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. Author manuscript; available in PMC: 2011 Nov 25.
Published in final edited form as: Virology. 2010 Sep 9;407(2):206–212. doi: 10.1016/j.virol.2010.08.014

Requirement of UAP56, URH49, RBM15, and OTT3 in expression of Kaposi sarcoma-associated herpesvirus ORF57

Vladimir Majerciak 1, Merlyn Deng 1, Zhi-Ming Zheng 1,*
PMCID: PMC2952739  NIHMSID: NIHMS230850  PMID: 20828777

Abstract

Transport of mRNA from the nucleus to the cytoplasm is mediated by cellular RNA export factors. In this report, we examined how RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, function in modulating KSHV ORF57 expression. We found that knockdown of each factor by RNAi led to decreased ORF57 expression. Specifically, reduced expression of either UAP56 or RBM15 led to nuclear export deficiency of ORF57 RNA. In the context of the KSHV genome, the near absence of UAP56 or RBM15 reduced the expression of both ORF57 and ORF59 (an RNA target of ORF57), but not ORF50. Collectively, our data indicate that the expression of KSHV ORF57 is regulated by cellular RNA export factors and cofactors at the posttranscriptional level.

Introduction

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, a homologue of HSV-1 ICP27, EBV EB2, and HCMV UL69, is a viral early protein which plays important roles at the posttranscriptional level for virus gene expression (Majerciak & Zheng, 2009). When the KSHV genome contains a disrupted ORF57, it results in both inefficient expression of a subset of viral lytic genes and poor production of infectious virions (Majerciak et al., 2007). ORF57 is important for the viral life cycle because it promotes viral RNA splicing and enhances the expression of viral intronless genes (Majerciak et al., 2008; Majerciak et al., 2010). It is also known to form specific associations with RNA, which is facilitated by cellular proteins (Majerciak & Zheng, 2009). Thereby, it was proposed that ORF57 promotes the expression of its target genes by stimulating RNA export via its interaction with Aly/REF, a cellular RNA-binding protein serving as an adaptor to interact with an RNA export factor NXF1/TAP (Malik et al., 2004). However, recent data from our lab, as well as others, indicate that the Aly/REF-ORF57 interaction does not appear to play a significant role in ORF57-mediated enhancement of ORF59 expression (Majerciak et al., 2006a; Nekorchuk et al., 2007). Other reports indicate that Aly/REF is not essential for nuclear export of bulk mRNA as Aly/REF knockout in Drosophila cells or C. elegans displayed no defect in RNA export (Longman et al., 2003; Gatfield & Izaurralde, 2002). Instead, a recent report describes ORF57 being able to recruit the entire TREX through its interaction with Aly/REF to then facilitate export of a viral late transcript, ORF47 (Boyne et al., 2008). Therefore, we were interested in examining the influence of TREX complex members on KSHV expression.

UAP56 (BAT1) and its close (90% identical residues) member URH49 (DDX39) are two DExD/H box helicases that have important roles in pre-mRNA splicing and nuclear export of mature mRNA (Kapadia et al., 2006; Shen et al., 2008; Kota et al., 2008). The role of UAP56 and URH49 in the TREX complex is to recruit the Aly/REF protein onto a region nearby the 5' end of mRNA by facilitating the interaction between CBP80/20 and Aly/REF (Taniguchi & Ohno, 2008; Luo et al., 2001; Cheng et al., 2006; Nojima et al., 2007). Both UAP56 and Aly/REF accompany the bound mRNA to the nuclear periphery where Aly/REF then interacts with NXF1/TAP to displace UAP56 from the mRNA, and subsequently transfers the mRNA from Aly/REF to NXF1/TAP for nuclear export (Hautbergue et al., 2008). The importance of UAP56, along with URH49, is exemplified by its interaction with the N-terminal half of HCMV UL69 (Lischka et al., 2006), which, independent of UL69-RNA binding, was found to be involved in UL69-mediated nuclear export of unspliced RNA (Toth et al., 2006).

RNA export cofactors RBM15 (OTT1, RBM15A) and its close member OTT3 (RBM15B) are members of the SPEN protein family that associate with spliceosomes and mediate RNA export function (Hiriart et al., 2005; Lindtner et al., 2006; Uranishi et al., 2009). RBM15 and OTT3 bind RNA, interact directly with NXF1/TAP via their C-terminal region, and function as cofactors to the nuclear export receptor NXF1/TAP (Hiriart et al., 2005; Lindtner et al., 2006; Uranishi et al., 2009). In the present study, we examined the function of RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, in KSHV ORF57 expression. We determined that these cellular factors are essential for efficient ORF57 expression. Both UAP56 and RBM15 are required for expression of ORF57, but not ORF50, during KSHV lytic induction.

