Rta, the gene product of Kaposi’s sarcoma-associated herpesvirus (KSHV) encoded mainly in open reading frame 50 (ORF50), is capable of activating expression of viral lytic cycle genes. In order to study the life cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV), we developed a gene expression system in KSHV-infected primary effusion lymphoma cells. This usually involves a very small number of infected cells in a particular tissue where the virus is present at very low copy number and there is limited or no viral gene expression. The Brg1 subunit of SWI/SNF and the TRAP230 subunit of TRAP/Mediator were shown to interact directly with RTA. Ectopic, immediate-early Us11 expression prevents eIF2alpha phosphorylation and the inhibition of translation observed in cells infected with a gamma(1)34.5 mutant by inhibiting activation of the cellular kinase PKR and the subsequent phosphorylation of eIF2alpha; however, a requirement for the Us11 protein, produced in its natural context as a gamma(2) polypeptide, remains to be demonstrated. We validated the interaction between LANA and KAP1 in vivo and in vitro, as well as their colocalization in the nucleus. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted.
Multiple viral proteins, such as vIRFs, ORF45, RTA and K-bZIP, are involved in this process 6, 7, 8, 9. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle. The majority of KSHV exists in a latent form in tumor cells, although a small population undergoes lytic replication. Taken together, our data suggest that LANA interacts with KAP1 and represses lytic gene expression to facilitate the establishment of KSHV latency. Activation of MAPK pathways was necessary and sufficient for activating the promoter of RTA. It is the most recently identified oncogenic DNA herpesvirus. However, the mechanism controlling KSHV latent versus lytic replication remains unclear.
In the presence of ICLs or stalled replication forks, the FA core E3 ligase complex (comprised of FANCA, B, C, E, F, G, L, and M) catalyzes the lysine site-specific monoubiquitination of two FA effector proteins, FANCI and FANCD2 (FANCI-D2), which promotes their recruitment to DNA damage repair sites in nuclear foci (Garcia-Higuera et al., 2001; Sims et al., 2007; Smogorzewska et al., 2007). This suggests that some miRNAs serve a primary role as subtle regulators to “fine tune” or “balance” levels of gene expression. In addition, antibodies against lytic proteins are significantly elevated in the sera of patients developing KS (54). The switch between latency and lytic-cycle gene expression of KSHV is initiated by a single immediate-early protein encoded by open reading frame 50 (ORF50) of the viral genome (30, 46). RBP-Jκ binding sites are present in a number of EBNA2- and RTA-regulated viral promoters. The RSKs are serine-threonine kinases and direct substrates of ERK1/ERK2. During viral reactivation, the K8 gene gets expressed in both the IE and delayed-early stages, driven by two distinct promoters (24).
While latent proteins have oncogenic properties, latency alone does not appear to be strongly immortalizing, suggesting that lytic replication has a role in KS pathogenesis. It has been shown that the latency-associated nuclear antigen (LANA) encoded by KSHV plays an important role in the establishment of KSHV latency (13, 17,–20). So far, KSHV miRs have been shown to regulate the viral life cycle and host pathways, which might contribute to viral persistent infection and development of KSHV-induced malignancies (1, 5, 17, 18, 23, 25, 26, 31, 32, 36, 38, 39, 42, 46, 53). In addition to the well-established significance of viral latency (7, 62, 125), the KSHV lytic phase contributes significantly to viral tumorigenesis by spreading viruses to target cells, thus serving to propagate the infection and providing paracrine regulation for KS development (22, 126). At the early stage of KSHV primary infection, lytic gene expression is shut down, along with increasing LANA expression (14, 20). LANA can inhibit RTA expression by repressing its promoter (13, 17). (D) Primer extension analysis of miR-K3 (lower band) and its variant miR-K3+1 (upper band) in the KSHV-negative BJAB cell line, KSHV-infected PEL cell lines (BC-1, JSC-1, BCBL-1, BCP-1, VG-1, BCLM, and BC-3), and 293T cells that were transfected with the parental lentiviral vector pLCE or with pLCE expressing a 250-bp fragment of pri-miR-K3 containing the GSNP or the ASNP.
