The virion cores of the replication competent type 1 human immunodeficiency virus (HIV‐1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV‐1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV‐1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100‐fold molar excess of other tRNAs. Cross‐linking analysis of this interaction locates the contact site to a region within the heavily modified anti‐codon domain of tRNALys,3.

We have purified and determined functional parameters of reconstituted, recombinant HIV‐1 reverse transcriptase (RT) heterodimers within which either the p66 or p51 polypeptide was selectively mutated in one or both aspartic acid residues constituting the proposed polymerase active site (‐Y‐M‐D‐D‐). Heterodimers containing a mutated p51 polypeptide retain almost wild type levels of both RNA‐dependent DNA polymerase and ribonuclease H (RNaseH) activity. In contrast, heterodimers whose p66 polypeptide was likewise mutated exhibit wild type RNaseH activity but are deficient in RNA‐dependent DNA polymerase activity. These results indicate that in heterodimer RT, the p51 component cannot compensate for active site mutations eliminating the activity of p66, indirectly implying that solely the p66 aspartic acid residues of heterodimer are crucial for catalysis.

Two single site substitutions (E478 → Q and H539 → F) were introduced into the C‐terminal RNase H domain of HIV‐1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+‐nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

We have analysed the mechanism of ribonuclease H (RNaseH) induced cleavage of a defined RNA‐DNA hybrid by human immuno‐deficiency virus (HIV‐1) reverse transcriptase (RT). An in vitro transcribed RNA labelled at the 3′ end was hybridized to a pentadecameric DNA oligonucleotide complementary to an internal region of the RNA. Upon incubation of this RNA‐DNA hybrid with recombinant p66 or p66/p51 HIV‐1 reverse transcriptase, RT‐RNaseH mediated cleavage is observed at most nucleotides within the short hybridized stretch, resulting in a spectrum of RNA fragments extending from the 3′ label to this region and differing in length by one nucleotide. The same RNA, this time labelled at the 5′ end, yields only one or two major cleavage products corresponding to RNA species extending from the 5′ label to the middle of the hybridized region. Such a result can be explained by the action of both endonuclease and 3′‐‐‐‐5′ exonuclease activities inherent to the C‐terminal domain of p66 RT. To investigate how RNaseH cleavage is coupled to reverse transcription, a combination of deoxynucleoside triphosphates was used which allowed controlled extension of the primer DNA. Concomitantly with the elongation of the oligonucleotide primer, RNaseH cleavage proceeds towards the 5′ end of the RNA with identical increments, suggesting a simultaneous action of both activities.

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