Results

UAP56, URH49, RBM15, and OTT3 are required for ORF57 expression

As HCMV UL69 has been reported to promote the expression of its RNA targets via interaction with UAP56 and URH49 (Lischka, et al., 2006), we wished to determine whether UAP56 and URH49 have a similar role in KSHV ORF57-enhanced expression of ORF59 (Majerciak et al., 2006a; Majerciak et al., 2007; Kirshner et al., 2000), an intronless gene encoding a viral DNA polymerase processivity factor. Using RNAi, we independently knocked down the expression of UAP56 or URH49 in both HeLa and HEK293 cells along with cotransfection of KSHV ORF57 and its target ORF59 (Kirshner et al., 2000; Majerciak et al., 2006a). Double knockdown of both UAP56 and URH49 in these cells was detrimental to cell growth (data not shown). Our immunoblotting results indicated an efficient knockdown of UAP56 or URH49 expression in both cell lines (Fig. 1A and 1B, compare lanes 1, 3, 5, and 7 with lanes 2, 4, 6, and 8). Consistent with our previous observation (Majerciak et al., 2006a), the expression of ORF59 in both cell types treated with non-specific (NS) siRNAs was significantly enhanced by ORF57 (Fig. 1A and 1B, compare lane 1 with lane 3 and lane 5 with lane 7). However, ORF57-mediated enhancement of ORF59 expression was largely diminished when the cells had a reduced expression of UAP56 or URH49 (Fig. 1A and 1B, compare lanes 3 and 7 with lanes 4 and 8). As the presence of ORF57, which is required to promote ORF59 expression, was also reduced in the knockdown conditions (Fig. 1A and 1B, compare lanes 3 and 7 with lanes 4 and 8), this indicated that the observed down-regulation of ORF59 expression by knocking down UAP56 or URH49 expression was a result of reduced presence of ORF57. We observed that GFP expression was not affected under the knockdown conditions. The negative effect on ORF57 expression by knocking down UAP56 or URH49 was unexpected, but since UAP56 and URH49 are thought to function downstream of ORF57 to promote ORF59 expression, we were unable to address how these factors are involved in regulation of ORF59 expression in the absence of ORF57.

Fig. 1.

Fig. 1

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UAP56 and URH49 regulate ORF57-enhanced expression of ORF59 by control of ORF57 production. (A and B) Knockdown of UAP56 and URH49 affects the expression of ORF57 and ORF59. HeLa (A) and HEK293 (B) cells were cotransfected with 40 nM of synthetic siRNAs (Dharmacon, Lafayette, CO) to knockdown cellular UAP56 (left panel) and URH49 (right panel) expression together with vectors expressing FLAG-tagged KSHV ORF59 and GFP-tagged KSHV ORF57. HPV16 E6 specific siRNA 209 (Tang et al., 2006) was used as a non-specific (NS) siRNA and a vector expressing GFP protein alone was used as a negative control. Protein samples were prepared 24 h after transfection and the expression of UAP56, URH49, ORF59-FLAG and ORF57-GFP was immunoblotted with anti-UAP56, anti-URH49, anti-FLAG, anti-GFP, or anti-tubulin antibody. Cellular tubulin served as a sample loading control.

To further examine this observation, we compared RNA export factors UAP56 and URH49 with RNA export cofactors RBM15 and OTT3 for the expression of ORF57 in HeLa cells. As shown in Fig. 2, knocking down either one of the tested RNA export factors or cofactors by a gene-specific siRNA led to a great reduction of ORF57 expression when compared with the cells transfected with a NS siRNA (Fig. 2A, compare lanes 2–5 with lane 1). When GFP was used as a control, the siRNA-mediated reduction of UAP56 or RBM15, a representative member of each RNA export factor family, had no effect on GFP expression (Fig. 2B). It was also noticed that knockdown of one factor had no effect on the expression of other factors, indicating a specific role of individual cellular factors in regulation of ORF57 expression.

Fig. 2.

