A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins. Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts. Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA. Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3. We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores. Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs. Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.

Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We report here that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs.

Short interfering RNAs (siRNAs) have been used to inhibit HIV-1 replication. The durable inhibition of HIV-1 replication by RNA interference has been impeded, however, by a high mutation rate when viral sequences are targeted and by cytotoxicity when cellular genes are knocked down. To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. We used this library to select a comprehensive population of Jurkat T-cell clones, each expressing a single discrete shRNA. The Jurkat clones were then infected with HIV-1. Clones that survived viral infection represent moieties silenced for a human mRNA needed for virus replication, but whose chronic knockdown did not cause cytotoxicity. Overall, 252 individual Jurkat mRNAs were identified. Twenty-two of these mRNAs were secondarily verified for their contributions to HIV-1 replication. Five mRNAs, NRF1, STXBP2, NCOA3, PRDM2, and EXOSC5, were studied for their effect on steps of the HIV-1 life cycle. We discuss the similarities and differences between our shRNA findings for HIV-1 using a spreading infection assay in human Jurkat T-cells and results from other investigators who used siRNAbased screenings in HeLa or 293T cells.

Nuclear factor kappa B (NF‐κB) is a key mediator of inflammation. Unchecked NF‐κB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1‐binding protein 1 (TAX1BP1) is a negative regulator of TNF‐α‐ and IL‐1β‐induced NF‐κB activation and that binding to mono‐ and polyubiquitin by a ubiquitin‐binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF‐κB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age‐dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF‐α and IL‐1β. TAX1BP1−/− cells are more highly activated for NF‐κB than control cells when stimulated with TNF‐α or IL‐1β. Mechanistically, TAX1BP1 acts in NF‐κB signalling as an essential adaptor between A20 and its targets.

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ∼24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ∼50%. We hypothesize that the interaction between RUNX1 and CBFβ is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFβ interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFβ directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFβ interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.T ranscription factors RUNX1 and CBFβ form a heterodimer for DNA binding and regulation of gene expression. Genes encoding both proteins play key roles in hematopoiesis (1) and are involved in leukemogenesis through recurrent chromosome abnormalities (2), such as a chromosome 16 inversion [(inv)16] that generates a fusion gene between CBFB and MYH11 (encoding the smooth muscle myosin heavy chain, SMMHC) in acute myeloid leukemia (AML) subtype M4Eo (3, 4), a translocation between chromosomes 8 and 21 that generates a fusion gene between RUNX1 and ETO in AML subtype M2 (5), and a translocation between chromosomes 12 and 21 that generates a fusion gene called TEL-RUNX1 in pediatric precursor B-cell acute lymphocytic leukemia (ALL) (6). All together, the CBF leukemias, which contain translocations involving RUNX1 or CBFB, account for 24% of adult AML cases (7) and 25% of pediatric ALL cases (8). Although core binding factor (CBF) leukemias are generally associated with relatively favorable prognoses, long-term survival for adult patients with CBF AML is only about 50% (9). Although children with CBF leukemias have survival rates of >80% (8, 10), standard therapy takes years to complete. Moreover, the current standard of care for all patients is frequently associated with significant morbidity and mortality. Therefore, targeted treatments for CBF leukemia with high efficacy and low toxicity are clearly desirable.Previous studies suggest that the physical interactions between RUNX1 fusion proteins (RUNX1-ETO and TEL-RUNX1) and CBFβ, and between the CBFβ fusion protein (CBFβ-SMMHC) and RUNX1 are critical for the pathogenesis of CBF leukemias (11-13). We therefore hypothesize that inhibitors of the RUNX1-CBFβ interaction will be therapeutic for all...

