KSHV ORF57 interacts with CBP80 and the hTREX complex but not the EJC
The hTREX complex contains several nuclear export proteins. Given that KSHV ORF57's primary role is attributed to the nuclear export of intronless viral mRNA, we first assessed if ORF57 interacted with hTREX components using co-immunoprecipitation assays. Moreover, as hTREX forms a complex with the 5′-cap protein CBP80 
, we were interested if ORF57 also interacted with CBP80. 293T cells were transfected with pGFP or pORF57GFP and untreated or RNase treated total cell lysate was used in co-immunoprecipitation experiments with CBP80-, Aly-, UAP56-, fSAP79- and hHpr1- specific antibodies in addition to an unrelated antibody control (a p53-specific antibody). Each of the hTREX proteins and CBP80 co-precipitated with ORF57, in an RNA-independent manner (). Moreover, indirect immunofluorescence showed that a proportion of ORF57GFP co-localised with hTREX proteins (Fig. S1
KSHV ORF57 interacts with hTREX.
To assess whether ORF57 also interacts with the EJC, co-immunoprecipitation assays were repeated using an antibody specific for eIF4A3, a core EJC component 
and a hHpr1-specific antibody, serving as a positive control. No interaction was observed with the EJC core component, eIF4A3, in contrast, ORF57 was readily detectable in the hHpr1 immunoprecipitation (). A control immunoprecipitation was performed to confirm that the eIF4A3 antibody precipitated EJC components (Y14) in this assay (data not shown).
In order to address potential overexpression artefacts and to assess whether ORF57 interacts with hTREX core components during lytic replication, KSHV-latently infected BCBL-1 cells were reactivated using the phorbol-ester, TPA, and lytic gene expression confirmed by detection of the ORF57 protein in TPA-treated cells by western blot analysis (). Reactivated BCBL-1 cell lysate remained untreated or was treated with RNase and co-immunoprecipitations performed using an ORF57-specific antibody. Western blot analysis using CBP80- and hHpr1- specific antibodies revealed that ORF57 interacts with CBP80 and hHpr1 during lytic replication, however ORF57 did not precipitate with either eIF4A3 (the EJC core component) or the cellular intronless mRNA-export protein, SRp20 (). Moreover, to confirm that ORF57 failed to interact with additional components of the EJC, co-immunoprecipitations were repeated using reactivated BCBL-1 cell lysates and Y14- and Magoh-specific antibodies. Results demonstrate that ORF57 did not precipitate with these additional EJC components (). A control immunoprecipitation was also performed to confirm that the Y14- and Magoh-specific antibodies precipitated eIF4A3 in this assay (Fig. S2
). Therefore, these data provide the first direct evidence of a viral protein associating with CBP80 and all the core components of the hTREX complex.
ORF57 loads hTREX, but not the EJC, onto intronless viral mRNA transcripts
One possible explanation for how herpesvirus intronless mRNAs undergo nuclear export is that ORF57 mimics splicing by loading key mRNA export proteins, such as hTREX, onto the intronless viral mRNA. In order to test if intronless KSHV transcripts were associated with hTREX proteins and if ORF57 was necessary for this interaction, RNA-immunoprecipitation (RNA-IP) assays were performed. We chose to perform this assay using 2 intronless KSHV mRNAs, specifically ORF47 and gB. RT-PCR and sequence analysis confirmed that both of these ORFs do not contain introns (data not shown). To perform the RNA-IPs, a vector expressing KSHV ORF47 (a late structural intronless gene) was transfected into 293T cells either alone or in the presence of pORF57GFP. Total cell lysates were then used in immunoprecipitations performed with either CBP80-, Aly-, UAP56- or hHpr1-specific antibodies. RNA-IPs performed on cell extracts transfected with ORF47 alone failed to show an interaction between Aly, UAP56 or hHpr1 and the viral ORF47 mRNA (). In contrast, extracts from cells transfected with both pORF47 and pORF57GFP displayed a clear interaction between Aly, UAP56 and hHpr1 and the intronless viral ORF47 mRNA (). CBP80 was found to bind to the intronless ORF47 viral mRNA independently of ORF57 (). Moreover, this analysis was repeated with a second intronless KSHV mRNA, namely the late structural glycoprotein gB, and similar results were observed (). These data show that ORF57 is required for the recruitment of core components of hTREX onto intronless viral mRNA.
