The strict conservation of the N-terminal H-X3-7H-X23-32C-X2-C and the central catalytic DxD35E motifs in retroviral and retroelement INs suggests that these motifs carry out similar functions in the retroelement and retroviral life cycles. However, a comparison between the roles of the N-terminal histidine/cysteine ZBRs in retroviruses and LTR retrotransposons has been limited due to the lack of information regarding the role of the IN ZBR in retrotransposons. Moreover, the intervening Ty1 IN X23-32 sequence contains several residues that are conserved among retroviruses and retrotransposons, suggesting similar and/or important functions, but this region has not been extensively scrutinized by mutagenesis for defects in integration, retroviral infectivity, or virus production.
The transposition defect exhibited by the four X32 mutants in-I28A, in-L32A, in-I37A, and in-V45A, as well as in-I16A, suggested a function related to the hydrophobic nature of these residues, as alanine substitution for nucleophilic, acidic, basic, or aromatic residues resulted in WT levels of transposition. Additional characterization of these transposition-compromised mutants revealed WT levels of IN, RT, and fully processed Gag in VLPs and WT levels of Ty1 cDNA, but there were deficiencies in processes that require the interaction of IN molecules, such as GST pulldown and intragenic complementation, as well as activity in an in vitro integration assay. The in-L32A mutant, representing the most highly conserved residue in this region among retrotransposons and retroviruses, exhibited the least severe transposition defect. Additionally, this mutant appeared to retain the capacity to interact with both WT and other in-L32A molecules but was still profoundly deficient in an in vitro integration assay. With this possible exception, these results suggest that the determinant for Ty1 IN-IN interaction resides, at least in part, in the N-terminal region of IN and is comprised of hydrophobic residues.
In the intragenic complementation analysis, the mutants in-I28A and in-V45A demonstrated nearly normal levels of transposition when coexpressed with WT IN. Although this result seems contradictory to the GST pulldown experiments, especially in the case of in-V45A, it may reflect a difference between the in vivo and in vitro integration reactions. In the in vivo complementation analysis, WT IN should be able to form WT-WT multimers and carry out integration even in the presence of a mutant IN, regardless of the mutation. With IN mutants that are defective in IN-IN interaction, this activity may, in fact, be enhanced as the defective IN molecules fail to occlude interaction sites on WT molecules, unlike the in-2600 and in-K596,597G mutants, which are WT with respect to their N termini. A prediction of this hypothesis is that in-I28A or in-V45A coexpressed with in-2600 should exhibit similar levels of complementation as in-2600 coexpressed with itself, since the IN N-terminal mutant is refractory in the reaction. A similar prediction could also be made for the IN N-terminal mutant coexpressed with in-K596,597G. Comparison of in-V45A plus in-2600 with in-2600 plus in-2600 and of in-V45A plus in-K596,597G with in-K596,597G plus in-K596,597G (Fig. ) indicates that this prediction holds, not only for this example but also for in-I28A paired with either in-2600 or in-K596,597G.
Although retroviral integration requires homomeric IN complexes (7
), the interacting domains vary among retroviruses. For Rous sarcoma virus and avian sarcoma virus INs, the multimerization determinants have been mapped to the core and C-terminal domains (2
). For HIV IN mapping, the multimerization domain is not straightforward, as all three domains assume a dimer conformation in solution (17
). However, structural studies indicating that specific hydrophobic residues in the N termini of HIV-1 and HIV-2 IN, including four in the X27
sequence, are found at the IN dimer interface support our hypothesis that Ty1 IN-IN interaction relies on specific hydrophobic residues in the N terminus. As our analysis was confined to the region between IN-I16 and IN-I60, residues outside these boundaries may contribute to intermolecular interactions, but our observation that mutants in-I51A
, and in-I60A
demonstrated WT transposition levels suggested that downstream residues were not involved in interactions, and the alignment of Ty and retroviral INs (Fig. ) revealed no remarkable similarities N terminal to I16. Interestingly, another hydrophobic residue (position 46 in Fig. , the residue following Ty1 IN V45) is highly conserved among retroviral INs but not in retrotransposons. Although this residue was not implicated in IN-IN interactions (13
), the degree of conservation invites evaluation for other retroviral defects.
In addition to similarity, we also based our choice of candidates for mutation on the inclusion of different classes of amino acids with the expectation that other IN functions might be attributed to this region. However, our results revealed only a minor, if any, reduction in transposition efficiency with these mutants, suggesting that multimerization is the primary, and perhaps the only, function of this sequence.
A mutagenesis study of the charged residues in X32
of Mo-MuLV demonstrated that alanine substitutions at K66/68 and K73 were impaired for in vitro 3′ processing and strand transfer and in vivo infectivity, but these mutants retained their ability to form dimers (65
). By comparison, we examined one charged residue, K33, which did not show a significant decrease in transposition when mutated. A comprehensive survey of HIV-2 IN examined the 3′ processing, strand transfer, and disintegration activities of 36 IN mutants including S39, which is adjacent to the ZBR residue C40 (57
). All the in vitro activities of this mutant were similar to those of the WT.
