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.