Fig. 2

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Knockdown of RNA export cofactor RBM15 or OTT3 also inhibits KSHV ORF57 expression. HeLa cells were cotransfected with synthetic siRNAs (40 nM) targeting to individual factors, together with an ORF57-GFP expression vector (A) or an empty GFP expression vector (B). Protein samples were collected 24 h after transfection. KSHV ORF57 expression and knockdown efficiency of individual gene expression were determined by western blot with anti-GFP for ORF57 or a specific antibody for individual cellular factor. Tubulin served as a sample loading control. (A) Efficient expression of KSHV ORF57 requires cellular RNA export factors or cofactors. (B) Knockdown of UAP56 and RBM15 expression in HeLa cells had no effect on GFP expression.

UAP56 and RBM15 are important for export of ORF57 RNA

Since UAP56 and RBM15 as well as their close members are cellular RNA export factors and cofactors, we hypothesized that the reduced expression of ORF57 protein upon knockdown of each tested factor might be attributed to defects in ORF57 mRNA export. To test this hypothesis, we examined the levels of total ORF57 RNA and fractionated nuclear and cytoplasmic ORF57 RNA after siRNA knockdown of UAP56, RBM15, or Aly/REF in HeLa cells. Aly/REF was included in this study since this RNA export factor plays no significant roles in efficient ORF57 expression, nor ORF57-mediated expression of ORF59 (Majerciak et al., 2006a). As shown in Figs. 1 and 2, our initial studies were based on a GFP-tagged ORF57 cDNA clone lacking a native intron from the primary ORF57 transcript. Since RNA splicing and export are coupled events, we constructed an expression vector, pVM87, containing the genomic region of ORF57 with its native small intron to mimic ORF57 expression from the KSHV genome in the RNA export assays. Thus, ORF57 expressed from pVM87 has a small intron within its pre-mRNA that can be spliced out. With this strategy, we determined that siRNA knockdown of UAP56 and RBM15 reduced the expression of both ORF57 protein and RNA, whereas siRNA knockdown of Aly/REF did not (Fig 3A–B). RNA fractionation analysis revealed that both UAP56 and RBM15, but not Aly/REF, were important for efficient export of nuclear ORF57 RNA (Fig. 3C–E). When the nuclear to cytoplasmic ratio of ORF57 RNA was taken into account, a prominent nuclear retention of ORF57 RNA, but not cellular cyclophilin RNA, was observed upon knockdown of either UAP56 or RBM15 (Fig. 3D–E). There was no detectable ORF57 pre-mRNA (unspliced) in the RPA assays, indicating that knockdown of UAP56 and RBM15 did not prevent ORF57 RNA splicing. As expected, knockdown of Aly/REF showed no significant effect on the export of ORF57 RNA (Fig. 3D–E), consistent with our previous observations on ORF59 RNA (Majerciak et al., 2006a). In a separate experiment, we also found that knocking down expression of UAP56 and RBM15, but not Aly/REF, not only affected ORF57 RNA export, but subsequently reduced total RNA level of ORF57, whereas cellular cyclophilin RNA remained exported efficiently and its total RNA production was not affected by short-term (24–48 h) of UAP56 or RBM15 knockdown (data not shown). Based on these results, we conclude that UAP56 and RBM15 are important for the efficient export of nuclear ORF57 RNA.

Fig. 3.

Fig. 3

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UAP56 and RBM15 are important for KSHV ORF57 mRNA expression and export. (A) Expression of ORF57 depends on UAP56 and RBM15 but not Aly/REF. HeLa cells pre-treated with the corresponding siRNA duplexes (40 nM) for 24 h were cotransfected with individual siRNAs (40 nM) together with an expression vector (pVM87) carrying an intron-containing genomic clone of ORF57 fused to FLAG-tag on its C-terminus. The cells at 24 h after transfection of ORF57 expression vector were harvested for protein expression as determined by western blot (WB) as in Figs. 1 and 2 and for RNA expression as determined by northern blot (NB). Tubulin served as a loading control in WB. Total RNA (5 µg/lane) in NB was blotted with the following oligo probes labeled with γ-32P: oVM11 for ORF57 and oZMZ270 for GAPDH as an RNA loading control. (B) A relative level of ORF57 total RNA in each sample after being normalized to GAPDH RNA was shown in the bar graph as a mean ± SD from three independent experiments, with the ORF57 level in the cells transfected with NS siRNA as 1. Stars indicate the significant changes (p<0.05, student t-test). (C) Diagram of an antisense riboprobe spanning over the exon junction of ORF57 exon 1 and exon 2 designed to detect ORF57 mRNA in (D) by RNase protection assays (RPA). Dashes on the left end of the probe are non-complementary sequences. The resulting protected products of ORF57 mRNA and pre-mRNA are shown below the probe. (D and E) UAP56 and RBM15 are required for the export of ORF57 RNAs. Fractionated nuclear (N) and cytoplasmic (C) RNAs (15 µg each) from HeLa cells with the individual siRNA knockdown in (A) were analyzed by RPA with the ORF57-specific riboprobe, together with a cyclophilin probe (Majerciak et al., 2007) for a loading control (D). A relative RNA level of ORF57 mRNA in each sample was quantified after being normalized to RNA export factors and ORF57 cyclophilin mRNA for sample loading and expressed as an N/C ratio (E). One representative experiment of two is shown.

