analyzed by SDS-PAGE and visualized using histones and HA-specific antibodies.

FIG 4

ORF59 deletion mutants retain the ability to localize to the nucleus during reactivation. (A) iSLK cells were transfected with ORF59-HA full-length or Δ351-396 plasmid and induced with doxycycline where indicated. IFA was performed to visualize the localization of ORF59 full-length and ORF59Δ351-396. Cells were visualized at ×40 magnification. (B) IFA was performed to visualize ORF59 localization in noninduced and induced iSLK BAC16 ORF59-HA WT and deletions. Cells were incubated with ORF59 antibody followed by secondary antibody labeled with Alexa Fluor 594. Cells were visualized at ×40 magnification.

FIG 5

The amino-terminus 2 to 300 aa of ORF59 are necessary for formation of dimers and tetramers. (A to C) (A) iSLK BAC16 ORF59-HA WT and (C) deletions were either noninduced or induced for 24 h, and (B) 293L cells transfected with pXI ORF59-HA were harvested and treated with 500 μM DSS or DMSO vehicle for 30 min, and cross-linking was quenched by addition of 1 mM tris-HCl pH 7.5. Protein complexes were separated by SDS-PAGE gel and analyzed by Western blotting using HA-specific antibody or specific histones H2B and H3. ORF59 (M) monomer, ∼55 kDa, (D) dimer, ∼110 kDa, and (T) tetramer, ∼220 kDa are shown.

FIG 6

ORF59 deletion mutants do not affect expression of IE and E viral transcripts. RNA was harvested and purified from iSLK BAC16 ORF59-HA WT and deletions at 6 and 24 hpi. Purified RNA was DNase treated and used for cDNA reactions, followed by qPCR using primers and probes specific to ORF50, the 3′ UTR region of ORF50 transcript from the dox-inducible promoter of the integrated gene in iSLK, ORF9, ORF58/59, ORFK2 (vIL-6), and ORF57 transcripts. Cycle threshold (C_T) values were normalized to cellular control untreated samples. Results represent three independent experiments. Bar graphs represent means ± the standard deviation (SD). Two-way analysis of variance (ANOVA) analysis was performed (P < 0.05).

FIG 7

The carboxy-terminus 301 to 396 aa are not required for viral DNA synthesis. (A) Total DNA was harvested at 48 and 72 hpi and purified for qPCR using TaqMan primers and probes to determine the relative levels of viral DNA accumulation. C_T values were normalized using cellular DNA and compared to control untreated samples. (B) ORF59 mutants produce infectious virus (GFP+/ml) at lower levels than WT infectious virus production of WT and deletions. Infectious virus is reported as described in Materials in Methods. Statistical significance was determined by unpaired t test; *, P < 0.05. (C) Rate of replication of WT and deletions. Induced cells at indicated time points were harvested, and viral genomes were quantified by qPCR. (D) For ORF59 to bind to DNA at the RRE region of oriLyt, 301 to 350 aa are not required. iSLK BAC16 ORF59-HA WT and ORF59 deletion mutants were either uninduced or induced for 24 h and harvested, cross-linked, and used for a ChIP assay to determine the ability of ORF59 and deletions to bind to the RRE region of the origin of viral replication. The first amino-terminus 300 aa of ORF59 do not show a strong association to RRE compared to WT and 301 to 350 aa at the carboxy terminus after lytic reactivation.