The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

In eukaryotes, the export of mRNA from the nucleus to the cytoplasm is a complex and carefully orchestrated process. In mammalian cells, the major mRNA export receptor is termed TAP/NXF1 (1, 4, 11, 16, 20, 49), and the homologue in Saccharomyces cerevisiae is Mex67p (14, 18, 40, 42, 45). TAP/NXF1 possesses a C-terminal domain that interacts with FG-repeats present on nucleoporins, which comprise the nuclear pore complex (NPC), and these interactions move TAP/NXF1 and its bound cargo RNA through the NPC to the cytoplasm (1, 2, 11, 16, 21). TAP/NXF1 does not bind directly to most RNA transcripts; instead, it interacts with export adaptor proteins that bind RNA and interact directly with TAP/NXF1 (47, 51). Aly/REF and its yeast homologue Yra1 have been shown to bind poly(A)+ RNA and to interact directly with TAP/NXF1 and Mex67p, respectively (18, 37, 40, 42, 47). Aly/REF was originally reported to be part of a complex of proteins termed the exon junction complex, which is deposited just upstream of exon junctions during a late stage of pre-mRNA splicing to mark the mRNA for export, mRNA surveillance, and localization in the cytoplasm (9, 24-26, 35, 50). Aly/REF requires UAP56, a splicing factor, for its recruitment to mRNA (13, 30, 31). More recent studies have provided evidence that Aly/REF has a only transient association with the exon junction complex and instead forms part of the TREX complex, which binds the 5′ end of the mRNA (8, 30, 33, 34, 46) through the cap binding protein CBP80 and which is essential for the export of both spliced and intronless mRNAs (8, 33, 46). UAP56 recruits Aly/REF and forms part of the TREX complex (3). The herpesvirus family of multifunctional regulatory proteins, typified by the herpes simplex virus type 1 (HSV-1) protein ICP27, have been reported to interact with Aly/REF (3, 6, 7, 17, 32) and/or UAP56 (28, 48) and to use the TAP/NXF1 pathway for viral mRNA export (3, 6, 7, 19, 22). We have reported that ICP27 interacts directly with both Aly/REF and TAP/NXF1 (6, 7) and that ICP27 recruits Aly/REF but not TAP/NXF1 to viral replication compartments (6). Overexpression of Aly/REF stimulated export of several viral transcripts (7, 22). We have also shown that bulk poly(A)+ RNA and the majority of HSV-1 transcripts were retained in the nuclei of cells infected with viral ICP27 mutants that either cannot bind RNA or that do not interact with TAP/NXF1 (19), demonstrating that ICP27 accesses the TAP/NXF1 pathway to mediate viral RNA export. These studies did not address whether Aly/REF is also required for viral RNA export. Another complication is that both the N and C termini of ICP27 must be intact for interaction with TAP/NXF1 (6), and these regions have also been found to be required for ICP27's interaction with RNA polymerase II (10) and Hsc70 (27) so that deletion mutations in the N and C termini may have pleiotropic effects on HSV-1 replication. To directly monitor the dynamics of ICP27 localization and its interactions with Aly/REF and TAP/NXF1 in living infected cells, we constructed viral recombinants in which ICP27 was either N-terminally tagged or C-terminally tagged with yellow fluorescent protein (YFP). N-terminally tagged ICP27 was seen to be exported to the cytoplasm around 5 h after infection, and Aly/REF was recruited by N-terminally tagged ICP27 into large globular structures resembling replication compartments, as we determined previously by staining fixed cells that had been infected with wild-type (WT) HSV-1 (6, 7). In contrast, C-terminally tagged ICP27 remained nuclear, and export to the cytoplasm was seen only when TAP/NXF1 was overexpressed, suggesting that the C-terminal tag, but not the N-terminal tag, interfered with the interaction of ICP27 and TAP/NXF1. To determine the role of Aly/REF and TAP/NXF1 in RNA export during viral infection, these factors were knocked down using small interfering RNA (siRNA). These studies showed that TAP/NXF1, but not Aly/REF, is required for RNA export during HSV-1 infection.