While the interaction between IN and viral DNA was extensively investigated by in vitro studies [18, 19, 22, 36–39], less was known for IN interaction with host chromatin under physiological conditions. Interestingly, a large number of recent studies have demonstrated that the cellular factor LEDGF/p75 plays an important role in tethering HIV-1 IN to the transcriptionally active units of host chromatin [40, 41]. However, how IN alone, in the absence of LEDGF/p75, plays a role in chromatin binding needs to be fully understood. In this study, we performed site-directed mutagenic analysis at the C-terminal region of the IN CCD for IN/chromatin binding and IN/LEDGF/p75 interaction. Results showed that several IN mutants including K159P, V165A, V176A, A179P, KR186,7AA and I203P were unable to bind both LEDGF/p75 and host chromatin. The mutants H171A, L172A and EH170,1AA, located in a loop region 170EHLK173 of IN, severely impaired their interaction with LEDGF/p75 but were still able to bind chromatin. Also, our data showed that LEDGF/p75 depletion in cells failed to dissociate IN from chromatin. Furthermore, the single-round HIV-1 replication assay results showed that the viruses harboring IN mutants capable of LEDGF/p75-independent chromatin binding still sustained a low level of infection. All of these data indicate that while LEDGF/p75 is important for productive HIV-1 replication, IN has the ability to bind chromatin in a LEDGF/p75-independent manner and sustain a low level HIV-1 infection.
The results showed that IN mutants K159P, A179P and I203P located at the α-helices 4, 5 and 6 specifically affected both chromatin- and LEDGF/p75-binding abilities. Since introducing proline often bends the amino-acid backbone and affects the secondary structure of the protein, it could be possible that introducing proline mutations disrupts α-helix formation and hampers both chromatin- and LEDGF/p75-binding abilities. Indeed, this could be the case for the IN mutant I203P because another mutant I203A was able to efficiently bind host DNA and LEDGF/p75 (Figs. 1 and 2 compare lane 14 to 15). However, given the fact that both A179P and A179I lost binding to host chromatin, the A179 residue may be directly involved in interacting with host chromatin (Fig. 1 and data not shown for A179I). Nevertheless, the chromatin-binding phenotype of K159P, A179P and I203P IN mutants suggest the involvement of α-helices 4, 5 and 6 of IN in host DNA recognition. Two other IN mutants that need to be addressed are KR186,7AA and F185A. We have previously shown that the IN mutant KR186,7AA was severely impaired in both chromatin- and LEDGF-binding affinities . In this study, we identified another mutant F185A that displayed a significant reduction in the interaction with LEDGF and chromatin, but to a lesser extent than that of KR186,7AA. The K186 and R187 of IN, by crystallographic studies, are known to lie in the dimer-dimer interface of IN [42, 43] and F185 has been implicated for tetramerization of IN . So, mutations at F185, K186 and R187 might affect IN oligomerization and further impair its chromatin binding affinity. In addition, a recent study by Merad H et al. revealed that a helix-turn-helix (HTH) (residues 149-186) motif consists of two helices (helix 4 and helix 5) and that the loop in between is involved in recognition of viral DNA . Interestingly, in our study, IN mutants K159A, V165A, V176A, A179P, KR186,7AA are located within this region and were identified as chromatin-binding defective mutants. Thus, the chromosomal attachment site within the IN CCD may also center on IN α-helix 4 to α-helix 5, and this HTH motif could be critical for the recognition of both viral and host DNA. However, how IN recognizes and binds both viral and host DNA sequence to form an active integration complex remains an open question and requires more detailed computational, experimental and structural investigations.
