The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Retroviruses must bypass the tight coupling of splicing and nuclear export of mRNA in their replication cycle because unspliced genomic RNA and incompletely spliced mRNA must be exported to the cytoplasm for packaging or translation. This process is mediated by a cis-acting constitutive transport element (CTE) for simple retroviruses and by the trans-acting viral protein Rev in concert with its response element (RRE) for complex retroviruses (e.g., HIV). Recently, we identified RNA helicase A (RHA) as a potential cellular cofactor for CTE. Here, we report that RHA also plays a role in Rev͞RRE-mediated gene expression and HIV replication. RHA binds weakly to HIV-1 RRE independently of Rev. Overexpression of RHA, but not of an RHA mutant lacking helicase activity, increased both Rev͞RRE-and CTEdependent gene expression and the levels of unspliced HIV mRNA. Microinjection of antibodies to RHA into nuclei dramatically inhibited both CTE-and Rev-dependent gene expression in human cells. Exogenous RHA cDNA, but not the mutant RHA, rescued this inhibition. We propose that RHA is required to release both CTE-and RRE-containing mRNA from spliceosomes before completion of splicing, thus freeing them for nuclear export.HIV uses complex regulatory mechanisms to control gene expression. Such mechanisms involve the interdigitation of viral and cellular elements. Rev (regulator of virion protein expression) is a trans-acting viral protein that recognizes a cis-acting RNA element, the Rev response element (RRE), on the viral genome (see ref. 1 for review). Extensive studies to date revealed that Rev͞RRE interaction facilitates the nuclear export of unspliced or singly spliced viral mRNA (2-4). However, Rev binding to RRE alone is insufficient. An activation domain on Rev, distinct from the RRE binding domain, is essential for function, presumably through binding of cellular effector molecules (2-6). This activator domain comprises a nuclear export signal (NES), which can be replaced functionally by the NES of some known export proteins (7-9). Several cellular proteins reportedly bind specifically to the Rev NES, including a nucleoporin-like protein called Rev activation domain-binding protein (10) or human Rev interacting protein (11), and the protein eIF-5A (12). More recently, Rev-NES was found to bind to the nuclear export receptor CRM-1͞exportin-1 (13, 14), and it is likely that this interaction bridges the indirect binding of Rev-NES to Rev activation domain-binding protein͞human Rev interacting protein (15,16). Cellular proteins involved in RNA splicing͞ processing also have been found to bind directly to RRE (17) or the Rev͞RRE complex (18). These proteins repressed Rev activity when overexpressed in cells. Other complex retroviruses, such as human T-cell leukemia virus and animal lentiviruses, also encode Rev-like proteins for post-transcriptional regulation (19).In contrast to complex retroviruses, simple retroviruses, such as the Mason-Pfizer monkey virus, do not encode a Rev-like protein, even though there ...

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein.

Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkBa/NF-kB pathway in limb-girdle muscular dystrophy type 2A

No abstract