UAP56 and RBM15 are required for expression of ORF57 and ORF59, but not ORF50, in the context of the KSHV genome during viral lytic infection

To further confirm the roles of UAP56 and RBM15 in ORF57 expression in the context of the KSHV genome, we choose Bac36 cells, a stable cell line derived from HEK293 cells, which carries a wt KSHV genome constructed in a bacterial artificial chromosome (Majerciak et al., 2007), for the study. In these cells, the KSHV is present in the latent stage, but lytic replication can be reactivated by treatment with various chemicals, such as butyrate, valproate, or TPA, or by ectopic expression of the viral transcactivator ORF50 (RTA). We demonstrated that siRNA knockdown of either UAP56 or RBM15 expression in Bac36 cells led to decreased expression of ORF57, but not ORF50 (an ORF57 transactivator), from the KSHV genome during virus lytic induction (Fig. 4A and B). This observation is consistent with the data obtained in transient transfection assays (Figs. 1 and 2), indicating that both UAP56 and RBM15 function in Bac36 cells on ORF57 expression from its native promoter. When ORF59 was detected by immunofluorescence labeling of lytically infected cells, both UAP56 and RBM15 knockdown were found to reduce ORF59 expression (Fig. 4C and D, top panels). We compared to NS siRNA treated cells, the total number of ORF59+ cells in 3 optical microscope fields was decreased by 53% in the cell population treated with UAP56 RNAi (93 vs 44), and by 59% in the cell population treated with RBM15 RNAi (117 vs 48). This effect may be due to the decreased expression of ORF57 (Kirshner et al., 2000; Majerciak et al., 2006a; Majerciak et al., 2007) (Fig. 4A and 4B). It is worth noting that both UAP56 and RBM15 knockdown at this regiment neither affected GFP expression (Fig. 4A–D) from the Bac cassette inserted in the KSHV genome, nor cell viability as shown by nuclear DAPI staining or optical microscopy in trypan blue exclusion assays (Fig 4C and 4D). Collectively, the data obtained from Bac36 cells provide further evidence that the expression of KSHV ORF57 from the KSHV genome during lytic induction is also dependent on cellular RNA export factors.

Fig. 4.

Fig. 4

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UAP56 and RBM15 are important for the expression of ORF57 and ORF59 during virus lytic replication. (A and B) Knockdown of UAP56 or RBM15 expression in Bac36 cells reduces the expression of ORF57, but not ORF50. A stable cell line latently infected with wild type KSHV genome (Bac36-wt) (Majerciak et al., 2007) was treated with 40 nM of NS, UAP56 (A), or RBM15 siRNA (B) for 24 h before virus lytic induction by butyrate (3 mM) for an additional 24 h. The efficiency of UAP56 or RBM15 knockdown and the levels of ORF57 and ORF50 expression from lytic KSHV induction were determined by immunoblotting with anti-ORF57 or anti-ORF50 antibodies. Tubulin served as a sample loading control. GFP expression in each sample indicates copy numbers of the KSHV genome in the cells. (C and D) Knockdown of UAP56 or RBM15 expression in Bac36 cells decreases O RF59 expression. ORF59 expression was measured by immunofluorescence staining (Majerciak et al., 2008) with anti-ORF59 antibody. GFP marks cells carrying the KSHV genome. The nuclei were stained with DAPI. Cell morphology was visualized with phase microscopy.