However, the functional roles of LEDGF/p75 and its potential correlation with chromatin binding of IN are of interest in our present study. It is well established that LEDGF/p75 serves as an IN-to-chromatin tethering factor, driving PICs to transcriptionally active regions of host chromosomes [5, 14]. Our previous results showed that chromatin binding defective IN mutants (V165A, A179P, KR186,7AA) also fail to interact with LEDGF/p75, suggesting that LEDGF-binding of IN might be linked to the chromatin-binding affinity of IN . Here, we attempted to select more IN mutants to map both chromatin- and LEDGF/p75-binding sites within the CCD of IN. Results showed that most of the IN mutants tested in this study lost both chromatin-binding and LEDGF/p75-interacting abilities, highlighting the importance of LEDGF/p75 as a tethering factor for IN chromatin targeting. Interestingly, two IN mutants, H171A and L172A within the CCD of IN, displayed a different phenotype; they could not efficiently interact with LEDGF/p75 yet still could bind chromatin (Fig. 1 and 2, see also Table 1). This raises the possibility that the HIV-1 IN may still be able to target chromatin in the absence of LEDGF/p75 association. Because H171 and L172 are located within or close to the loci of IN/LEDGF interface (α4/5 connector residues 166-171) , we next focused on detailed chromatin- and LEDGF-binding affinities within the IN region 170EHLK173. For this purpose, four IN double mutants, EH170,1AA, EK170,3AA, HL171,2AA and HK171,3AA, were tested. Indeed, it was shown again that the IN mutant EH170,1AA showed relatively high affinity with host chromatin but was unable to bind LEDGF/p75 effectively, while the IN mutant HL171,2AA had defects on both the chromatin- and LEDGF-binding affinities (Fig. 3). These results suggest that the HIV-1 IN is able to bind chromatin independently of LEDGF/p75.
Because the IN mutants H171A, L172A and EH170,1AA bound to chromatin but not LEDGF/p75, we further reconfirmed the LEDGF/p75 independent chromatin binding of wild type IN using the LEDGF/p75-KD cells. Our results showed that the efficient knockdown of LEDGF/p75 had no significant effect on IN to chromatin-association, suggesting that the chromatin binding of IN might still take place in the absence of LEDGF/p75. Meanwhile, we ruled out the possible effect of LEDGF/p75 knockdown on nuclear translocation of HIV-1 with wild type IN by observing the intracellular localization of all the IN fusion proteins using immunostaining, which is indeed consistent with the previous observation . Most likely, the IN is still able to target chromatin without preferential targeting sites in the absence of LEDGF/p75. Consistently, previous studies have highlighted that the role of LEDGF/p75 during HIV-1 integration is advantageous to HIV-1 integration but could be nonessential to the process of integration [5, 45]. We speculate that, without the LEDGF/p75 tethering, IN might still be able to bind chromatin, but it might lack the preferential selection site. Also, it is possible that other unknown cellular factor(s) might contribute to the chromatin targeting of IN; such proteins should harbor both DNA-binding and IN-binding domains similar to that of LEDGF/p75. Further efforts are underway to seek new cellular partners involved in IN-to-chromatin targeting.
In an attempt to correlate IN chromatin-binding ability to its effect on virus infection, we introduced IN mutants EH170,1AA, EK170,3AA or HL171,2AA into a VSV-G pseudotyped HIV-1 single cycle replicating virus and investigated their effects on HIV-1 infection. As expected, viruses containing the IN HL171,2AA mutation, which lost both LEDGF/p75- and chromatin-binding abilities, are unable to replicate (Fig. 6). This result is consistent with previous reports in which the impaired integration of proviral DNA into host cell chromatin accounted for the replication defect of the L172 mutant virus [46, 47]. Interestingly, another batch of viruses harboring the IN EH170,1AA mutation, which fail to associate with LEDGF/p75 but are still able to interact with chromatin, retain the infectivity towards the susceptible cell lines, although at a low efficiency. This suggests that the chromatin association of IN, rather than LEDGF/p75 binding, is essential for HIV-1 infection. These results are consistent with the previous study by Shun and his co-workers in which the LEDGF-null mouse embryo fibroblasts were able to support approximately 10% of HIV-1 integration compared to control cells . These results again highlighted the importance of LEDGF/p75-binding property of IN during HIV-1 replication. It is possible that the LEDGF-independent chromatin binding of IN is still able to target viral PICs to host chromatin, but, without the escort of LEDGF/p75, such IN-mediated "nonspecific" chromatin binding is less efficient and/or could not efficiently target viral PICs to transcriptionally active sites in the chromatin and mediate a productive viral replication. Another interesting question is how IN is still able to interact with host chromatin under a very low level of LEDGF/p75. Whether it is through IN directly binding to host DNA or whether it requires other undefined cofactor(s) for this process remains unclear and requires more detailed study. Successful elucidation of the mechanism underlying how HIV-1 IN possesses a LEDGF/p75-independent chromatin binding and identification of other IN-interacting cofactors involved in this process will contribute to a better understanding of the action of IN during HIV-1 replication and aid in development of efficient and comprehensive anti-HIV strategies.