KSHV ORF45 is involved in evasion of the host innate antiviral responses by inhibiting interferon regulatory factor 7 (IRF7) (9–11). It has been reported that LANA can accumulate heterochromatin components on the terminal repeat (TR) of the KSHV genome to repress viral gene expression and recruit Dnmt3a or other repressors to silence host gene expression (25,–27), but how LANA represses the transcriptional activity of the RTA promoter remains largely unknown. While CIP29 can bind UAP56 simultaneously with Aly, Chtop binding can be outcompeted by the presence of Aly, indicating consecutive loading of both proteins onto TREX. In an artificial system, it has been shown that the N-terminal domain can interact with the mSin3A complex to repress gene expression (30), yet little is known about the C-terminal domain of LANA. In screening, we identified a host protein, Krüppel-associated box domain-associated protein 1 (KAP1), that binds to LANA. KAP1 is also known as tripartite-motif-containing protein 28 (TRIM28) and transcription intermediary factor 1β (TIF1β) (31). KAP1 functions as a transcriptional repressor and can change epigenetic state by recruiting a histone deacetylase (HDAC) and methyltransferase complex (31,–33).
These results demonstrate that the molecular mechanisms that underlie RTA-mediated transcriptional activation require a large number of transcriptional cofactors and that their actions ultimately direct well-controlled viral gene expression and thereby viral lytic reactivation. The repression mediated by the KRAB-KAP1 complex can exert a long-range effect on the genome by spreading H3K9me3 and heterochromatin protein 1β (HP1β) (34). Also, the KRAB-KAP1 complex can mediate the repression of episomal gene expression of adeno-associated viral and nonintegrated lentiviral vectors, which implies a functional role in the herpesvirus family (35). A previous study has demonstrated that KAP1 regulates KSHV latency by association with transcriptional promoters in the viral genome, which is modulated by the viral protein kinase (vPK) (36). (A) Diagram of two miRNA responding elements (MREs) within the 3′ UTR of IKKε. Western blotting was performed as described previously (1). Therefore, we sought to develop a more robust assay system (vero-rKSHV.219/PAN-LUC) for easy and quantitative measurement of lytic induction of KSHV.
Based on the interface determined, we proved that LANA can recruit KAP1 to the KSHV genome to repress gene expression. We found multiple cooccupation sites of LANA and KAP1 on the whole KSHV genome and demonstrated that LANA-recruited KAP1 plays a critical role in the shutdown of lytic gene expression during the early stage of KSHV primary infection. Our results indicate that LANA interacts with the KAP1 protein and represses lytic gene expression to facilitate the establishment of KSHV latency. KSHV-positive B lymphoma cell lines (BCBL1, JSC-1, and BC-3) were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). 1A). As a result, BLV transcripts are not cleaved by Drosha, and only subgenomic small RNAs are processed into miRNAs. 293T (a human embryonic kidney cell line transformed with the E1 region of adenovirus and the simian virus 40 [SV40] T antigen) and 293 cells were cultured in Dulbecco’s modified Eagle’s medium (Cellgro) containing 10% fetal bovine serum (FBS) and antibiotics (50 U of penicillin/ml and 50 μg of streptomycin/ml).
HKB5/B5 is an EBV-negative cell line formed by fusion of HH514-16 cells with 293T cells (kindly provided by M.-S. Flow cytometry.Cells (5 × 105) were washed with RPMI 1640 medium containing 10% fetal calf serum and incubated for 30 min with antibodies. Ricciardi, respectively. To generate a K8 deletion mutant, we replaced the K8 coding sequence of BAC36 with a kanamycin (Kan)/SacB cassette by homologous recombination. Therefore, our results provide another example of RTA-mediated downregulation of a repressor protein, which may contribute to the switch between KSHV latency and lytic replication. Hemagglutinin (HA)-tagged KAP1 truncated constructs (ΔRBCC, ΔPB, and M) were constructed by cloning the corresponding fragments into the pCMV-HA vector in frame at the 5′ EcoRI and 3′ XhoI sites. miR mimics were commercially synthesized based on the sequences of mature miRs (Table 1) (Ambion, Life Technologies, Grand Island, NY).
