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We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease ; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.Reverse transcriptase (deoxynucleoside-triphosphate: DNA deoxynucleotidyltransferase, RNA-directed) of human immunodeficiency virus, type 1 (HIV-1) is presently under extensive study as a target for antiviral drugs. Reverse transcriptase constitutes one component of the HIV-1 pol open reading frame [l] and is processed, in either virions or heterologous systems expressing this polyprotein, to polypeptides of 66 kDa and 51 kDa, through the action of the polcodcd protease [2 -61. Although expression systems for highlevel synthesis of the enzymatically active 66-kDa reverse transcriptase (p66) have been documented [7, 81, the occurrence, nevertheless, of a 51 -kDa polypeptide (p51) suggests that a p66/p51 heterodimer may be the relevant enzyme form. To elucidate this, large quantities of the p66/p51 enzyme are required to determine whether its enzymatic or physical properties differ from those of p66. Since p51 reverse transcriptase is produced from p66 via C-terminal processing [9], and its C-terminus (and hence p51 /p66 cleavage recognition site) is not clearly defined, it is impossible by conventional cloning technology to produce a p.51 polypeptide with an authentic C-terminus. We have addressed this problem by over-expressing p66 reverse transcriptase and the pol-coded protease as separate proteins on the same vector, under the control of similar transcription and translation signals. In the absence of protease, p66 reverse transcriptase accumulates to approximately 10% of the total cellular protein. In the dual expression system, a 17-kDa precursor HIV-1 protease (p17) is cleaved to the enzymatically active 10-kDa molecule, which Correspondence to S. F. J. Le Grice, Central Research Units, F. Hoffmann-La Roche & Co. Ltd, CH-4002 Basel, SwitzerlandAhhreviutians. HIV-1, human immunodeficiency virus, type 1 ; p66, 66-kDa HIV-1 reverse transcriptase polypeptide; p51, 51-kDa proteolytic product of p66; p17, 17-kDa precursor polypeptide of HIV-1 protease; His-p66, p66 with (His), added at the amino terminus; His-p51, p51 with (His)6 ...

The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

SUMMARY Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an “A”-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the “A” and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.