5′-mRNA capping is an early modification that affects pre-mRNA synthesis/splicing, RNA cytoplasmic transport, and mRNA translation and turnover. In eukaryotes, a 7-methylguanosine (m7G) cap is added to newly transcribed RNA polymerase II (RNAP II) transcripts. A subset of RNAP II-transcribed cellular RNAs, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and telomerase RNA, is further hypermethylated at the exocyclic N2 of the guanosine to create a trimethylguanosine (TMG)-capped RNA. Some of these TMG-capped RNAs are transported within the nucleus and from the nucleus to the cytoplasm by the CRM-1 (required for chromosome region maintenance) protein. CRM-1 is also used to export Rev/RRE-dependent unspliced/ partially spliced HIV-1 RNAs. Here we report that like snRNAs and snoRNAs, some Rev/RRE-dependent HIV-1 RNAs are TMG-capped. The methyltransferase responsible for TMG modification of HIV-1 RNAs is the human PIMT (peroxisome proliferator-activated receptor-interacting protein with methyltransferase) protein. TMG capping of unspliced/partially spliced HIV-1 RNAs represents a new regulatory mechanism for selective expression.required for chromosome region maintenance | RNA export | peroxisome proliferator-activated receptor-interacting protein with methyltransferase P osttranscriptional processing of RNA plays a critical role in gene expression (1-3). An early mRNA modification is the formation of a 7-methylguanosine (m7G) cap (4-6). In mammals, the acquisition of an m7G cap requires two enzymes (7-9), a capping enzyme (triphosphatase and guanylyltransferase activity) and an RNA-(guanine 7)-methyltransferase. These proteins are essential for cell growth, and mutations in the triphosphatase, guanylyltransferase, or methyltransferase components of the capping apparatus are lethal in vivo (10).Cellular RNAP II transcripts are mostly m7G-capped (1-3, 11). An m7G cap facilitates the initiation of translation in mammalian cells, and a failure to cap premRNAs results in their accelerated decay by 5′ exoribonuclease degradation (2,(12)(13)(14). Capping of viral RNAs has been less extensively investigated. It has been generally assumed that like cellular RNAs, many viral RNAs have a 5′ m7G cap, and that viruses either use the cellular capping machinery (e.g., viruses that replicate in the nucleus) or encode their own capping enzymes (e.g., viruses that replicate in the cytoplasm) (15, 16). Interestingly, there is a surprisingly diverse range of cap modifications; for instance, Mimivirus, a large DNA virus of amoeba, encodes an RNA cap guanine-N2 methyltransferase that hypermethylates the viral RNA cap (17); influenza virus "snatches" caps from cellular mRNAs (18, 19); West Nile fever virus has two methyl additions on its RNA cap (20); Sindbis virus produces mRNAs that have dimethylguanosine and trimethylguanosine caps [hypermethylated caps/m 2,2,7 G caps (21)]; and Semliki forest virus late mRNAs also have hypermethylated caps (22). On the other hand, poliovirus, encephalomyelitis virus, foot and mouth...

A series of ring expanded nucleoside (REN) analogues were synthesized and screened for inhibition of cellular RNA helicase activity and human immunodeficiency virus type 1 (HIV-1) replication. We identified two compounds 1 and 2 that inhibited the ATP dependent activity of human RNA helicase DDX3. Compounds 1 and 2 also suppressed HIV-1 replication in T cells and monocytederived macrophages. Neither compound at therapeutic doses was significantly toxic in ex vivo cell culture or in vivo in mice. Our findings provide proof-of-concept that a cellular factor, an RNA helicase, could be targeted for inhibiting HIV-1 replication.

Human T-cell leukemia virus type 1 (HTLV-1) encodes a 40-kDa Tax phosphoprotein. Tax is a transcriptional activator which modulates expression of the viral long terminal repeat and transcription of many cellular genes. Because Tax is a critical HTLV-1 factor which mediates viral transformation of T cells during the genesis of adult T-cell leukemia, it is important to understand the processes which can activate or inactivate Tax function. Here, we report that ubiquitination of Tax is a posttranscriptional mechanism which regulates Tax function. We show that ubiquitination does not target Tax for degradation by the proteasome. Rather, ubiquitin addition modifies Tax in a proteasome-independent manner from an active to a less-active transcriptional form.