ORF57 recruits hTREX to intronless viral mRNA but does not recruit the EJC.
To determine whether EJC components are recruited to intronless viral transcripts prior to export, RNA-IP assays were also performed using eIF4A3-, Y14- and Magoh-specific antibodies. Results failed to show any interaction between the EJC core components and viral intronless ORF47 and gB mRNAs in the absence or presence of ORF57 (). These results show that the EJC is not recruited to intronless viral transcripts by ORF57 and suggests that the EJC is not required for KSHV intronless viral mRNA nuclear export.
To determine whether the hTREX and EJC components were recruited to a spliced viral transcript, RNA-IPs were also performed using a vector expressing the genomic (intron-containing) KSHV ORF50 gene. 293T cells were transfected with pORF50 in the absence or presence of ORF57. Total cell lysates were then used in immunoprecipitations performed with either CBP80-, Aly-, UAP56-, hHpr1-, eIF4A3-, Y14- or Magoh-specific antibodies. Results demonstrated that CBP80, hTREX and EJC components were recruited to the spliced ORF50 mRNA in an ORF57 independent manner (). This suggests that splicing of a viral transcript is sufficient to recruit the cellular proteins necessary for nuclear export. In contrast, ORF57 is required for the recruitment of the hTREX proteins to an intronless viral transcript.
UAP56 functions as a bridge between Aly and the hTHO-complex to facilitate assembly of hTREX
Currently, while it is known that hTREX recruitment to a mammalian mRNA is both 5′-cap- and splicing-dependent, the protein-protein interactions that govern assembly of the hTREX complex itself are not fully understood. As ORF57 functions to recruit hTREX onto the intronless viral mRNA in a splicing independent manner we assessed whether this viral-system could be used to investigate hTREX assembly in more detail. To this end, we sought to determine if any hTREX proteins directly interacted with ORF57. Radio-labelled ORF57 was generated by in vitro
coupled transcription/translation (ITT), RNase treated, and used in GST pull-down experiments using constructs expressing GST-, GST-Aly, GST-UAP56 and GST-hHpr1 fusion proteins. Equal amounts of each expressed protein were used in each pulldown experiment (). Analysis showed that ORF57 bound directly to GST-Aly but not to any other hTREX component (). Due to the instability of GST-CBP80, a reverse pulldown experiment was performed using GST-ORF57 () and radio-labelled ITT CBP80, a GST-Aly pulldown with ITT CBP80 served as a positive control 
. Results also revealed a direct interaction between CBP80 and KSHV ORF57 ().
ORF57-hTREX complex formation requires both Aly and UAP56.
These data suggest that ORF57 only interacts directly with Aly and CBP80, therefore the question remains how the complete hTREX complex associates with ORF57. It has previously been suggested that the hTREX complex is formed by UAP56 bridging the interaction between Aly and the hTHO-complex 
. Therefore, to further investigate ORF57-hTREX assembly, we assessed which hTREX components were required to reconstitute the ORF57-hHpr1 interaction. GST pulldown experiments were performed using GST-hHpr1 and ITT ORF57 alone or combinations with ITT Aly or recombinant UAP56. When the GST-hHpr1 ITT ORF57 pulldown was repeated in the presence of both ITT Aly and purified UAP56, analysis revealed a clear interaction between hHpr1 and ORF57 (), suggesting that ORF57 requires both Aly and UAP56 to recruit the hTHO-complex, thus facilitating formation of the ORF57-hTREX complex. These findings provide the first direct evidence that UAP56 functions as a bridge between Aly and the hTHO-complex component hHpr1 to facilitate assembly of hTREX. However, at present we cannot exclude the possibility that ORF57 interacts directly with other hTHO-complex components.