Our analysis of the ZBR mutants in-H17A, in-H22A, in-C55A, and in-C58A was limited by the instability and processing defects exhibited by these mutants whether expressed in the context of element or expressed independently as a recombinant protein. Although the instability of the recombinant protein could be partially relieved by the addition of an N-terminal GST moiety, this synthetically stabilized protein was still inactive in GST pulldown and in vitro integration assays.
The high degree of conservation of an N-terminal histidine/cysteine motif among retroviral INs has fostered numerous studies of the effects of mutation or deletion of these residues. In vitro experiments with purified recombinant mutant INs and linear DNA substrates representing both donor and target molecules have shown that, in general, the steps most affected by these mutations are the initial 3′ dinucleotide cleavage and the subsequent strand transfer. The reverse disintegration reaction is usually unaffected (7
). The abundance of in vitro data obtained with purified recombinant retroviral INs underscores the difference in stability of retroviral and Ty1 INs. In addition, our observation that the instability of the downstream RT and defects in protein maturation by the Ty1 PR are associated with N-terminal ZBR mutations suggests that this region plays different roles in transposition than the HHCC domain plays in retroviral infectivity, as these extended effects on retroviral RT stability and protein maturation are not observed in retroviruses. The difference in retroviral and retrotransposon IN ZBR effects may reflect the difference in pol
organization, as the IN and RT are reversed relative to each other in retroviral and Ty/copia
elements. This notable difference is particularly intriguing in light of the report by Bizub-Bender et al. (4
) that experiments with monoclonal antibodies to different epitopes of HIV-1 IN indicate an association between the N and C termini. If a similar intramolecular interaction occurred with Ty1 IN after Gag/Pr cleavage but before PR/IN processing, this interaction could position PR near the IN/RT cleavage site. Alternatively, if the interaction occurred after PR/IN cleavage, the folded IN might recruit PR to the IN/RT cleavage site. In retroviruses, the IN N-C interaction would not place either the IN N terminus or PR near a cleavage site, due to IN being the most distal protein in the retroviral Pol arrangement. Whether an IN N-C terminus interaction occurs is not known, but this possibility illustrates how the different pol
arrangement of Ty1 could affect transposition. In fact, we constructed a Ty1 element in which the positions of IN and RT were reversed while preserving all the PR cleavage sites (43
). Expression of the resulting “flipped” element revealed no Pol proteins and a partially processed Gag similar to that of the HHCC mutants reported here (S. P. Moore and D. J. Garfinkel, unpublished results). This result supports the hypothesis that the order of the Pol proteins in the Ty1/copia family is functionally significant and that these elements have evolved in a manner that ensures the maintenance of this order.
While our assays do not permit us to directly address the influence of IN on RT stability, a required postproteolytic interaction between Ty1 IN and RT has been reported (51
) as well as several observations of retroviral IN-RT interactions (31
). Wilhelm and Wilhelm (62
) have proposed that Ty1 RT is a component of a heteromeric complex that includes IN and element cDNA in a preintegration complex and that RT remains associated with IN even after nuclear import of the preintegration complex. Their experiments included an IN-RT PR cleavage site mutant in which IN residues 45 to 520 are also deleted, removing both cysteines of the ZBD and the entire central catalytic core domain. This mutant produces an 80-kDa fusion protein which is detectable by immunoblotting, and VLPs containing this fusion possess RT activity. This result is interesting compared with our observation that mutation of either of these cysteines leads to both IN and RT instability, but the deletion of an additional 462 residues beyond the ZBD in their construct precludes an extensive comparison.
Another interesting observation is that in HIV-1 a premature stop codon at the beginning of IN, or at position 107, results in decreased virion production as measured by release of capsid protein into the culture supernatant, but this effect is reversed when PR is inactivated either by mutation or by a PR inhibitor (9
). Considering the even closer proximity of the IN ZBD to PR in Ty1 than in retroviruses, it is possible that IN mutations that disrupt the conformation of this region of IN also lead to aberrant PR function prior to PR cleavage of the PR-IN-RT polypeptide. Although it would be possible to examine the effect of PR inactivation on the ZBR mutants, a direct comparison could not be made as Ty1 VLPs are not released into the culture supernatant, and the HIV-1 mutant whole-cell lysates did not exhibit the defective phenotype (9
Until the structure of Ty1 IN is solved, it is not possible to definitively determine the roles of specific amino acids in IN structure or to propose structure-function relationships. However, our results indicating that I28 and V45 mutants are compromised for GST pulldowns when coexpressed with their cognate mutant IN but less so when coexpressed with WT IN, and our transposition complementation data with mutations in other domains of IN suggest that the hydrophobic residues function in IN-IN interaction, while the histidine/cysteine residues profoundly affect one or more early steps in retrotransposition, resulting in a subsequent failure in the cascade of events required for reverse transcription, proteolytic processing, and integration. Overall, our work illustrates the importance of the N-terminal domain of IN for Ty1 transposition, delineates different roles played by the histidine/cysteine residues compared to the hydrophobic X32 residues, and highlights the structural similarities and differences in retroviral and retrotransposon mechanisms.