Reverse transcriptase (RT) and integrase (IN) play a central role in the replication and integration of retroviral and retrotransposon genomes into the host cell chromosomes. Several studies suggest that functional interactions between these two enzymes occur during replication. For the Saccharomyces cerevisiae retrotransposon Ty3, mutations in the nonconserved amino- and carboxy-terminal domains of IN affect multiple stages of the retrotransposon's life cycle, including RT and IN expression, 3′-end processing of the cDNA, and the amount of DNA associated with virus-like particles (VLPs) (15, 26, 27). These results, coupled with the observation that two forms of Ty3 RT (the mature 55-kDa RT species and a 115-kDa RT-IN fusion protein) were detected by immunoblot analyses (7, 14), imply a functional role of the interaction between IN and RT. Moreover, mutations of Ty3 IN that caused a reduced accumulation of full-length cDNA in VLPs could be rescued only by an RT-IN protein (expressed as a capsid [CA]-IN-RT fusion protein) delivered in trans, but not by a CA-IN protein; this observation supports a model where RT and IN domains function closely or even in cis during Ty3 replication (27). A model where IN and RT are closely associated and might be components of a Ty3 RT/RT-IN heterodimer has been proposed. Precedents of an association between RT and IN proteins exist in retroviral systems (4, 8, 9, 11, 20, 28, 29, 31, 33, 39, 41, 42). In avian leukosis retroviruses, where this association has been well studied, IN is an integral part of an α-β heterodimeric form of RT, where α is the RT protein and β is an RT-IN fusion protein (6, 13, 34, 35). In human T-cell leukemia virus type 1, the RT and RT-IN proteins are likely associated in an oligomeric structure of the α3/β type (32). In human immunodeficiency virus type 1 (HIV-1) and murine retroviruses, RT and IN are fully separated by proteolytic processing during virion maturation. However, an important biological role for IN in the initiation of reverse transcription has been demonstrated for HIV-1. Several mutations of IN displayed an in vivo DNA synthesis defect and a block of viral replication at the level of reverse transcription (18, 39, 42), while other mutations increased virus fitness by augmenting the initiation of reverse transcription (29). Similarly, mutations of Moloney murine leukemia virus IN have been reported to affect cDNA production (17). Wu et al. (39) showed previously that a direct physical interaction between the IN and RT of HIV-1 could explain the role of IN in the increase of HIV-1 DNA synthesis. Pulldown assays with antibodies generated against RT or IN have also demonstrated that the HIV-1 or murine leukemia virus proteins interact physically in vitro (9, 11, 31). Furthermore, biochemical analyses showing that HIV-1 RT and IN inhibit each other suggest a regulatory role for the interaction between these two proteins (28, 31). More recently, Zhu et al. (42) have found that a C-to-S substitution at position 130 in HIV-1 IN conferred an inability to initiate reverse transcription and that IN with the C130S substitution failed to interact with RT, resulting in a defect of reverse transcription. Hehl et al. (8) have mapped the domains of interaction on both protein partners and shown that both monomeric and heterodimeric forms of HIV-1 RT can interact with IN. In the yeast retrotransposon Ty1, IN and RT are expressed and assembled in the VLPs as part of a large Gag-Pol-p199 precursor protein (Fig. (Fig.1)1) (1, 5, 19, 22, 23, 25, 40). After the assembly of VLPs, the precursor is processed by the pol-encoded protease to liberate the mature Gag-p45, PR-p20, IN-p71, and RT-RNase H (RH)-p63 proteins. In protease-deficient VLPs, Ty1 RT is functionally active as part of the Gag-Pol-p199 precursor (40). By systematically mutagenizing the cleavage sites between the protein domains of the Ty1 Gag-Pol precursor, Merkulov et al. (22) have shown that PR-IN-RT and IN-RT fusion proteins make amounts of Ty1 cDNA similar to the amounts produced by wild-type (WT) proteins. This indicates that Ty1 RT retains its full activity in vivo when it is fused to the IN domain. These results and the observation that an active Ty1 recombinant protein can be obtained only after including amino acid residues encoded by the IN gene (37) suggest that interactions between IN and RT might also be important for the function of Ty1 RT. FIG. 1. (A) Schematic representation of the Ty1 genome, mRNA, and proteins. The portions of the Gag-Pol-p199 polyprotein containing the GAG protein, protease (PR), IN, and RT-RH are indicated. LTR, long terminal repeat; PBS, primer binding site. (B) Schematic ... In the present study, IN deletion mutants were used to investigate the role of IN on the activity of RT in Ty1 VLPs. Ty1 IN, a 71-kDa protein containing 635 amino acid residues, has two phylogenetically conserved regions. The N-terminal region contains a conserved zinc binding sequence, HHCC, which is likely involved in substrate binding. The central core domain contains the conserved catalytic DDE motif required to bind divalent metal ions and to perform the DNA cleavage and joining reactions during transposition. The C-terminal region of Ty1 IN is not as well conserved as and is larger than the C-terminal region of retroviral RTs, and its function is not well understood. The IN deletion mutants studied here were able to synthesize minus-strand strong-stop DNA (−sssDNA) and showed RNase H activity. However, they failed to perform the first-strand transfer or to synthesize plus-strand DNA. Moreover, the exogenous polymerase activities as measured by a standard oligo(dG)/poly(rC) primer template assay were very different for WT and mutant VLPs. The deletion of half of the C-terminal region (residues 233 to 520) or a larger deletion including the C-terminal end of the zinc binding sequence, the core domain, and half of the C-terminal region (residues 45 and 520) did not impair the polymerase activity of Ty1 RT. On the contrary, these deletions increased the exogenous polymerase activity of Ty1 RT up to 20-fold. In contrast, the deletion of a domain comprising amino acid residues 521 to 605 strongly decreased the specific activity of RT. A striking feature of this domain is its high content of acidic amino acid residues which might be involved in the formation of a complex between IN and RT through some ionic interactions. Notably, in a previous report (37) we showed that an active recombinant RT could be obtained only if the 115-amino-acid contiguous C-terminal portion of the IN spanning this acidic domain was fused to the RT-RH domain. We have now expressed an active recombinant fusion protein bearing a much shorter IN domain (36 amino acid residues), in which a few acidic residues mimicking the acidic domain of IN are added at its N terminus. This suggests that the acidic amino acid residues in the C-terminal region of IN might be involved in the proper folding of the protein and could play a role in the activity of Ty1 RT.