Discussion

In this study, we have shown that cellular RNA export factors UAP56 and URH49, and RNA export cofactors RBM15 and OTT3, are important for efficient ORF57 expression. Although we initially thought that KSHV ORF57, like HCMV UL69 (Lischka et al., 2006), would require UAP56 to function in promoting ORF59 expression, our finding that ORF57 itself is controlled by UAP56, URH49, RBM15, and OTT3 was unexpected. UAP56 and RBM15 affect ORF57 expression by promoting export of the nuclear ORF57 transcripts. When nuclear RNA export became less efficient, the ORF57 RNA retained in the nuclear compartment may undergo degradation and resulted in reduction of ORF57 total RNA similar to other report (Herold et al., 2003). There are perhaps other possibilities leading to reduction of ORF57 total RNA in the absence of UAP56 or RBM15. For example, the accumulated nuclear ORF57 RNA may trigger a feedback suppression on ORF57 transcription, and alternatively, degradation of the cytoplasmic ORF57 RNA might be increased in the absence of an RNA export factor.

The function of UAP56 and RBM15 in promotion of viral lytic gene expression appears selective since a decreased level of either protein affected the expression of ORF57 and ORF59 (a downstream target of ORF57), but not ORF50, in the context of the wt KSHV genome during lytic induction. Similar to viral ORF50, expression of cyclophilin, tubulin, GAPDH, and GFP in transient transfection or from Bac36 stable cells remained unchanged in the cells with knockdown of UAP56 and RBM15. Together, these data argue the possibility that the selective effect on ORF57 expression resulted from an off-target effect of UAP56 or RBM15 siRNAs. However, how these proteins function selectively toward individual RNA species remains to be understood. Whether ORF57 RNA contains a cis-element for an RNA export factor or cofactor to bind needs to be determined. A recent report (Read & Digard, 2010) also showed differential dependence of various influenza A virus mRNAs on cellular export machinery. UAP56, when expressed ectopically above its endogenous level, showed no effect on the expression of ORF57 or ORF59 (data not shown). In addition, UAP56 appeared no additive effect on ORF57 to promote ORF59 expression when all three expression vectors were cotransfected (data not shown). Nevertheless, our data from this study indicate that KSHV ORF57 might utilize a distinct mechanism from its homolog CMV UL69 (Toth et al., 2006; Lischka et al., 2006) in regulation of the expression of its RNA targets.

Although expressed from two different genes positioned on different chromosomes, UAP56 and URH49 which are 90% identical did not compensate each other in ORF57 expression when one was knocked down. This interesting observation seems in conflict with the predicted redundant functions of UAP56 and URH49. While our study was under revision, Yamazaki et al reported that UAP56 and URH49 preferentially form distinct mRNA export machineries and do exhibit separate functions (Yamazaki et al., 2010). However, another study showed that HeLa cells with transient knockdown of UAP56 or URH49 appeared to proliferate normally and expressed a co-transfected gene well (Kapadia et al., 2006). Consistent with this report, HeLa cells with transient knockdown (24–48 hrs) of UAP56 or URH49 in our study also displayed no growth defect by microscopy or by trypan blue exclusion assays when compared with the cells receiving non-specific siRNAs. In contrast, we and others (Hautbergue et al., 2009) also noticed that the cells with double knockdown of both UAP56 and URH49 proliferated poorly, with low viability. Taken together, these data suggest that UAP56 and URH49 may exhibit some overlapping functions, but their other functions appear separable.

Aly/REF is an RNA binding protein and was thought to play an important role in viral RNA export (Malik et al., 2004). A recent report describes ORF57 being able to recruit the entire TREX through its interaction with Aly/REF to facilitate export of a viral late transcript, ORF47 (Boyne et al., 2008). However, recent data from our lab, as well as others, indicate that the Aly/REF-ORF57 interaction does not appear to play a significant role in ORF57 expression, nor ORF57-mediated enhancement of ORF59 expression (Majerciak et al., 2006a; Nekorchuk et al., 2007). In this study, we provide further evidence showing that knocking down Aly/REF expression did not decrease RNA export of ORF57, nor cellular cyclophilin. Our data is consistent with the reports that Drosophila cells and C. elegans with Aly/REF knockout exhibits no defect in RNA export of bulk mRNA (Longman et al., 2003; Gatfield & Izaurralde, 2002). A recent study suggests that mammalian cells may have adapted a compensatory mechanism for cell survival by increasing expression levels of alternative export factors (UIF, UAP56, URH49) following Aly/REF knockdown (Hautbergue et al., 2009).

Materials and methods

Cell lines

HEK293 and HeLa cells obtained from ATCC were cultivated in DMEM with 10% FBS. HEK293-derived cell line carrying latent KSHV-wild type genome (Bac36-wt) was described (Majerciak et al., 2007) and KSHV lytic infection was induced by sodium butyrate (n-butyrate) treatment (3mM final). All transfections were performed with Lipofectamine2000 (Invitrogen, Carlsbad, CA) as recommended by manufacturer.

