Analysis of Intracellular Trafficking and Interactions of Cytoplasmic HIV-1 Rev Mutants in Living Cells

Abstract

The HIV-1 Rev protein is an essential nuclear regulatory viral protein. Rev mutants that are able to block wild-type (WT) Rev activity in trans have been reported and used in antiviral approaches. Not only nuclear but also cytoplasmic Rev mutants were described and suspected to be transdominant by retaining WT Rev in the cytoplasm. To investigate their potential for cytoplasmic retention, we studied the localization, trafficking, and interactions of cytoplasmic Rev mutants containing mutations in the N-terminal multifunctional domain. Using a novel dual-color autofluorescent protein-tagging system, we found that coexpression of the nucleolar blue-tagged WT Rev protein together with green-labeled cytoplasmic Rev mutants did not result in the retention of WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants to the nucleolus. A combination of mutations abolished the interaction with WT Rev, defining two domains important for Rev protein interaction. The identified domains were also essential for specific Rev responsive element (RRE) RNA binding and nuclear retention. Inactivation of the nuclear export signal shifted the steady-state distribution of the mutants from the cytoplasm to the nucleus, indicating their capability for nucleo-cytoplasmic shuttling. The cytoplasmic mutants were not transdominant compared to the nuclear mutant RevM10BL. These results emphasize that efficient oligomerization with WT Rev combined with RRE-specific RNA binding are prerequisites for effective transdominance.

Rev‐GFP transgenic lines for studies of nucleocytoplasmic transport in drosophila

“…1C; compare Zimowska and Paddy, 2002). In summary, the subnuclear distribution of Rev::GFPsg in the different cell types of Drosophila examined in this study appeared to be comparable to its localization in mammalian cultured cells (Stauber et al, 1998b). This demonstrates that Drosophila does possess, as in mammalian cells, the essential cellular machinery for Rev subcellular localization, supporting the notion that the Rev::GFPsg reporter can be used to delineate nuclear trafficking in different cell types in vivo.…”

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2

“…The steady-state localization of HIV-1 Rev is nuclear/nucleolar although the protein is constantly shuttling between the nucleus and the cytoplasm (Stauber et al, 1995). Since E1B-GFP did not colocalize with HIV-1 Rev to the nucleoli ( Figure 5a,b) the inhibitory e ect of Rev appeared not to be mediated by direct protein-protein interaction as demonstrated for trans-dominant Rev mutants (Stauber et al, 1998a). In contrast, overexpression of E1B-55K or infection with adenovirus type 5 did not a ect the tra cking of Rex-GFP or a cytoplasmic shuttling HIV-1 Rev mutant (Rev38-GFP) (Figure 5c,d and data not shown).…”

“…The position of the identi®ed NES is marked by the shaded box. Note that the NES is absent in the Ad strains type 40, 41 and 12 independent of domains in the protein potentially interfering with export by mediating nuclear/cytoplasmic retention (Stauber et al, 1998a;Stauber and Pavlakis, 1998). It is striking that in the de®ned minimal NES the absence of the leucine residue at position 83 completely abolished nuclear export (Figure 4).…”

“…Third, E1B-55K has been reported to possess RNA-binding activity (Horridge and Leppard, 1998), and fourth, nucleocytoplasmic shuttling of E1B-55K would be compatible with recent observations showing that mRNA export of activated cellular genes in the late phase of Ad infection is dependent on the expression of a functional E1B-55K protein (Huang and Flint, 1998;Yang et al, 1996). Some of these features are reminiscent of retroviral posttranscriptional regulators like the HIV-1 Rev or the HTLV-1 Rex proteins also characterized by an NLS/NES, a RNA-binding domain and e cient nucleo-cytoplasmic tra cking (Felber, 1997;Stauber et al, 1998a). In contrast to the Rev/ RRE or Rex/RxRE axis, no speci®c RNA structure has been de®ned in Ad mRNAs.…”

“…The same localization and tra cking pattern was observed in the p53 negative cell line H1299 (e,f) or the p53 and Mdm2 negative cell line X174 (g,h). The presence of p21Rex-BFP in the same cell was veri®ed using the appropriate ®lter to detect BFP and mediated by the CRM1 export pathway (Fornerod et al, 1997;Heger et al, 1999;Stauber et al, 1998a). To understand how adenoviral or retroviral shuttle proteins exploit this export pathway, we reasoned that a shuttle protein with a high a nity for common export factors should be able to a ect the tra cking of a shuttle protein with a lower a nity in trans.…”