Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3 splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3 splice site together act to inhibit splicing at the 3 splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3 splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3 splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus whose RNA splicing is dependent on the host cell splicing machinery (12, 50). HIV-1 pre-mRNA contains five 5Ј splice sites and nine 3Ј splice sites which are used to generate more than 30 different mRNA species by alternative splicing. In addition, a pool of unspliced RNA must be maintained to serve as genomic RNA and as mRNA for the gag and pol gene products (18,21,32,34,35,38). To achieve the balance between spliced and unspliced RNAs, splicing of HIV-1 RNA is inefficient, a feature which is shared with all other retroviruses (4,7,11,20,22,(45)(46)(47)52). In contrast to the major HIV-1 5Ј splice sites, which are strong, the 3Ј splice sites are weak (33). These are characterized by the presence of nonconsensus polypyrimidine tracts and branch point sites other than the eukaryotic consensus adenosine residue (2,13,17,33,43). In addition, exon sequences downstream of the 3Ј splice sites enhance or inhibit splicing at the 3Ј splice sites (2,3,39,42,51). Furthermore, the virus-encoded protein Rev plays an essential role in facilitating the transport of unspliced and singly spliced mRNAs, thus preventing the viral RNA from splicing to completion (19, 37; for a review, see reference 12).HIV-1 tat mRNA is formed by the splicing of two coding exons (exon 2 and exon 3) and one or more upstream noncoding exons. Exon 3 is also joined to rev exon 2 and is therefore referred to as tat/rev exon 3 (34). The 3Ј splice sites flanking exon 2 and exon 3 (3Ј splice sites 3 and 7, respectively [ Fig. 1]) contain suboptimal polypyrimidine tracts, and the 3Ј splice site flanking exon 3 has been reported to contain a nonconsensus bra...

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

Early events in tumor development are spontaneous, microscopic, and affected by the microenvironment. We developed a mouse model of spontaneous lymphoma in which malignant transformation is coupled with light emission that can be detected noninvasively using bioluminescent imaging. The human T-cell leukemia virus (HTLV) type 1 transcriptional transactivator Tax is an oncogene sufficient to produce lymphoma in transgenic animal models. Using the granzyme B promoter to restrict Tax expression to the mature natural killer (NK)/T-cell compartment, we have reproduced many elements of HTLV-associated adult T-cell leukemia/lymphoma. Tax activates signaling cascades associated with transformation, inflammation, and tumorigenesis. Here, we report that Tax-mediated activation of luciferase in long terminal repeat-luciferase (LTR-LUC) mice serves as a reporter for imaging these processes in vivo. Using bioluminescent imaging (BLI), we discovered that microscopic intraepithelial lesions precede the onset of peripheral subcutaneous tumors, tumorigenesis progresses through early reversible stages, and Tax is sufficient for inducing tumors. Based on these findings, we propose that Tax expression in activated lymphocytes initiates a cascade of events that leads to NK/T cell recruitment, activation, and transformation. The use of BLI expands our ability to interrogate the role of Tax in tumorigenesis in vivo and has made the association of inflammation with tumor initiation amenable for study.