NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases

Abstract

Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3′-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. IMPORTANCE Intensive studies on HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatments escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens on the possibility of adapting to the optimisation of further functionalities exerted by the same protein. Such property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

The HIV-1 Integrase C-Terminal domain induces TAR RNA structural changes promoting Tat binding

“…1a ). The boundaries between CTD and CT were defined according to sequence alignment and previous structural studies 16 43,44 45 . The IN-CT is not conserved among lentiviruses ( Supplementary Fig.…”

“…Construction of plasmid pET15b, encoding N-terminal 6XHis tagged IN-CTD (aa 220-270) and IN-CTD-ΔCT (aa 220-288) were previously reported 44 . IN-FL gene from pNL4-3 were cloned in pPROEX-HTa vector in frame with 6XHis tag at N-terminus.…”

Biological and structural analysis of new potent Integrase-LEDGF allosteric HIV-1 inhibitors

“…Interestingly, M. Kanja et al . identified, in the IN CTD, a new functional motif constituted by four noncontiguous amino acids (N 222 K 240 N 254 K 273 ) [33], including this position N222. This NKNK motif strictly conserved in all natural sequences of HIV-1 group M, generates a positive surface potential that is important for the nuclear import of reverse-transcribed genomes.…”

The C-Terminal Domain of HIV-1 Integrase: A Swiss Army Knife for the Virus?

“…This interface is a structural hallmark of the HIV-1 IN CTD fragment in solution [ 33 , 42 , 43 ]. One exception is the crystal structure of a trimeric CTD harbouring an N-terminal polyhistidine-tag that coordinated Ni2+ ions along a 3-fold molecular axis [ 37 ] ( Figure 3 C). The W243 is not accessible within this trimer, impeding the formation of any canonical dimeric interface in the crystal.…”

“…( C ). Crystal structure of a trimeric CTD (PDB: 6T6J; [ 37 ]) harbouring an N-terminal polyhistidine-tag that coordinates Ni2+ ions (in green) along a three-fold molecular axis. ( D ).…”