Interaction of the HCMV pUL84 with Importina Proteins

The second cytomegalovirus protein for which we detected a nonconventional interaction 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 multifunctional 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 transcription-activating protein of HCMV (Spector and Tevethia 1994). Studies concerning 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 transient 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-a protein family as strong interaction partners of this viral protein (Lischka et al. 2003a). Since importin-a proteins function as adapter molecules bridging NLS-containing import cargo proteins to the import receptor importin-P (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-a and -P proteins we were indeed able to show that pUL84 nuclear import occurs via the well-characterized importin-a/p pathway (Lischka et al. 2003a). Intriguingly, however, the domain of pUL84 interacting with importin-a proteins turned out to be unconventional. While most nuclear proteins dock to importin-a 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-a binding (see Fig. 5). This domain serves as a transferable, importin-a 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

Fig. 5 Schematic representation of the pUL84 NLS/importin-a binding domain. The localization of the two autonomous leucine-rich nuclear export signals within the NLS domain is shown on gray background. Leucine residues with a critical function for the nuclear export activity are underlined

may depend on protein dimerization generates the functional pUL84 NLS (Lischka et al. 2003a).

Interestingly, sequence inspection of the UL84-importin-a interaction domain revealed the presence of two small leucine-rich regions that exactly match the consensus sequence of a classical nuclear export signal, suggesting that the pUL84 NLS domain may also be able to mediate nuclear export, thus serving as a complex bidirectional transport domain (Fig. 5). Further experimentation revealed that both leucine-rich regions are able to function as autonomous nuclear export signals and are required for CRM-1 dependent nucleocytoplasmic shuttling of pUL84 (Lischka et al. 2006a). This suggests that pUL84, in addition to its role within the nucleus as an initiator protein of origin-dependent viral DNA replication, may carry out an unexpected function within the cytoplasm that has yet to be defined. However, given the recent description of a sequence-specific RNA-binding activity of pUL84 (Colletti et al. 2007) as well as its homology to DExD/H box RNA helicases (Colletti et al. 2005), it is tempting to speculate that pUL84, similar to the UL69 protein, may be able to enhance the accumulation of specific viral transcripts within the cytoplasm of infected cells.

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