?(Fig.5).5). isolate proteins that bind to KSHV DNA fragments. This study led to the identification of several cellular replication, repair, and recombination factors, such as topoisomerases (Topo) I and II, MSH2/6, RecQL, DNA-PK, poly(ADP-ribose) polymerase 1 (PARP-1), and Ku autoantigens. These cellular proteins build up in viral replication compartments (VRCs) during viral DNA replication, suggesting their possible functions in KSHV replication. Additionally, we found that a nuclear scaffold/matrix protein (scaffold attachment factor A, or SAF-A) bound to the viral DNA, suggesting that attachment of DNA to the nuclear scaffold/matrix structure may be necessary for efficient viral DNA replication. MATERIALS AND METHODS Cell culture. The primary effusion lymphoma cell collection BCBL-1, which carries latent KSHV and was established by Ganem and his colleagues (30), was obtained from the NIH AIDS Research and Reference Reagent Program. The cells were produced in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco-BRL), penicillin-streptomycin (50 models/ml), and fungizone (1.25 g/ml amphotericin B and 1.25 g/ml sodium deoxycholate). Plasmids and constructs. Plasmids pOri-A and its mutants (pOri-15.7, pOri-M12, pOri-M1256, etc.) were explained previously (37). pCR3.1-ORF50 was constructed by cloning the cDNA sequence of the ORF50 coding region into the pCR3.1 vector (Invitrogen). The construct was explained in Lin et al. (25). DNA affinity purification and assay. Numerous biotinylated DNA fragments (+) PD 128907 were synthesized using PCR with pOri-A DNA or its mutants as themes and two oligonucleotides as primers. The two oligonucleotides for 3F and its derivative DNA fragments were 3F (5-CGGCAAAGCTAATTTGCATG-3) and Biotin-7R (5-biotin-ACTGGAATAGGGGCTGCGATGACTC-3). The oligonucleotides for 9F and its derivative DNAs were 9F (5-CAATTCTATAATTAAACAAGGTAGAA-3) and Biotin-ID13R (5-biotin-CGCCACCGAACAACCCCGTGGACAG-3). The oligonucleotides for 11F and its derivative DNAs were 11F (5-TAGGGCCCGATGAGTCATGGGGTT-3) and Biotin24280R (5-biotin-ACGGGTAAATCCAAGAGATCCGTCCC-3). The resultant biotinylated PCR fragments were coupled to streptavidin-conjugated magnetic beads (Dynal, Oslo, Norway) and then mixed with nuclear extracts prepared from tetradecanoyl phorbol acetate (TPA)-induced (and uninduced) BCBL-1 cells. In each reaction mixture, 2/3 volume of DNA-coupled beads in a solution [20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM (+) PD 128907 phenylmethylsulfonyl fluoride (PMSF), 0.05% NP-40, 15 mM MgCl2, 75 g/ml salmon sperm DNA, and 0.2 DNA. To find proteins that bind to KSHV DNA, we designed a DNA affinity purification process. Three overlapping DNA fragments, representing the core domain name of KSHV DNA were confirmed by Western (+) PD 128907 bolts with specific antibodies. Prominent bands were excised from each lane and subjected to in-gel trypsin digestion. A portion of each peptide digest was injected onto a nanocapillary reverse-phase high-performance liquid chromatograph coupled to a nanoelectrospray ionization source of an ion trap mass spectrometer (ThermoFinnigan LCQ). Mass spectrometry steps peptide masses and then fragments individual peptides to produce liquid chromatography-MS/MS spectra of fragments that reflect the peptide sequence. The MS/MS spectra were run against a nonredundant database with the program SEQUEST. The identities of the proteins that were recognized by this proteomic approach are indicated in Fig. ?Fig.1B.1B. The mass spectrometry spectra and the sequences of the corresponding peptides of each of the proteins are outlined in Table ?Table11. TABLE 1. Tryptic peptides of KSHV DNA in the computer virus context in cells. In brief, protein-DNA cross-linking was induced by addition of formaldehyde (+) PD 128907 to living TPA-induced BCBL-1 cells. Chromatin from these cells was fragmented by sonication, and the producing material was immunoprecipitated with specific antibodies against each of the viral and cellular proteins to be tested. The protein-bound DNAs were quantified by PCR using SMOC2 two pairs of primers designed to amplify the KSHV sequences from nucleotides 23147 to 23405 and from nucleotides 24020 to 24155 within the (L). The former region includes the K8 binding sites, and the latter region is near the RRE. Findings revealed that (i) both sequences could be coprecipitated by antibodies against three viral proteins, namely, K8, RTA, and ORF57, which are known to be involved in lytic viral DNA replication, but not by preimmune sera (either mouse or rabbit) or antibodies against ORF45 and ORF64 (Fig. ?(Fig.2);2); (ii) all the cellular proteins tested were found to be associated with.
