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 cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+ hematopoietic progenitor cells were transduced with a retroviral vector carrying RSRbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+ cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of RSRbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+ cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+ cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+ thymocyte subsets derived from RSRbz-transduced CD34+ cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-R (HIF1R). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand-and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

The CCR5 beta-chemokine receptor is the coreceptor for macrophage-tropic (M-tropic) strains of HIV-1 and appears to be the principal coreceptor during early stages of human immunodeficiency virus-1 (HIV-1) infection. Approximately 1%-2% of the Western European Caucasian population is homozygous for a 32-bp deletion in the coding region of the CCR5 gene, rendering them less susceptible to HIV infection. These individuals still harbor a normal immune response, thereby making CCR5 an attractive cellular target for anti-HIV therapies. Based on the natural population studies, reduction in CCR5 expression should not affect the physiologic function of the modified cells but should interfere with their susceptibility to HIV-1 infection. To downregulate this receptor, we have designed a hammerhead ribozyme (RZ) that specifically targets the CCR5 mRNA and lacks complementarity to other members of the chemokine receptor gene family. For expression of this highly specific ribozyme, we have taken advantage of the stable transcripts afforded by transcription from the RNA polymerase III (pol III)-based adenoviral VA1 gene. Importantly, the VA1-chimeric ribozyme is stably expressed with a half-life of almost 6 hours. Using this expression system, we show up to 70% downregulation of the elevated levels of CCR5 receptor in the HOS-CD4.CCR5 cell line. The monocytic cell line PM1 was stably transduced with the chimeric VA1 ribozyme constructs. In these cells, substantial resistance to challenge with an M-tropic but not a T-tropic HIV viral strain was observed, demonstrating specificity in downregulating the CCR5 coreceptor. The VA1-CCR5 ribozyme chimeras described in this study should prove useful in both studies of CCR5 receptor function and therapeutic intervention of monocytotropic HIV-1 infection. The VA1 vector described in this study is well suited for the stable cytoplasmic expression of other ribozyme constructs as well.

The highly sensitive and specific methods of molecular biology emphasize the frequency of subclinical infections in the genital tract tissues by the human papillomaviruses (HPVs). The purpose of this work was to investigate occult viral infections by the HPV type 6, 11, 16, and 18 in the gingival tissues. The Southern blot method with 32P-radiolabeled DNA probes applied under stringent conditions to 20 interproximal gingival papilla specimens revealed homologous viral sequences in 1 of 6 cases of adult periodontitis (HPV 16), 1 of 2 cases of rapidly progressive periodontitis (RPP) (HPV 6/HPV 11), 2 of 2 cases of acute gingivitis in psychiatric institutionalized patients (HPV 6; HPV 6/HPV 11), and 2 of 10 cases of acute gingivitis in AIDS patients (HPV 6/HPV 11/HPV 16; HPV 6). No periodontal or extra-periodontal specimen hybridized with the HPV 18 probe. Simultaneous hybridization with two or three HPV types was common (3/6 cases). The present detection of HPV 6, 11, 16 DNAs or related-DNAs in periodontal tissues without obvious clinical signs of viral infection suggests that the gingival epithelium may act as a reservoir.