Role of IN in retrovirus replication cycle . (A) Classical model of early steps in retrovirus replication. IN plays a role in the 3' processing as well as in the integration itself, these two steps being separated both in time and in space. Following synthesis of linear blunt-ended DNA in the cytoplasm (step 1 in Fig. 7A), IN cleaves their 3' termini, thus eliminating the terminal two bases from each 3'end (step 2). The resulting recessed 3'OH groups provide the attachment sites of the provirus to host DNA, an attachment which is performed only after import of 3'processed DNA into the nucleus where the final step of the integration process occurs (step 3). Circular DNA carrying LTR-LTR junctions are reportedly formed from linear DNA via the action of cellular ligases (step 4). The circularization is considered to be an alternate fate of linear DNA that has not integrated, and may indirectly explain why DNA bearing LTR-LTR junctions accumulates to high levels in cells harboring integration-defective viruses. This classical model considers that functions of IN in processes other than integration are secondary. (B) Alternate retrovirus replication model. IN cleaves the LTR-LTR junction generated at the reverse transcription step (step 1) to produce 3'end-processed linear DNA (step 2). This specific activity of the IN explains the pleiotropic effects of this protein and the phenotypes associated with its mutagenesis. First, since linear DNA is the direct product of a reaction that is catalyzed by IN, its levels would decrease under IN-defective conditions. Moreover if LTR-LTR junction molecules indeed constitute the substrate for IN, their amount would increase as a direct consequence of defective IN. Second, decreased levels of integrated proviruses would be an indirect result of the decreased pool of 3'processed IN-catalyzed linear DNA molecules that are available for integration (step 3). In this model, 2-LTR molecules are a replication-intermediate. Low levels of these molecules would be due to their rapid processing by IN in the wild-type infections. Rapid processing might also explain the presence of linear molecules with 3' processed ends in the cell cytoplasm during diverse retroviral infections, even though no blunt-ended linear molecules can be recovered from infected cells. Thus, apart from participating in retroviral DNA integration per se, IN would act upstream by controlling linear DNA production. This function of IN, as included in the modified replication model presented here, provides a parsimonious interpretation of the pleiotropic effects observed in cells infected with IN mutants.