Plasmids

Mammalian expression vectors pEGFP-N1 was purchase from Clontech (Mountain View, CA) and pFLAG-CMV-5.1(Sigma, St Louis, MO). Constructs pVM18 expressing KSHV ORF59-FLAG fusion and pVM8 with ORF57 cDNA-GFP fusion were described (Majerciak et al., 2006a). Plasmid pVM87 was constructed by insertion of ORF57 genomic clone amplified with oVM68 (5'-TACTCAGAATTCACC/ATGGTACAAGCAATGATAGACATGG-3') and oVM69 (5'-ATCGTGGATCC/AGAAAGTGGATAAAAGAATAAACCCTTG-3') on Bac36-wt DNA and cloned into pFLAG-CMV-5.1.

RNAi

Synthetic SMARTpools siRNAs targeting human UAP56 (BAT1, M-003805-00), URH49 (DDX39, M-004920-00), RBM15 (RBM15, L-010854-00), OTT3 (RBM15B, L-018823-01) and Aly/REF (THOC4, M-012078-00) were purchased from Dharmacon (Lafayett, CO). SiRNA 209 (5'-UCCAUAUGCUGUAUGUGAUdTdT-3') targeting the HPV16 E7 coding region (Tang et al., 2006) was used as a non-specific siRNA. All knock down experiments were performed with siRNA final concentration of 40 nM.

Western blot

Protein samples were prepared by direct cell lysis in Laemmli SDS protein sample buffer, separated in SDS-PAGE, following standard Western blot protocol. Following antibodies were used in this study: anti-UAP56 (BAT1, Abnova, Taipei, Taiwan), anti-URH49 (DDX39, Aviva Systems Biology, San Diego, CA), anti-FLAG (M2, Sigma, St Louis, MO), anti-GFP (JL-8, BD Biosciences, San Jose, CA), anti-RBM15 (ProteinTech Group, Chicago, IL), anti-Aly/REF (11G5, Novus Biologicals, Littleton, CO), or anti-tubulin (CloneTub2.1, Sigma) antibody. Anti-OTT3 antibody was a generous gift of Dr. Evelyne Manet (INSERM, Lyon, France). Monoclonal anti-ORF50 antibody was a gift of Dr. Koichi Yamanishi.

Northern blot

Total RNA was extracted from cells by Trizol Reagent (Invitrogen) and analyzed in Northern blot as described (Majerciak et al., 2006a). Individual transcripts were detected with γ-32P-labeled DNA oligo probes (oVM11, 5'-CTCGTCTTCCAGTGTCGGTG-3', for KSHV ORF57 and oZMZ270, 5'-TGAGTCCTTCCACGATACCAAA-3', for cellular GAPDH).

RNase protection assay (RPA)

Nuclear and cytoplasmic RNA were prepared as described (Majerciak et al., 2006b). RPA was carried out with RPA III kit (Ambion, Austin, TX) as recommended. Antisense RNA probes were synthesized by in-vitro transcription in the presence of [α-32P]GTP with Riboprobe System-T7 (Promega, Madison, WI). The templates included PCR product with build-in T7 promotor amplified on ORF57 cDNA by oligos oVM68 and oVM14 (5'-TAATACGACTCACTATAGG/GCTCGTCTTCCAGTGTCGGT-3') and human antisense cyclophilin A template (Ambion). Protected products were separated in 8% urea-denaturating PAGE, captured using a Molecular Dynamic PhosphorImager Storm 860, and analyzed with Image-Quant Software.

Indirect immunofluorescent staining

Cells cultivated on cover slides were fixed and intracellular staining was carried out as previously described (Majerciak et al., 2007) using anti-KSHV ORF59 (Advanced Biotechnologies, Columbia, MD) in combination with AlexaFlour546-conjugated secondary antibody (Invitrogen). Cell nuclei were counterstained by DAPI.

Acknowledgments

We thank Evelyne Manet for providing anti-OTT3 antibody, Koichi Yamanishi for anti- ORF50 antibody, and Barbara Felber for providing UAP56-HA plasmid. We thank Michael Kruhlak for his critical reading of our manuscript. This study was supported by the Intramural Research Program of NIH, National Cancer Institute, Center for Cancer Research.

Footnotes

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