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

The human immunodeficiency virus (HIV-1) differentially controls viral protein expression at the level of splicing as well as nuclear export of incompletely spliced viral RNA. This process, mediated by the Rev protein, interfaces with cellular components involved in post-transcriptional gene regulation. While a number of reviews have focused on the host proteins (i.e., Crm1, importin-beta and nucleoporins) that specifically regulate shuttling of Rev between the nucleus and cytoplasm, we could find no systematic review of other cellular proteins implicated in Rev function. Therefore, we will here focus on other Rev cofactors (eIF5a, hRIP, Sam68, RNA helicases, etc) and the role they play in Rev/RRE function and HIV-1 replication.

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Summary Schistosomiasis mansoni, a tropical helminthic disease, is caused by disseminated worm eggs that induce CD4+ T‐cell mediated granulomatous inflammation and fibrosis. T suppressor cell activity has been proposed as one of the mechanisms active in the down‐modulation of the murine disease during the chronic stage (16–20 weeks of the infection). In recent years a new category of the CD4+ CD25+ T regulatory (Treg) lymphocyte has been identified that maintains immune tolerance to self, and also functions in the regulation of parasite‐induced immunopathology. The Foxp3 gene which encodes the transcription factor Scurfin was found to be expressed by and required for the generation of CD4+ CD25+ T reg. At 8 weeks of the infection Foxp3 gene expression of splenocytes was similar to that of naïve mice, but increased fourfold by 16 weeks. In contrast, granulomatous livers at 8 and 16 weeks showed 10‐ and 30‐fold increases, respectively, in gene expression compared with normal liver. The percentage of granuloma CD4+ CD25+ T cells rose from 12% at 8 weeks to 88% at 16 weeks of the infection. Foxp3 expression was 3·5‐fold higher in the CD4+ CD25+ versus the CD4+ CD25– T cells in the 8 week infection granulomas. As a novel observation neuropilin‐1 membrane expression, a recently identified marker for Treg, was correlated with Foxp3 expression in the granuloma CD4+ CD25+ but not the CD25– cells. Co‐incubation with polyclonal stimulation of CD4+ CD25+ splenic cells with CD4+ CD25– cells suppressed proliferation of the latter. Retroviral transfer of the Foxp3 gene at the onset of granuloma formation enhanced fourfold Foxp3 expression in the granuloma CD4+ CD25+ T cells and strongly suppressed full granuloma development. Gene transfer also significantly enhanced transforming growth factor‐β, interferon‐γ and interleukin‐4 but not interleukin‐10 expression. It is concluded, that CD4+ CD25+, Foxp3+ Treg cells also regulate schistosome egg‐induced immunopathology.

The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1.While only fully spliced cellular mRNAs are exported from the nucleus to the cytoplasm, replication of retroviruses requires the nuclear export of partially spliced and unspliced viral RNA transcripts. These transcripts serve as templates for the synthesis of a subset of viral proteins and as genomes for progeny viral particles. The complex retroviruses, exemplified by human immunodeficiency virus type 1, employ a special, virally encoded protein to mediate this export process (for a recent review, see Ref. 1). The Rev protein of human immunodeficiency virus type 1 binds to its cognate viral RNA response element, RRE, and the export receptor CRM-1 to form an active export complex (2). In contrast, the simian type D retroviruses do not encode a Rev-like protein but rather act through a cis-acting RNA element termed the constitutive transport element (CTE) 1 (3, 4). Thus, CTE is functionally equivalent to the Rev/RRE of human immunodeficiency virus type 1. However, the export receptor for CTE is not known.We recently showed that RNA helicase A (RHA) specifically binds to functional CTE RNA (5) and is required for CTE activity (6). RHA was identified previously as a nuclear protein capable of unwinding RNA duplexes in an ATP-dependent manner (7). We first observed that RHA shuttles between the nucleus and cytoplasm despite its predominant nuclear localization at steady state (5) and subsequently mapped a bidirectional nuclear transport domain at the carboxyl terminus (CTD) of the protein (8). A second CTE-binding protein, TAP, has also been identified and implicated in CTE-mediated RNA nuclear export and gene expression (9, 10).The evidence that RHA plays a role in post-transcriptional regulation of gene expression prompted us to search for RHAinteracting proteins that might also be involved in this process. Here we report the isolation of a novel human protein, named helicase A-binding protein 95 (HAP95), found by virtue of its binding to the CTD of RHA in a yeast two-hybrid screening of a human cDNA library. HAP95 has extensive homology with AKAP95, a member of the A-kinase-anchoring protein family. However, HAP95 ...