hTREX recruitment to intronless viral mRNA is essential for their nuclear export
To assess whether hTREX is essential for viral mRNA nuclear export we produced an ORF57 mutant protein which was unable to interact with Aly and as such would be predicted to prevent the recruitment of the complete hTREX complex onto intronless viral mRNA. A minimal region responsible for Aly-binding has been identified in ORF57 and spans 35aa between residues 181 and 215 
. Upon closer examination of this sequence, we identified a PxxP-polyproline motif. To assess whether this motif was important for Aly-binding, both proline residues were substituted with alanine residues by site-directed mutagenesis to generate pORF57PmutGFP. To determine if mutating the PxxP-motif in ORF57 led to a loss of Aly binding, GST-Aly pulldown assays were performed using ITT ORF57 or ITT ORF57Pmut. Results demonstrated that the mutant ORF57 protein was unable to interact with GST-Aly, in contrast to the wild type protein (). Moreover, similar results were observed using pull-down assays with pGFP-, pORF57GFP- or pORF57PmutGFP-transfected 293T cell lysates (). These data demonstrate that the ORF57 PxxP-motif is required for the direct interaction with Aly. To confirm that the mutagenesis of the PxxP motif had no effect on ORF57 protein stability or other reported functions, several independent experiments were performed to assess the ability of ORF57PmutGFP to localise to nuclear speckles, homodimerise, directly interact with ORF50 and bind viral intronless mRNA (Fig. S3
), all of which are features of the wild type ORF57 protein. In each case the ORF57PmutGFP phenotype was indistinguishable from that of wild type ORF57.
The ORF57 PxxP motif is required for direct interaction with Aly.
Having established that ORF57PmutGFP is unable to interact with Aly and that the mutation does not affect other ORF57 functions, we then asked if, in the absence of Aly-binding, ORF57 was still able to complex with CBP80 and hTREX components. 293T cells were transfected with pGFP, pORF57GFP or pORF57PmutGFP and total cell lysates were used in co-immunoprecipitation experiments, using CBP80-, Aly-, UAP56-, and hHpr1-specific antibodies. In each case the hTREX antibody immunoprecipitated ORF57GFP but not ORF57PmutGFP, demonstrating that in the absence of the Aly-interaction ORF57 was unable to form a complex with hTREX (). In addition, the ORF57PmutGFP exhibited a reduced but specific binding to CBP80 (). This reduced binding may be due to the mutation of the PxxP-polyproline motif either affecting CBP80 binding directly or the loss of hTREX binding affects the stability of the CBP80-ORF57 complex. To further investigate whether the mutation of the PxxP-polyproline motif affected direct binding to CBP80, GST pulldown assays were performed using GST-ORF57 and GST-ORF57PmutGFP. Equal amounts of each expressed protein was incubated with radio-labelled ITT CBP80. Results demonstrated that ORF57 and ORF57PmutGFP bound to CBP80 with similar affinity (Fig. S4
). This suggests that the reduced binding observed between ORF57PmutGFP and CBP80 may be due to the loss of hTREX, which is possibly required to stabilise the export competent vRNP.
To determine if ORF57PmutGFP was unable to recruit hTREX proteins to KSHV intronless mRNA transcripts in the absence of Aly binding, RNA-IP assays were performed using CBP80-, Aly-, UAP56- or hHpr1-specific antibodies. These data demonstrate that in contrast to pORF57GFP, pORF57PmutGFP is unable to recruit hTREX components to intronless viral mRNA (). This suggests that a direct interaction between Aly and ORF57 is required for hTREX recruitment onto intronless viral transcripts.
