Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1.

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

We report that the third intron of the Li ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human Li ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the Li ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components.

T he expression of HIV type 1 (HIV-1) is controlled by a posttranscriptional mechanism. From a single primary transcript several mRNAs are generated. These RNAs can be divided into three main classes: unspliced 9-kb, singly spliced 4-kb, and the multiply spliced 2-kb RNAs. Each of these RNAs is exported to the cytoplasm for translation and, in the case of the 9-kb RNA, for packaging into virions (1). Normally, pre-mRNAs must undergo a splicing process to remove one or more introns before being exported to the cytoplasm. HIV-1 overcomes this limitation, allowing singly spliced and unspliced RNA to be exported via interaction with its own encoded Rev protein. This regulatory protein binds an RNA stem-loop structure termed the Rev response element located within the env coding region of singly spliced and unspliced HIV RNAs (2-5). Binding of Rev to this element promotes the export, stability, and translation of these HIV-1 RNAs (6-15). The export process is mediated by the nuclear export signal of Rev, which binds the receptor exportin 1͞CRM1. It is believed that CRM1 bridges the interaction of Rev with the nucleoporins required for export to the cytoplasm (16).When Rev and Tat are expressed independently of other HIV transcripts, these proteins localize within the nucleolus of human cells (17)(18)(19)(20)(21)(22). The simultaneous presence of a nuclear export signal as well as a nuclear import͞localization signal confers upon Rev the ability to shuttle between the nucleus and the cytoplasm (16). It has recently been reported that in HeLa cells, the expression of Rev induces the relocalization of the nucleoporins Nup98 and Nup214, along with a significant fraction of CRM1, into the nucleolus (23). This result has led to the hypothesis that formation of the Rev-CRM1-nucleoporin complex targeted to the nuclear pore complex occurs in the nucleolus. It can be similarly hypothesized that HIV RNAs are also relocalized to the nucleolus before cytoplasmic export. Previous studies, which used in situ hybridization assays to define the subcellular localization of HIV RNAs, failed to detect these RNAs in the nucleoli (24)(25)(26)(27). This failure to detect these RNAs is most likely due to the dynamic process of RNA transport, making it difficult to identify discrete nucleolar localization. Therefore we have investigated the same problem, using an alternative strategy based on the use of nucleolar localized ribozymes.

The Tat protein is a key regulator of HIV-1 replication (1). Its major function is to transactivate RNA polymerase (pol) II transcription from the viral LTR promoter. The binding of Tat to a transactivation response (TAR) element in the 5Ј UTR of the viral RNAs stimulates the processivity of RNA pol II, greatly increasing HIV-1 RNA transcription (2-6). In the absence of Tat the transcription complex is able to initiate transcription from the LTR, but elongates very inefficiently (3). The transactivation activity of Tat is mediated by the interaction with a positive transcription elongation factor, P-TEFb (7-9), composed of CDK9 and cyclin T1 (10-16). The binding of Tat with cyclin T1 increases its affinity for the TAR element and induces the cooperative binding of the P-TEFb complex to TAR (13). CDK 9 can phosphorylate the carboxyl-terminal domain of the largest subunit of RNA pol II, stimulating its processivity (10,16,17). Because Tat affects one of the earliest stages of HIV-1 gene expression and may be involved in other critical steps in virus replication (reverse transcription for example, ref. 18), it has been considered a good candidate for therapeutic intervention against HIV-1. Among the different gene therapy strategies that have been tested to block Tat activity in human cells one of the most successful is the use of small RNA molecules that work as ''decoys.'' These RNAs mimic the specific RNA binding element for Tat, subsequently leading to its titration. TAR RNA or in vitro-evolved Tat binding aptamers have been previously used as decoys for Tat and have resulted in inhibitory effects on HIV-1 replication (19-23).In addition to its known localization in the nucleoplasm, Tat has been shown to have nucleolar localization properties (24-28). The functional role of Tat nucleolar trafficking is unclear, but it may associate with cyclin T1 in this compartment (29). We have previously used strategies for localizing an anti-HIV ribozyme as well as a Rev binding element in the nucleolus and demonstrated that both of these strategies inhibit HIV-1 replication (30, 31). To test whether Tat nucleolar localization is functionally important, we have used a similar strategy to direct a TAR element into this compartment. We demonstrate here that human T-lymphoblastoid CEM cells stably expressing a nucleolar-localized TAR element are highly resistant to HIV-1 infection. Using in situ hybridization analyses we also show that a Tat-enhanced GFP (EGFP) fusion protein colocalizes with the nucleolar-localized TAR element. The present observations taken together with the nucleolar localization properties of HIV-1 Rev (32-34), as well as some HIV RNAs (30,35,36), represent an additional paradigm for the role of the nucleolus in HIV-1 replication. The potent inhibition of HIV-1 replication mediated by the nucleolar-localized TAR decoy also suggests a strategy for genetic therapy of HIV-1 infection. Materials and MethodsPlasmid Constructs. The U16TAR DNA was prepared synthetically by PCR (37) using the primers A, B, C, D, E...

Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc-a MYC-derived polypeptide interfering with MYC activity-taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC. This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.

At present, treatment for HIV-1 infection employs highly active anti-retroviral therapy (HAART), which utilizes a combination of RT and protease inhibitors. Unfortunately, HIV can escape many therapies because of its high mutation rate and the complexity of its pathogenesis. HIV-1 integrates into the cellular genome, which facilitates persistence and acts as a reservoir for reactivation and replication. As an alternative or adjuvant to chemotherapy we have been developing an RNA-based gene therapy approach for the treatment of HIV-1 infection. This article summarizes the various RNA based technologies that we have developed for potential application in a gene therapy setting.