Two viral encoded enzymes play central roles in the early stages of the replication of retroviruses and retrotransposons. The first one, reverse transcriptase (RT), 1 converts the singlestranded viral RNA into double-stranded DNA in a relatively complex process, reverse transcription. This step is catalyzed by the two catalytic activities of RT, the DNA polymerase (capable of copying both RNA and DNA into DNA) and the RNase H activity, which concomitantly hydrolyzes the RNA strand in the DNA-RNA heteroduplex formed (1). Subsequently, the RT-produced double-stranded DNA is transported into the nucleus, as part of the nucleoprotein complex (designated the preintegration complex), where it integrates into the genomic target DNA by the second viral enzyme, the IN. IN identifies the ends of the linear viral DNA, trims them (by removing two or three extra nucleotides located 3Ј to the highly conserved CA 3Ј termini), and then accompanies the DNA into the nucleus to catalyze integration into the target cellular DNA (1-3). There are several examples for potential linkages between RT and IN. First, the DNA product of RT is the substrate for IN, the next enzyme in the line of the viral replication cycle. Second, both proteins are proteolytic products of the same polyprotein precursor encoded by a single retroviral gene, the pol (1). In some cases, as in avian sarcoma leukosis virus, the IN sequence appears in two forms, one as part of the large ␤-subunit of the RT and the other as a free IN protein designated pp32 (1, 4). Moreover, PICs, which are capable of performing in vitro integration, contain the viral DNA, IN, RT, and other proteins (5-8). Third, the INs and RTs of HIV-1 and MLV were shown to exhibit physical interactions (9 -11). These direct contacts between the IN and RT of HIV-1 were recently confirmed by us by using surface plasmon resonance technology. 2 Finally, we and others (12, 13) have shown recently that RT can inhibit in vitro the enzymatic activities of IN, suggesting functional roles for these interactions.After the completion of reverse transcription in the cytoplasm of the infected cell, there is a significant delay in the process of integration depending on the rate by which the PICs are transported into the nucleus (1). Because all possible catalytic components for integration are likely to be present in the PICs, the viral DNA can serve as donor DNA as well as the target DNA for integration. Such a potential auto-integration process is suicidal for the virus, as it destructs the viral genome. For that reason, it is imperative to understand the mechanisms that regulate the integration of the retroviral genome and learn how to control it. Several cellular proteins are known to be involved in the integration process in HIV-infected cells (14 -17). Most interestingly, one of these factors serves also as a barrier to auto-integration in MLV-infected cells (18).

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We present evidence that the integrases (INs) of HIV types 1 and 2 are inhibited in vitro by the reverse transcriptases (RTs) of HIV-1, HIV-2 and murine leukaemia virus. Both 3′-end processing and 3′-end joining (strand transfer) activities of IN were affected by the RTs. Full inhibitions were accomplished with most RT and IN combinations tested at around equimolar RT/IN ratios. The disintegration activity of IN was also inhibited by RTs. Neither DNA synthesis nor the ribonuclease H (RNase H) domain of RT were involved in IN inhibition, since specific DNA polymerase inhibitors did not affect the level of IN inhibition, and the p51 isoform of HIV-1 RT (which lacks the RNase H domain) is as effective in inhibiting IN as the heterodimeric p66/p51 isoform. On the other hand, the catalytic activities of HIV RTs were not affected by the INs, showing that RTs can inhibit IN activities, whereas INs do not inhibit RTs. We postulate that sequences and/or three-dimensional protein structures common to RTs interact with INs and inhibit their activities. We show evidence for this hypothesis and discuss the possible sites of IN involved in this interaction.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two niutantc of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leul84) RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, 0. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-2621. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Va174]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis : 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wild-RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.