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Interaction of the HCMV pUL84 with Importin-α Proteins
The second cytomegalovirus protein for which we detected a nonconventional inter-
action with components of nuclear transport pathways is the gene product of the open
reading frame UL84. The open reading frame UL84 of HCMV encodes a multifunc-
tional protein with nuclear localization that appears to be absolutely essential for viral
replication (Xu et al. 2002, 2004a; Lischka et al. 2003a; He et al. 1992; Yu et al.
2003) (see also the chapter by G. Pari, this volume). Initially, pUL84 was identified
as a direct binding partner of the regulatory protein IE2-p86, which is the major tran-
scription-activating protein of HCMV (Spector and Tevethia 1994). Studies concern-
ing the functional consequence of the pUL84-IE2 interaction revealed on the one
hand that this interaction downregulates the transactivation activity of IE2 on some
early promoters (Gebert et al. 1997). On the other hand, it has been reported that this
pUL84-IE2 complex is required for the activation of a bidirectional promoter located
within the origin of lytic DNA replication (ori -Lyt) (Xu et al. 2004b). Since pUL84
is the only non-core protein required for origin-dependent DNA replication in a tran-
sient replication assay (Pari and Anders 1993; Sarisky and Hayward 1996), pUL84
was proposed to act as an initiator protein for viral DNA synthesis of HCMV (Xu
et al. 2004b). Initiator proteins of some other herpesviruses were demonstrated to
exert an inherent catalytic activity that may unwind a specific region of DNA within
ori -Lyt, thus allowing the assembly of the DNA replication machinery. In line with
this, pUL84 has been shown to display UTPase activity and to exhibit homology to
the DExD/H box family of helicases (Colletti et al. 2005).
In a yeast two-hybrid screen that was performed in order to identify cellular
binding proteins of pUL84, we were able to select four members of the importin-α
protein family as strong interaction partners of this viral protein (Lischka et al.
2003a). Since importin-α proteins function as adapter molecules bridging NLS-
containing import cargo proteins to the import receptor importin-β (see Fig. 1), this
finding suggested that pUL84 may either access the nuclear import pathway via this
interaction or may even be able to modulate this pathway. By performing in vitro
import assays using digitonin-permeabilized cells together with purified importin-α
and -β proteins we were indeed able to show that pUL84 nuclear import occurs via
the well-characterized importin-α/β pathway (Lischka et al. 2003a). Intriguingly,
however, the domain of pUL84 interacting with importin-α proteins turned out to
be unconventional. While most nuclear proteins dock to importin-α via short, kary-
ophilic amino acid sequences corresponding either to a classical, basic-type NLS
(Lange et al. 2007) or to other, short NLS-like sequences (Wang et al. 1997; Wolff
et al. 2002), we determined that a long UL84 protein domain comprising 282 amino
acids was required for importin-α binding (see Fig. 5). This domain serves as a
transferable, importin-α dependent NLS, which was demonstrated by fusing this
sequence with a nonkaryophilic protein, resulting in its nuclear translocation
(Lischka et al. 2003a). Since we observed that further N- or C-terminal as well as
internal deletions abrogated the nuclear translocation as well as the dimerization/
multimerization capacity of this domain, we propose that, similar to the cellular
transcription factor STAT1 (Fagerlund et al. 2002), a complex overall structure that
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