KSHV ORF57 may preferentially bind Aly over UIF.

<p>(A) Competition assays were performed using recombinant GST-ORF57 bound to glutathione agarose beads and incubated with (i) a constant amount of purified Aly-His (1 µg) and increasing amounts of purified UIF-His (0, 0.5, 1, 2, 3 µg), (ii) a constant amount of purified UIF-His (1 µg) and increasing amounts of purified Aly-His (0, 0.5, 1, 2, 3 µg), Precipitated protein and inputs were analysed by western blotting using a His-specific antibody. (B) Dose-dependent co-immunoprecipitations were performed using 293T cells cotransfected with (i) 0.5 ug of pORF57GFP and 0.5 ug of pAly-myc, in addition to increasing amounts (0, 0.1, 0.5, 0.8, 1.2 ug) of pUIF-Flag or (ii) 0.5 ug of pORF57GFP and 0.5 ug of pUIF-Flag, in addition to increasing amounts (0, 0.1, 0.5, 0.8, 1.2 ug) of pAly-myc, empty vector was also added to ensure a similar amount of DNA was transfected in each sample. After 24 hours, cell lysates were incubated with GFP-TRAP-Affinity agarose beads and the amount of precipitated (i) Aly or (ii) UIF was identified by immunoblotting with Myc- or Flag-specific antibodies, respectively. Western blots for input loading are also shown for ORF57, Aly and UIF constructs.</p

ORF57 is required for the recruitment of UIF to a KSHV intronless mRNA.

<p>RNA immunoprecipitations were performed in 293T cells cotransfected with KSHV pORF47 in the presence of either pEGFP or pORF57GFP. After UV crosslinking cell lysates were immunoprecipitated using GFP- or UIF-specific antibodies. In addition, no antibody and a negative control Y14 antibody were also used as controls. Protein was then digested, and immunoprecipitated RNA was analysed by qRT-PCR.</p

ORF57 interacts directly with UIF during KSHV lytic replication.

<p>Immunoprecipitations were performed on unreactivated or reactivated BCBL-1 cells using no antibody control, ORF57- or UIF-specific antibodies. Precipitated proteins were detected by western blot using UIF- and ORF57-specific antibodies. BCBL-1 cells were reactivated by the addition of TPA (20 ng/ml).</p

KSHV ORF57 colocalises with UIF in the nucleus and nucleolus.

<p>293T cells were either mock (Ai, Ci), or transfected with pORF57-mCherry (Aii, Cii) or pUIF-GFP (Bi, Di) and in combination (Bii, Dii), incubated for 24 h, fixed and immunofluoresence staining performed. ORF57 and UIF were visualised by direct fluorescence of Cherry and GFP, respectively. Subcellular localisation within the nuclear speckles or nucleolus was confirmed using antibodies specific to SC-35 (A, B) or B23 (C, D), respectively. A merge of the mCherry/GFP channels is also included for all images. White arrows indicate nuclear speckles (Bii) or nucleolar localisation (Dii).</p

ORF57 is linked to the hTREX complex by UIF; however, ORF57 preferentially interacts with Aly over UIF.

<p>(A) Recombinant GST, and GST-ORF57 were bound to glutathione agarose beads and GST Pull-down assays performed using purified recombinant UIF-His or control ORF73-His proteins. Precipitated proteins and inputs were analysed by western blotting using a His-specific antibody. (B) Reconstitutive GST pulldowns were performed using GST or GST-UAP56 bound to glutathione agarose beads and incubated with recombinant purified ORF57-His or Aly-His alone or in combination. Precipitated proteins and inputs were analysed by western blotting using a His-specific antibody.</p

Knockdown of both Aly and UIF impairs the ability of ORF57 form an export competent viral ribonucleoprotein particle.

<p>(A) 293, 293ΔAly, 293ΔUIF and 293ΔAlyΔUIF cells were mock treated or treated with 2 µg/ml doxycyclin grown for 72 h. Cell lysates were analysed by western blotting using Aly-, UIF- and UAP56-specific antibodies. (B) (i) 293 cells were transfected with pEGFP and grown for a further 24 h. Cell lysates were incubated with GFP-TRAP-Affinity beads and after washing, the precipitated proteins were detected by western blotting using GFP-, UAP56-, FSAP79- and TAP-specific antibodies. (ii) 293, 293ΔAly, 293ΔUIF and 293ΔAlyΔUIF cells were treated with 2 µg/ml doxycyclin and grown for 48 h before being transfected with pGFP-ORF57 and grown for a further 24 h. Cell lysates were incubated with GFP-TRAP-Affinity beads and after washing, the precipitated proteins were detected by western blotting using GFP-, UAP56-, FSAP79- and TAP-specific antibodies. Transfected cell lysates were used as an input control.</p

Knockdown of both Aly and UIF impairs the ability of ORF57 to export KSHV intronless mRNA from the nucleus and reduces KSHV late protein production.

<p>(A) 293, 293ΔAly, 293ΔUIF and 293ΔAlyΔUIF cells were treated with 2 µg/ml doxycyclin and grown for 48 h before being transfected with pORF47 in the presence of either pEGFP or pGFP-ORF57. 24 h post-transfection RNA was isolated from total and cytoplasmic fractions and relative levels were analysed by qRT-PCR using GAPDH as a reference. Fold increase was determined by ΔΔcT and statistical significance by a non-paired <i>t</i>-test. Data from 3 independent experiments is presented as fold increase versus pGFP-transfected controls. (B) 293, 293ΔAly, 293ΔUIF and 293ΔAlyΔUIF cells were mock treated or treated with 2 µg/ml doxycyclin and grown for 48 h before infection with KSHV at a MOI = 1. At 48 h post-infection cells lysates were analysed by western blot using antibodies specific to KSHV (i) gB and (ii) ORF4 proteins. Results are shown of densitometry analysis of the western blots from 3 independent experiments carried out using the ImageJ software and is shown as expression of gB or ORF4 between uninduced and induced cell lines relative to β-actin.</p

KSHV ORF57 interacts directly with UIF.

<p>(A) (i) Bacterially expressed GST-, GST-UAP56-, GST-ORF57- and GST-ORF57Pmut-bound to glutathione agarose beads and separated by SDS-PAGE, proteins were visualised by coomassie staining. (ii) GST Pull-down assays were performed using pUIF-FLAG transfected cell lysates. Precipitated UIF protein was detected by western blot analysis using a FLAG-specific antibody. UIF-FLAG transfected cell lysate served as a loading control (Input). (B) Immunoprecipitations using GFP- or UAP56-specific antibodies were performed using cell lysates cotransfected with UIF-FLAG in the presence of either pEGFP, pUAP56-Myc, pORF57GFP or pORF57PmutGFP. Precipitated UIF-FLAG, UAP56-myc, GFP, ORF57GFP and ORF57PmutGFP were detected by western blot analysis using antibodies specific to FLAG, myc or GFP. Transfected cell lysates served as a loading control (Inputs). (C) Immunoprecipitations were performed in the presence or absence of RNase, using a no antibody control, (SC35)- or FLAG-specific antibodies on cell lysates cotransfected with pUIF-FLAG in the presence of either (i) pGFP and (ii) pORF57GFP. Precipitated proteins were detected by western blotting using GFP- or FLAG-specific antibodies. (iii) ORF57-transfected co-immunoprecipitations were also immunoblotted with an Aly-specific antibody to confirm the activity of the RNase digestion to abolish the RNA-dependent interaction between Aly and UIF.</p