Emergence of compensatory mutations reveal the importance of electrostatic interactions between HIV-1 integrase and genomic RNA

HIV-1 integrase (IN) has a non-catalytic function in virion maturation through its binding to the viral RNA genome (gRNA). Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit IN-gRNA binding and result in non-infectious viruses marked by mislocalization of the gRNA within virions. HIV-1 IN utilizes basic residues within its C-terminal domain (CTD) to bind to the gRNA. However, the molecular nature of how these residues mediate gRNA binding and whether other regions of IN are involved remain unknown. To address this, we have isolated compensatory substitutions in the background of a class II IN mutant virus bearing R269A/K273A substitutions within the IN-CTD. We found that the nearby D256N and D270N compensatory substitutions restored the ability of IN to bind gRNA and led to the formation of mature infectious virions. Reinstating the local positive charge of the IN-CTD through individual D256R, D256K, D278R and D279R substitutions was sufficient to restore IN-RNA binding and infectivity for the IN R269A/K273A as well as the IN R262A/R263A class II mutants. Structural modeling suggested that compensatory substitutions in the D256 residue created an additional interaction interface for gRNA binding. Finally, HIV-1 IN R269A/K273A, but not IN R262A/R263A, bearing compensatory mutations was more sensitive to ALLINIs providing key genetic evidence that specific IN residues required for RNA binding also influence ALLINI activity. Taken together, our findings highlight the essential role of CTD in gRNA binding and ALLINI sensitivity, and reveal the importance of pliable electrostatic interactions between the IN- CTD and the gRNA. IMPORTANCE In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues within its C-terminal domain (CTD) and regulates proper virion maturation. Here we show that compensatory mutations in nearby acidic residues (i.e. D256N and D270N) restore the ability to bind gRNA for IN variants bearing substitutions in these positively charged CTD residues. Similarly, charge reversals through individual D-to-R and D-to-K substitutions at these positions enabled the respective IN mutants to bind gRNA and restore virion infectivity. Further, we show that specific residues within the IN-CTD required for RNA binding also influence sensitivity to allosteric integrase inhibitors, a class of novel IN- targeting compounds that target the non-catalytic function of IN. Taken together, our findings reveal the importance of electrostatic interactions in IN-gRNA binding and provide key evidence for a crucial role of the IN-CTD in allosteric integrase inhibitor mechanism of action.

). In line with the titer data, D256K significantly increased IN-gRNA 239 binding whereas the D256R completely restored it in the context of the HIV-1 NL4-3 IN (R262A/R263A) 240 class II mutant virus (Fig 4E, S4N). D256N,D256K and D256R IN successfully 241 transcomplemented a class I IN mutant (D116N), suggesting that they did not distort the 242 catalytic activity of IN (Fig 4F).

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Charge reversal substitutions at acidic residues other than D256 can suppress the class 244 II phenotype 245 Given the above findings, we next determined whether charge reversal substitutions at other 246 nearby acidic residues could restore IN-gRNA binding and infectivity for the HIV-1 NL4-3 IN 247 (R269A/K273A) virus. Remarkably, the D279R substitution increased virus infectivity by 18-fold and 248 the D278R/D279R substitutions restored infectivity to WT levels (Fig. 5A). We also determined 249 whether substitutions of the original D256 and D270 residues into Ile would restore virus titers at 250 levels similar to the D-to-N substitutions. However, we found that neither D256I alone nor the 251 D256I/D270I (D2I) substitutions increased viral titers (Fig. 5A). Of note, these substitutions did 252 not impact Gag and Gag-Pol expression, processing, virion release or virion-associated IN 253 levels, though we noted the presence of an aberrantly processed IN with the D2I mutant (Fig. We have previously shown that recombinant IN binds to TAR RNA with high affinity and 288 provides a nucleation point to bridge and condense RNA (38). We next examined the ability of 289 class II IN mutants bearing compensatory mutations to bind and bridge TAR RNA. Consistent 290 with findings from CLIP, the D2N and D256R substitutions also enhanced or restored the ability 291 of IN R269A/K273 and IN R262A/R263A mutants to bridge between RNA molecules (Fig. 7I).

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Together, these data demonstrate that the compensatory mutations directly restore the ability of

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8C,F). Taken together, our findings provide key genetic, biochemical and virological evidence 316 that specific CTD residues required for RNA binding are also crucial for the ALLINI mechanism 317 of action.

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Persistence of HIV-1 in memory CD4+ T-cells as latent proviruses constitutes a major barrier to 320 HIV-1 cure. Although the majority of HIV-1 proviruses in these cells are defective (77)

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only the R224Q substitution resulted in loss of a positive charge, whereas the E246K and 328 G273R substitutions resulted in gain of positive charges. These mutations were introduced into 329 the NL4-3 proviral backbone with minimal effects on Gag expression and particle release ( Fig.   330   9B). Although the E246K virus was significantly less infectious ( Fig. 9C), we did not find any 331 evidence for loss of IN-gRNA binding (Fig. 9D), suggesting that this mutant likely displays a 332 class I phenotype. Thus, we conclude that the class II mutant viruses are rarely present in the 333 latently infected cells and therefore unlikely to contribute to chronic immune activation.

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The IN-CTD is decorated by numerous acidic and basic residues (Fig. 3A)

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CLIP experiments were conducted as previously described (15,72,73,99,100     sulfate (50 μg/mL). The titers were normalized relative to particle numbers as assessed by an

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RT activity assay and are presented relative to WT (set to 1). Also see Fig. S1 for titer and RT