Architecture of an HIV-1 reverse transcriptase initiation complex.

Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a central step in viral infection 1 and a common target of antiretroviral drugs 2 . The reaction is catalysed by viral reverse transcriptase (RT)3,4 that is packaged in an infectious virion with two copies of viral genomic RNA 5 each bound to host lysine 3 transfer RNA (tRNALys3), which acts as a primer for initiation of reverse transcription6,7. Upon viral entry into cells, initiation is slow and non-processive compared to elongation8,9. Despite extensive efforts, the structural basis of RT function during initiation has remained a mystery. Here we use cryo-electron microscopy to determine a three-dimensional structure of an HIV-1 RT initiation complex. In our structure, RT is in an inactive polymerase conformation with open fingers and thumb and with the nucleic acid primer-template complex shifted away from the active site. The primer binding site (PBS) helix formed between tRNALys3 and HIV-1 RNA lies in the cleft of RT and is extended by additional pairing interactions. The 5' end of the tRNA refolds and stacks on the PBS to create a long helical structure, while the remaining viral RNA forms two helical stems positioned above the RT active site, with a linker that connects these helices to the RNase H region of the PBS. Our results illustrate how RNA structure in the initiation complex alters RT conformation to decrease activity, highlighting a potential target for drug action.

In-silico Studies Show Potent Inhibition of HIV-1 Reverse Transcriptase Activity by a Herbal Drug.

Acquired immunodeficiency syndrome (AIDS) is a life threatening disease of the human immune system caused by human immunodeficiency virus (HIV). Effective inhibition of reverse transcriptase activity is a prominent, clinically viable approach for the treatment of AIDS. Few non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the United States Food and Drug Administration (US FDA) as drugs for AIDS. In order to enhance therapeutic options against AIDS we examined novel herbal compounds of 4-thiazolidinone and its derivatives that are known to have remarkable antiviral potency. Our molecular docking and simulation experiments have identified one such herbal molecule known as (5E)-3-(2-aminoethyl)-5-benzylidene-1, 3-thiazolidine-2,4-dione that may bind HIV-1RT with high affinity to cause noncompetitive inhibition. Results are also compared with other US FDA approved drugs. Long de novo simulations and docking study suggest that the ligand (5E)-3-(2-aminoethyl)-5-benzylidene-1, 3-thiazolidine-2,4-dione (CID: 1656714) has strong binding interactions with Asp113, Asp110, Asp185 and Asp186 amino acids, all of which belong to one or the other catalytic pockets of HIV-1RT. It is expected that these interactions could be critical in the inhibitory activity of the HIV-1RT. Therefore, this study provides an evidence for consideration of (5E)-3-(2-aminoethyl)-5-benzylidene-1, 3-thiazolidine-2,4-dione as a valuable natural molecule in the treatment and prevention of HIV-associated disorders.

Crystal structures of HIV-1 reverse transcriptase complexes with thiocarbamate non-nucleoside inhibitors.

O-Phthalimidoethyl-N-arylthiocarbamates (TCs) have been recently identified as a new class of potent HIV-1 reverse transcriptase (RT) non-nucleoside inhibitors (NNRTIs), by means of computer-aided drug design techniques [Ranise A. Spallarossa, S. Cesarini, F. Bondavalli, S. Schenone, O. Bruno, G. Menozzi, P. Fossa, L. Mosti, M. La Colla, et al., Structure-based design, parallel synthesis, structure-activity relationship, and molecular modeling studies of thiocarbamates, new potent non-nucleoside HIV-1 reverse transcriptase inhibitor isosteres of phenethylthiazolylthiourea derivatives, J. Med. Chem. 48 (2005) 3858-3873]. To elucidate the atomic details of RT/TC interaction and validate an earlier TC docking model, the structures of three RT/TC complexes were determined at 2.8-3.0A resolution by X-ray crystallography. The conformations adopted by the enzyme-bound TCs were analyzed and compared with those of bioisosterically related NNRTIs.

Onconase action on tRNA(Lys3), the primer for HIV-1 reverse transcription.

Onconase, a cytotoxic and antitumor RNase inhibits viral replication in chronically HIV-1-infected human cells under sub lethal concentrations. Cellular tRNA has been implicated as the target for onconase action. We have recently shown that onconase cleaves selectively at GG residues in the UGG context in the variable loop and D-arm of the tRNA substrates. We therefore examined onconase cleavage specificity in in vitro transcribed tRNA(Lys3), which is the primer for HIV-1 reverse transcription but does not have UGG anywhere in its sequence. Onconase was found to cleave tRNA(Lys3) predominantly at the GG residues in the GGG triplet present in the variable loop. Mutations at this site did not effect onconase cleavages. Interestingly thus, onconase seems to cleave predominantly in the variable loop of tRNA(Lys3) regardless of the sequence context implying possible contribution of even structural determinants for its selective cleavages.

Structural analyses of purified human immunodeficiency virus type 1 intracellular reverse transcription complexes.

Retroviruses copy their RNA genome into a DNA molecule, but little is known of the structure of the complex mediating reverse transcription in vivo. We used confocal and electron microscopy to study the structure of human immunodeficiency virus type 1 (HIV-1) intracellular reverse transcription complexes (RTCs). Cytoplasmic extracts were prepared 3, 4, and 16 h after acute infection by Dounce homogenization in hypotonic buffer. RTCs were purified by velocity sedimentation, followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. RTCs had a sedimentation velocity of approximately 350 S and a density of 1.34 g/ml and were active in an endogenous reverse transcription assay. Double labeling of nucleic acids and viral proteins allowed specific visualization of RTCs by confocal microscopy. Electron microscopy revealed that RTCs are large nucleoprotein structures of variable shape consisting of packed filaments ca. 6 nm thick. Integrase and Vpr are associated with discrete regions of the 6-nm filaments. The nucleic acids within the RTC are coated by small proteins distinct from nucleocapsid and are partially protected from nuclease digestion.