Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication

Abstract

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.

Figure 1. KSHV ORF57 interacts with hTREX.

(A) Cells were transfected with pGFP or pORF57GFP and immunoprecipitations performed with the indicated antibody and analysed by western blot using a GFP-specific antibody. Total cell lysate from transfected cells served as positive controls (input). (B) Cells were transfected with pGFP or pORF57GFP and immunoprecipitations performed using hHpr1- and eIF4A3-specific antibodies. Western blot analysis was performed using a GFP-specific antibody. Total cell lysate from transfected cells served as a positive control (input). (C) BCBL-1 cells remained latent or reactivated using TPA, reactivation was confirmed by detection of the ORF57 protein (i). Immunoprecipitations using an ORF57-specific antibody were performed on reactivated BCBL-1 cell lysates that were either untreated or treated with RNase. Western blot analysis was then performed using hHpr1-, CBP80-, eIF4A3 and SRp20-specific antibodies, total reactivated cell lysate served as a positive control (input) (ii). Reactivated BCBL-1 cell lysate was used in immunoprecipitations with CBP80-, Aly-, UAP56-, hHpr1-, SRp20-, eIF4A3-, Y14 and Magoh-specific antibodies. Western blot analysis was performed using an ORF57-specific antibody (iii).

Figure 2. ORF57 recruits hTREX to intronless viral mRNA but does not recruit the EJC.

(A) Cells were transfected with pORF47 in the absence or presence of pGFP or pORF57GFP. Following UV crosslinking, RNA-IPs were performed using CBP80-, Aly-, UAP56- and hHpr1-specific antibodies. Total RNA extracted from mock and ORF47 transfected cells served as controls (input). (B) RNA-IPs were repeated using specific antibodies to the EJC components, eIF4A3, Y14 and Magoh, a hHpr1-specific antibody served as a positive control. (C) RNA-IPs were repeated using a second, intronless mRNA reporter, gB. RNA-IPs were performed using the labelled antibodies. Total RNA extracted transfected cells served as controls (input). (D) RNA-IPs were carried out on cells transfected with pORF50 in the absence or presence of ORF57. RNA-IPs were performed using the labelled antibodies. Amplification of the pORF50 vector using ORF50 RT oligos which span the ORF50 intron act as a positive control for PCR.

Figure 3. ORF57-hTREX complex formation requires both Aly and UAP56.

(A) Equal amounts of recombinant GST, GST-Aly, GST-UAP56, GST-hHpr1 bound to beads were separated by SDS-PAGE and proteins visualised by coomassie staining. (B) Bound recombinant GST-fusion proteins were incubated with ³⁵S-Met-labeled ORF57 produced by ITT. Following washes, bound proteins were separated by SDS-PAGE and the dried gel was exposed to autoradiograph film for 16 hrs. (C) Recombinant GST, GST-Aly, GST-ORF57 were bound to glutathione-agarose beads. Following washes, 10% of the beads used in the subsequent GST pull-down assays were separated by SDS-PAGE and proteins visualised by coomassie staining. (D) Bound recombinant GST-fusion proteins were incubated with ³⁵S-Met-labeled CBP80 produced by ITT. Following washes, bound proteins were separated by SDS-PAGE and the dried gel was exposed to autoradiograph film for 16 hrs. (E) Bead bound recombinant GST, GST-Aly and GST-hHpr1 fusion proteins were incubated with ³⁵S-Met-labeled ORF57 produced by ITT in the absence or presence of ³⁵S-Met-labelled Aly or purified His-tagged UAP56. Following washes, bound proteins were separated by SDS-PAGE and the dried gel was exposed to autoradiograph film for 16 hrs. Inputs for ORF57, Aly and UAP56 are shown.

Figure 4. The ORF57 PxxP motif is required for direct interaction with Aly.