To test if a failure in ORF57-mediated recruitment of hTREX to the intronless ORF47 mRNA prevented nuclear export of intronless KSHV transcripts, two independent mRNA export assays were performed. Firstly, northern blotting was used to detect if intronless ORF47 mRNA was present in the nuclear or cytoplasmic fraction of transfected cells. Very little ORF47 mRNA was detected in the cytoplasmic RNA fraction of cells transfected with pORF47 alone (9.9±4.9%), whereas cells co-transfected with pORF47 and pORF57GFP displayed a clear shift in ORF47 mRNA from the nuclear to the cytoplasmic fraction (81.5±1.0%), indicative of ORF57-mediated viral mRNA nuclear export. However, upon co-transfection with pORF47 and pORF57PmutGFP, the majority of ORF47 mRNA was no longer found in the cytoplasmic fraction (21.3±3.8%), instead it was retained in the nuclear pool at similar levels to those seen for the negative control, symptomatic of a failure in ORF57-mediated viral mRNA nuclear export (). To confirm that the ORF57 mutant did not affect mRNA stability, total RNA levels were assessed by northern blot analysis. No significant difference in ORF47 mRNA levels was observed between cells expressing wild type or mutant ORF57 proteins (). However, a slight decrease in total mRNA levels is seen in the presence of both the ORF57 or ORF57PmutGFP compared to the GFP control. At present, the reason for this is unknown, however, it could be due to the overexpression of the ORF57 protein.
hTREX recruitment to intronless viral mRNA is required for efficient nuclear export.
To confirm the above result, a fluorescent in situ hybridisation assay was utilised. 293T cells were transfected with pORF47, in addition to either pGFP, pORF57GFP or pORF57PmutGFP. 24 h post-transfection cells were fixed, permeabilised and incubated with a biotin-labelled oligonucleotide specific for the KSHV ORF47 mRNA. After a 4 hr hybridisation cells were washed and ORF47 mRNA subcellular localisation was visualised using Cy5-streptavidin. Cells transfected with pORF47 and GFP retained the ORF47 mRNA in the nucleus, whereas ORF47 mRNA was clearly visualised in the cytoplasm of cells transfected with pORF47 and pORF57GFP. However, upon transfection with pORF57PmutGFP, ORF47 mRNA was only observed in the nucleus, symptomatic of a failure in ORF57-mediated viral mRNA nuclear export (). Together, these two independent assays demonstrate that the ORF57-dependent recruitment of hTREX to intronless viral transcripts is essential for their efficient nuclear export.
We were also interested to determine whether the recruitment of the complete hTREX complex is required for virus replication and infectious virion production. To this end, we utilised a 293T cell line harbouring a recombinant KSHV BAC36-GFP genome 
. This KSHV-latently infected cell line can be reactivated releasing infectious virus particles in the supernatant which can subsequently be harvested and used to infect 293T cells 
. The 293T-BAC36 cell line was transfected with pGFP, pORF57GFP or pORF57PmutGFP and concurrently reactivated using TPA and incubated for 72 hours. The supernatants from each flask were then harvested and used to re-infect 293T cells and GFP positive cells were scored 48 h post-infection, as described above. Results revealed similar levels of lytic replication and virus production from cells expressing pGFP or pORF57GFP. However, virus production was significant reduced (P
0.018) upon the expression of the ORF57PmutGFP (Fig. S5
). Therefore, these results demonstrate that the ORF57-dependent recruitment of the complete hTREX complex to intronless viral transcripts is essential for efficient virus lytic replication and infectious virion production.
ORF57-mediated recruitment of Aly and TAP to intronless viral mRNA is not sufficient for efficient nuclear export and virus replication
The above data show that ORF57 binds viral intronless mRNA and directly interacts with Aly. Given that Aly is able to recruit the export factor TAP directly, it was of interest to determine if UAP56 and the hTHO-complex are required for viral mRNA export. In contrast to the cellular mRNA model, a major advantage of our viral system is that hTREX assembly on the viral mRNA is dependent upon an interaction with a virus-encoded protein, not splicing. Specifically, ORF57 binds viral mRNA, directly interacts with and recruits Aly which in turn then interacts with and uses UAP56 to bridge an interaction with the hTHO-complex. This ordered recruitment allows us to specifically disrupt the viral mRNA-ORF57-hTREX complex at different points and assess the functional significance on nuclear export. Furthermore, rather than using an artificial in vitro assay to investigate the functional significance of hTREX, we assessed this in the context of the virus replication cycle using the 293T-BAC36 assay described above.
