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is common to all ICP27 family members and is also shared by a large number of
proteins involved in nuclear mRNA export such as Tap and Aly/REF (Sandri-
Goldin 1998; Mears and Rice 1998; Farjot et al. 2000; Goodwin et al. 1999; Bello
et al. 1999; Lischka et al. 2001; Bear et al. 1999; Rodrigues et al. 2001). Although
it was initially proposed for ICP27 that a leucine-rich region interacting with
CRM1/exportin1 acts as a NES (Sandri-Goldin 1998), subsequent studies revealed
that a CRM1-independent export mechanism is crucial for the mRNA export activ-
ity (Koffa et al. 2001; Chen et al. 2002). This could also be confirmed for other
members of the ICP27 family, including pUL69 (Lischka et al. 2001; Hiriart et al.
2003; Williams et al. 2005). Interestingly, CRM1-independent nuclear export of
pUL69 is mediated via a novel, transferable nuclear export signal of 28 amino acids
that is located within the unique C-terminal domain of the β-herpesviral protein
(Lischka et al. 2001) (Fig. 3). Since we detected that UAP56 can also shuttle
between the nucleus and the cytoplasm (P. Lischka, M. Thomas, and T. Stamminger,
unpublished observations), we initially assumed that the nuclear export of pUL69
may be mediated via docking to UAP56. However, since mutation of the UAP56
binding motif did not abrogate the nucleocytoplasmic shuttling of pUL69, we
speculate that the nuclear export pathway accessed by the pUL69 NES still remains
to be defined.
In summary, our experiments revealed that interactions of pUL69 with both the
transcription elongation factor hSPT6 and the mRNA export factor UAP56 were
essential for the mRNA export activity. Since pUL69 binds to these cellular factors
via distinct protein domains, the interaction with hSPT6, traveling along with RNA
polymerase II, may increase the co-transcriptional loading of UAP56 onto intron-
less viral transcripts, ultimately leading to the formation of an export-competent
mRNP that associates with TAP-p15 (Fig. 4). However, several open questions
remain to be answered, in particular regarding the exact functions of pUL69 during
viral replication. It is not clear whether pUL69 affects the nuclear export of viral
RNAs in general or whether there is specificity for a distinct subgroup of tran-
scripts. Studies using an HCMV mutant virus with a deletion of the UL69 coding
region showed that the lack of pUL69 led to a substantially diminished level of
several viral late transcripts (Hayashi et al. 2000), suggesting that these RNAs may
be targeted by pUL69 for efficient nuclear export. Additionally, the role of RNA
binding by pUL69 requires further investigation. Since it was shown that a subset
of viral and cellular mRNAs is incorporated into HCMV virions by an as yet
unknown mechanism (Bresnahan and Shenk 2000; Greijer et al. 2000), it will be
interesting to investigate whether the tegument localization of pUL69 contributes
to this. This aspect also emphasizes that pUL69 is certainly a multifunctional pro-
tein during viral replication, which is also illustrated by the potential of this protein
to induce cell-cycle arrest (Hayashi et al. 2000; Lu and Shenk 1999). The construc-
tion and characterization of recombinant viruses expressing pUL69 mutants with a
loss of distinct protein functions (e.g., UAP56 binding, RNA binding, nucleocyto-
plasmic shuttling) will certainly contribute to a further definition pUL69 functions
during viral replication.