Interestingly, the type I IFNs elicited through this pathway presumably govern the induction of CD8+ T-cell responses contributing to adaptive immunity (48, 49). Plasmid pGL3p-TR was generated by subcloning the TR fragment from plasmid pBSpuroA3 (a kind gift from Subhash C. Verma, University of Nevada, Reno, NV, USA) into the pGL3p vector at the NotI site. The underlined nucleotides make up the minimal T7 promoter. The hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) against purified ORF45 proteins and by Western blotting to lysates of TPA-induced BCBL-1 cells and purified viruses. 4124) was purchased from Cell Signaling Technology. Recent research has also shown that Chtop is able to bind Nxf1/TAP, a process necessary for mRNA handover from Aly to Nxf1/TAP .
Anti-acetyl-histone H3 (anti-AcH3) rabbit polyclonal antibody (no. 06-599) was purchased from Merck Millipore. Anti-LANA mouse monoclonal antibody produced by 1B5 hybridoma was made in our laboratory (antigen source for immunization, LANA aa 900 to 1162). Tandem affinity purification-mass spectrometry (TAP-MS) was used to identify LANA protein complexes. Identification of RTA binding proteins.To identify cellular proteins interacting with RTA, bacterially expressed GST-RTA fusion proteins were used as an affinity column for 35S-labeled lysates of Raji B cells. Strep-Flag-tagged LANA was overexpressed in 293T cells. Specifically, Strep-Tactin Sepharose (IBA) and Flag M2 Sepharose (Sigma) were utilized to purify native protein complexes.
Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF]) for 15 min. The band intensities were quantified using NIH ImageJ. We next constructed a BgH-inserted mutant. The three compounds efficiently induced the expression of all tested lytic genes (RTA, PAN, ORF57, ORF21, and ORF36) and SB only showed slight induction of each gene (Figure ). The immunoprecipitates were washed four times with RIPA buffer and then boiled in SDS loading buffer for Western blot analysis. For Western blotting, protein samples were analyzed by SDS-PAGE and transferred onto nitrocellulose membranes, followed by blocking and probing with the indicated antibodies for detection. Escherichia coli strain BL21(DE3) expressing GST or GST fusion proteins was grown in Luria broth (LB) medium at 37°C to exponential phase and cultured overnight at 16°C after induction with isopropyl-thiogalactopyranoside.
Cells were harvested and resuspended in ice-cold phosphate-buffered saline (PBS), followed by sonication lysis (Sonics; cycle, 3-s on/5-s off pulses; amplitude, 35%). 2D). In the host network model on the left, some viral miRNAs function as analogs of host miRNAs through seed sequence similarity, thereby targeting transcripts through the same docking sites as the mimicked host miRNAs. The nuclei pellet was resuspended in 210 μl of nuclear storage buffer (50 mM Tris-HCl [pH 8.0], 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA) and stored at −80°C. All probes were labeled by the random-primed method (46). Whole-cell lysates were used for immunoblotting with anti-Flag (αNIC) and antitubulin (αTubulin) antibodies. After serum starvation for 24 h, the transfected cells were induced with 20% FBS for 10 min.
For reconstitution of recombinant viruses in B lymphocytes, freshly prepared BAC DNAs were introduced into BJAB cells by electroporation. Coimmunoprecipitations.Protein coimmunoprecipitation assays were carried out with exogenously expressed proteins from transfected HEK 293T cell lysates and endogenous proteins from BCBL1 cell lysates. Cells were cross-linked in the medium with 1% formaldehyde for 30 min (LANA and KAP1) or 5 min (acetyl-histone H3) at room temperature and quenched with 0.125 M glycine. Annexin V-FITC (5 μl) and PI (5 μl) were added to 100 μl of cells. ISGs such as myxovirus resistance A (MxA), 2′-5′oligoadenylate synthetase 1 (OAS1), RNA-specific adenosine deaminase (ADAR), double-stranded RNA (dsRNA)-dependent serine/threonine protein kinase R (PKR), ISG56, ISG54, and ISG60 target and disrupt the viral life cycle at distinct stages through pleiotropic effects (4, 42, 104). The nuclei were lysed in SDS lysis buffer (50 mM HEPES, 1 mM EDTA, 1% SDS, 1 mM PMSF) for 10 min on ice. The lysates were subjected to sonication to obtain 200- to 500-bp fragments of DNA (Sonics; cycle, 2 6-s pulses; amplitude, 30 to 35%) and then centrifuged at 12,000 × g at 4°C for 10 min to obtain the supernatants.