The trans-dominant mutant, pAlyΔC-myc, which has 20 residues deleted from the carboxy-terminus of Aly, is unable to interact with UAP56 
. We were interested in establishing if this mutant could be used to disrupt the assembly of UAP56 and hTHO-complex on an intronless viral mRNA and as such provide insights into whether these proteins are essential for nuclear export. However, prior to its use in the replication assay it was essential to confirm that AlyΔC-myc is still recruited by ORF57 to intronless viral mRNA and is able to interact with TAP. To this end, ORF57, UAP56 and TAP were expressed as GST fusion proteins and incubated with either pmyc, pAly-myc or pAlyΔC-myc transfected cell lysates and pulldown analysis performed. Western blotting using a myc-specific antibody demonstrated that Aly-myc interacted with ORF57, TAP and UAP56. In contrast, AlyΔC-myc is unable to associate with UAP56 but retains the ability to interact with both ORF57 and TAP (). These results suggest that AlyΔC-myc is an ideal mutant to inhibit the recruitment of UAP56 and hTHO-complex on the viral intronless mRNA. However, one caveat to this system is that expression of pAlyΔC-myc may also act in a dominant negative capacity to inhibit spliced mRNA nuclear export 
. Therefore it was important to allow expression of the spliced ORF57 protein prior to accumulation of pAlyΔC-myc. To this end, transient transfection of pAlyΔC-myc was performed concurrent with reactivation of the KSHV lytic replication cycle, and ORF57 protein levels assessed 24 h later. Results show that comparable amounts of ORF57 were expressed in untransfected, pmyc, pAly-myc and pAlyΔC-myc transfected cell lysates ().
ORF57-mediated recruitment of Aly and TAP to intronless viral mRNA is not sufficient for efficient nuclear export and virus replication.
To test if AlyΔC-myc inhibited the recruitment of UAP56 and the hTHO-complex onto KSHV intronless RNAs, RNA-IPs were performed on reactivated KSHV-infected 293T cells transfected with pmyc, pAly-myc or pAlyΔC-myc. We obtained similar results for pmyc and pAly-myc to those shown in , where recruitment of hTREX components onto the viral RNA was readily detected 48 h-post reactivation. However, RNA-IPs using cell extracts transfected with AlyΔC-myc showed a dramatic decrease in the recruitment of UAP56 and hHpr1 to viral mRNA (). RNA-IPs performed using a TAP-specific antibody showed that TAP is recruited to the intronless viral mRNA, irrespective of Aly status. Critically, RNA-IPs using an ORF57-specific antibody produced ORF47 RT-PCR products of a similar intensity, suggesting that ORF57 was not limiting in this assay (). It should also be noted that we observed a decrease in TAP recruitment to the viral mRNA in the presence of both pAly-myc and pAlyΔC-myc, compared to pmyc control. At present, we are unsure why TAP recruitment is reduced, however, no difference in mRNA nuclear export is observed between pmyc and pAly-myc transfected cells, suggesting that this reduction in TAP recruitment does not impede the nuclear export of intronless viral mRNAs.
To assess if the AlyΔC-myc mutant affected intronless viral mRNA export during replication, northern blot analysis was performed as described above. Results demonstrated that ORF47 mRNA nuclear export is impaired in reactivated cells that expressed AlyΔC-myc, but not in cells expressing myc or Aly-myc (). Moreover, to determine if expression of AlyΔC-myc had any effect on virus replication, the KSHV-latently infected 293T BAC36-GFP cell line was transfected with pmyc, pAly-myc or pAlyΔC-myc and concurrently reactivated using TPA and incubated for 72 hours. The supernatants from each flask were then harvested and used to re-infect 293T cells. The level of virus replication was determined by scoring the percentage of GFP positive cells 48 h post-infection, as previously described 
. Similar levels of lytic replication and virus production were observed from pmyc and pAly-myc pre-transfected cells. Strikingly, virus production from pAlyΔC-myc pre-transfected cells was reduced by approximately 10 fold (). These data demonstrate that ORF57-mediated recruitment of Aly and TAP to an intronless viral mRNA is insufficient for its nuclear export and that a lack of UAP56 and hTHO-complex on an intronless viral mRNA has a profound effect on intronless nuclear export and KSHV lytic replication.