Reverse transcription is a key event in the replication cycle of human immunodeficiency virus type 1 (HIV-1) that is initiated after the virus enters the cell. Events subsequent to reverse transcription include nuclear transport of the preintegration complex (PIC), targeting PICs to the sites of integration, proviral integration, and finally the repair of the termini by mechanisms not yet fully understood. Reverse transcription is initiated in the cytoplasm, and the viral cDNA is synthesized in the cytoplasmic compartment. However, it is unclear if reverse transcription is completed prior to proviral integration, as proviral DNA containing discontinuities in plus-strand DNA has been shown to efficiently integrate in vitro. Indeed, reverse transcriptase (RT) has been detected in PICs and intracellular reverse-transcription complexes isolated from the nuclei of HIV-1-infected cells (5, 11). Thus, it is possible that RT is required for polymerization subsequent to nuclear transport, including the completion of plus-strand DNA synthesis and for repair of the single-strand gaps that remain after integration. In certain retrotransposons, exemplified by R2Bm, reverse transcription begins after endonucleolytic cleavage of the genomic DNA by integrase (IN). The RNA is copied using the 3Ј-OH terminus generated by cleavage of the genomic DNA (24), which would necessitate the retention of RT in the nuclear PICs.Furthermore, in avian leukosis virus (14, 30) and in human T-lymphotropic leukemia virus type 1 (33), RT and IN are parts of a single polypeptide forming the ␣ subunit of the heterodimeric ␣␤ complex. In Rous sarcoma virus, where it has been well studied, the ␣␤ complex retains both RT and IN activities. In addition, the recent demonstration that the ␣␤ complex localizes to the nucleus suggests that the RT and IN proteins are likely present together in the nuclear PICs (37). In contrast, the RT and IN proteins of murine retroviruses and of HIV are fully separated by proteolytic cleavage during virion maturation (27). The observation that RT is present in the nuclear PICs (5) in addition to within reverse-transcription complexes (11) suggests that RT may be retained via proteinprotein or protein-nucleic acid interactions with other viral components.Previous studies led to the observation that the RT and IN proteins specifically interact with each other and that this interaction is not mediated by nucleic acid bridging (38). Here, we demonstrate that both monomeric and heterodimeric forms of RT can interact with IN, and we map the domains of interaction on both protein partners. Attempts to assess the effect of IN on RT function showed that RT function was unaffected by IN. In contrast, in the presence of RT, the IN-mediated joining reaction was stimulated significantly, while the 3Ј-end processing was unaffected. These results suggest functional interaction of the two proteins.

RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers Inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) used in current highly active antiretroviral therapy regimens can block viral replication and retard the onset of AIDS. However, toxicity (4) and the rapid appearance of drug-resistant viral strains (24) are serious concerns and diminish their merit. Current anti-RT drugs have exploited the presence of two binding pockets on this viral DNA polymerase. The nucleoside analog RT inhibitors (NRTI) bind to the deoxynucleoside triphosphate-binding pocket, which is formed partly by the template-primer nucleic acid and partly by the protein surfaces (29). The other site, the nonnucleoside RT inhibitor (NNRTI)-binding pocket, is a hydrophobic pocket exclusively present in the RT of the M subgroup of HIV-1 (29). A key surface on the RT, the templateprimer-binding cleft, in spite of its central importance in viral reverse transcription, has been minimally explored as a target to obstruct viral replication.Small nucleic acid aptamers with high affinity for HIV-1 RT were previously isolated in vitro from a library of randomized DNA and RNA sequences via the SELEX (systematic evolution of ligands by exponential enrichment) procedure (5,9,26,30,31). The anti-RT aptamers are small RNA molecules that lack primary sequence homology to each other, display high affinity and specificity for HIV-1 RT, and competitively inhibit its enzymatic activity in vitro. Thus, their three-dimensional structures all recognize the same surface on the RT, the template-primer-binding cleft. Some of the anti-HIV-1 RT aptamers have the potential to form pseudoknot-like secondary structures, often with a sharp bend reminiscent of the conformation of template-primer bound to HIV-1 RT (5). The crystal structure of HIV-1 RT bound to one of the RNA aptamers shows that the aptamer makes extensive contacts with the template-primer cleft of RT (12). It has been shown that the association constant of such aptamers for HIV-1 RT correlates with the degree of inhibition. Thus, these aptamers are termed here template analog RT inhibitors (TRTIs).Despite the unique nature of the anti-HIV-1 RT RNA aptamers, their utility as inhibitors of viral replication has remained unexploited till now. Therefore, we examined their suitability for intracellular expression via gene delivery and their ability to block HIV replication. In this report, we show that such aptamers efficaciously block HIV-1 replication in cell culture. The aptamers block an early stage of the...

Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34+ hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ−/−) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4+ T cells per μl 12 weeks after CD34+ injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) has a 3=-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIV JR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (C max ) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC 90 ) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment. N ucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are highly effective for both first-line therapy and preexposure prophylaxis (PrEP) of human immunodeficiency virus (HIV) infection. There are seven FDA-licensed single NRTIs, including the nucleoside emtricitabine (FTC) and the nucleotide tenofovir (TFV). Long-term use of these drugs has resulted in the emergence of drug-resistant variants in treated patients as well as in treatment-naive individuals due to transmission of these drugresistant variants (1, 2). New, more-potent compounds with activity against NRTI-resistant strains are needed. All currently approved anti-HIV NRTIs lack the 3=-hydroxyl group and therefore inhibit further DNA polymerization by HIV RT through immediate chain termination after incorporation into the nascent DNA. The absence of a 3=-OH, however, imparts unfavorable properties to these NRTIs by negatively impacting their binding affinity for RT compared to the natural deoxynucleoside triphosphate (dNTP) substrates and by reducing their binding affinity for the cellular nucleoside/nucleotide kinases responsible for intracellular phosphoryl...