Dual luciferase activities were read after 48 h with the Dual Luciferase Reporter assay system (Promega). After washing the beads 5 times with PBS, the bound proteins were eluted using glutathione elution buffer (10 mM gluthathione, 50 mM Tris-HCl, pH 8.5) and then dialyzed against PBS overnight. A small fraction of the supernatants were kept as input, and the remainder were divided into groups according to the experiment. The aliquots were incubated with pretreated protein A or G beads and the corresponding antibody overnight at 4°C. After extensive washing with RIPA buffer, wash buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 1 mM PMSF) and TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) (four times each), the beads were resuspended in TE buffer. The resuspended beads were subjected to RNase A and proteinase K digestion, and the cross-linking was reversed at 65°C for 8 to 10 h. DNA was recycled with a DNA purification kit (Tiangen).
KAP1 knockdown was achieved with short hairpin RNA (shRNA) (target sequence, GCGTCCTGGCACTAACTCA), as previously described (40). [35S]methionine/cysteine-labeled 293T cells transfected with the EBG or EBG-RTA vector were used for glutathione-Sepharose affinity chromatography (resin). All the procedures were described in the manual of the luciferase assay system (Promega). A dual luciferase reporter assay was not used because LANA may affect the expression of the control plasmid (29). The luciferase reporter plasmid was transfected into cells, along with the plasmid expressing HA-LANA, and the total amount of DNA was normalized with empty vector in the transfection. (E) Sponge/K12-11 can rescue IKKε expression in BCBL1 cells. The shaded residues are identical, and the two variants have 94.3% amino acid identity.
Moreover, the promoter activation was not dependent on viral protein(s) because there were no viral proteins in uninfected vero cells. Quantitative PCR (qPCR) was used to determine the relative quantities of RNA (cDNA) and DNA. Total RNA was extracted from harvested cells using TRIzol reagent (Life Technologies), and cDNA was obtained by reverse transcription with a genomic DNA (gDNA) eraser RT kit (TaKaRa). Total DNA (host and viral genomes) was extracted from harvested cells using the genome DNA extraction kit (Life Technologies). qPCR was performed with a SYBR green Real-Time PCR master mix kit (Toyobo). To compare between active viral RCs (WT) or pre-replicative foci (HP66), Vero cells were infected with either WT or the HP66 DNA Pol mutant virus and FANCD2 foci formation assessed by indirect immunofluorescence. Recently, in an orthotopic humanized mouse model, exogenous expression of KSHV mir-K12-11 was shown to be sufficient to drive hyperproliferation of B cells .
A set of double-stranded oligonucleotides, including pan1 (pPAN RRE), MJmulti (a mutant version of pPAN RRE), Kpsn (pKpsn RRE), Kpsn/TG (a mutant version of pKpsn RRE), K-ORF572 (pORF57 RRE), and K2p25 (pvIL-6 RRE) were used for electrophoretic mobility shift assays (EMSAs), (see Fig. (Left) Diagram of wild-type ORF50 and ORF50 deletion mutants used in EMSAs and reporter assays. Particularly, the induction kinetics of K5, K7, and Orf11 mRNAs were faster than those of other viral genes upon doxy treatment (Fig. For the double-label experiment, the primary mouse monoclonal and rabbit polyclonal antibodies were diluted together and incubated with cells on the slides at room temperature for 1 h. Gardella gel assay.Approximately 5 × 105 293T or BJAB cells carrying BAC36 and K8-null mutant viruses, respectively, were collected 3 days after induction for lytic KSHV replication. HEK 293T cells were cotransfected with Hey1-Myc and GFP-RTA or GFP, and the transfected cells were cultured in the absence or presence of the proteasome inhibitor, MG132. The enriched DNA was subjected to library preparation for high-throughput sequencing.
2A). These responses must be effectively curbed by KSHV to ensure successful viral particle assembly directed toward egress.