(A) Recombinant GST and GST-Aly were bound to glutathione-agarose beads and incubated with ³⁵S-Met-labeled ORF57 or ORF57Pmut produced by ITT. Following washes, bound proteins were separated by SDS-PAGE and the dried gel was exposed to autoradiograph film for 16 hrs. ITT input controls for ORF57 and ORF57Pmut are shown. (B) Recombinant GST- or GST-Aly-bound glutathione-agarose beads were incubated with pGFP-, pORF57GFP or pORF57PmutGFP-transfected cell extracts. Following washes, bound proteins were analysed by western blot using a GFP-specific antibody. Total cell lysates from transfected cells served as a positive control (input). (C) Cells were transfected with pGFP, pORF57GFP or pORF57PmutGFP and immunoprecipitations performed with the indicated antibody and analysed by western blot using GFP-specific antibody. Total cell lysate from transfected cells served as positive controls (input). (D) RNA-IPs were performed using the indicated antibodies and nested RT-PCR carried out on the extracted RNA using ORF47-specific oligonucleotides. Total RNA extracted from transfected cells served as control (input).

Figure 5. hTREX recruitment to intronless viral mRNA is required for efficient nuclear export.

(A) Cells were transfected with pORF47 in the presence of pGFP, pORF57GFP or pORF57PmutGFP and incubated for 24 h. Total RNA or RNA isolated from nuclear and cytoplasmic fractions was analysed by northern blot using an ORF47-specific radio-labelled probe. A probe to the 18S subunit of ribosomal RNA was used as a loading control. Densitometry analysis of three independent northern blot experiments determined relative levels of nuclear, cytoplasmic and total RNAs and standard error calculated (n = 3). (B) 293T cells were transfected with pORF47 in the presence of pGFP, pORF57GFP or pORF57PmutGFP. Cells were then fixed in paraformaldehyde and extracted in SDS buffer. In situ hybridisation was subsequently performed using a biotinylated oligonucleotide probe specific to ORF47 mRNA and the probe detected using Cy5-streptavidin. Images shown are a representative of all transfected cells.

Figure 6. ORF57-mediated recruitment of Aly and TAP to intronless viral mRNA is not sufficient for efficient nuclear export and virus replication.

(A) Recombinant GST-, GST-UAP56, GST-TAP or GST-ORF57-bound glutathione-agarose beads were incubated with mock-, pmyc-, pAly-myc- or pAlyΔC-myc-transfected cell extracts. Following washes, bound proteins were analysed by western blot using a myc-specific antibody. Total cell lysate from mock and transfected cells served as a positive control (input). (B) Total cell lysate from mock-, pmyc-, pAly-myc- or pAlyΔC-myc-transfected and reactivated 293T BAC36 cells were isolated at the 24 h time-point and analysed by western blot using an ORF57-specific antibody. Western blot analysis of B-actin levels served as a loading control. (C) RNA-IPs were performed using the indicated antibodies. (D) 293T BAC36 cells were transfected with the indicated vectors and concurrently reactivated using TPA. Northern blot analysis was performed using an ORF47-specific radio-labelled probe. (E) Lytic virus replication was assayed by harvesting the supernatant of transfected 293T BAC36 cells. Supernatant was used to infect 293T cells and 48 h later the level of virus infection was scored by direct-immunofluorescence, n = 3000.

Figure 7. Models for hTREX assembly.

(A) hTREX is recruited to spliced mRNA in a 5′ cap- and splicing dependent manner. (B) Our model for hTREX assembly on an intronless viral mRNA. The hTREX complex is recruited via a direct interaction between ORF57 and Aly. (C) Several models for intronless mammalian mRNA export. (i) Huang et al report that certain intronless mRNAs are targeted by SR-proteins that recognise specific sequences in the mRNA then directly access the TAP/p15 export factor. (ii) The Nojima et al model suggests that Aly is recruited via an interaction with the CBC. (iii) Taniguchi et al report that Aly is loaded onto the intronless mRNA by UAP56 in an ATP-driven mechanism.