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

Abstract

Independent of its catalytic activity, HIV-1 integrase (IN) enzyme regulates proper particle maturation by binding to and packaging the viral RNA genome (gRNA) inside the mature capsid lattice. Allosteric integrase inhibitors (ALLINIs) and class II IN substitutions inhibit the binding of IN to the gRNA and cause the formation of non-infectious virions characterized by mislocalization of the viral ribonucleoprotein complexes between the translucent conical capsid lattice and the viral lipid envelope. To gain insight into the molecular nature of IN-gRNA interactions, we have isolated compensatory substitutions in the background of a class II IN (R269A/K273A) variant that directly inhibits IN binding to the gRNA. We found that additional D256N and D270N substitutions in the C-terminal domain (CTD) of IN restored its ability to bind gRNA and led to the formation of infectious particles with correctly matured morphology. Furthermore, reinstating the overall positive electrostatic potential of the CTD through individual D256R or D256K substitutions was sufficient to restore IN-RNA binding and infectivity for the R269A/K273A as well as the R262A/R263A class II IN mutants. The compensatory mutations did not impact functional IN oligomerization, suggesting that they directly contributed to IN binding to the gRNA. Interestingly, 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. Structural modeling provided further insight into the molecular nature of IN-gRNA interactions and ALLINI mechanism of action. Taken together, our findings highlight an essential role of IN-gRNA interactions for proper virion maturation and reveal the importance of electrostatic interactions between the IN CTD and the gRNA.AUTHOR SUMMARYIn addition to its well-defined catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome and regulates proper virion maturation. Inhibition of IN binding to the HIV-1 genome through mutations of positively charged residues within the C-terminal domain (CTD, i.e. R269, K273) results in non-infectious particles in which the viral genomes are mislocalized in improperly matured virions. Here we have isolated compensatory mutations in the background of the class II IN (R269A/K273A) mutant virus that restored the ability of IN to bind RNA. We found that additional substitutions of nearby acidic residues (i.e. D256 and D270), which restored the overall positive charge of the CTD, rescued the ability of IN to bind RNA and thus resulted in formation of correctly matured, infectious virions. These compensatory substitutions also revealed the role of specific residues within the CTD that determine sensitivity to allosteric integrase inhibitors (ALLINIs), a class of compounds that indirectly target IN-RNA interactions. Taken together, our findings reveal the importance of the electrostatic interactions between the IN CTD and the gRNA and provide key genetic evidence for a crucial role of the CTD in antiviral activity of ALLINIs.

The Drug-Induced Interface That Drives HIV-1 Integrase Hypermultimerization and Loss of Function

“…Therefore, mutations within this patch of the CTD surface would necessitate simultaneous adaptive changes in several positions of the IN amino acid sequence. Moreover, this compact domain has been implicated in multiple HIV-1 IN functions outside the process of integration, which may additionally limit the mutational rates at some of its key positions ( 50 – 53 ). Amino acid substitutions within the HIV-1 IN CTD can cause class I or class II lethal phenotypes, and the replication-defective HIV-1 K266E and W235E mutant viruses ( Fig.…”

Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs

“…Immunoblotting was performed as previously de-scribed. 24 Primary antibodies used were rabbit polyclonal anti-SARS-CoV-2 nucleocapsid (Sino Biologicals/40588-T62), and mouse monoclonal anti-actin (SantaCruz/SC-8432). Membranes were probed with fluorophore-conjugated secondary antibodies (LI-COR) and scanned using a LI-COR Odyssey system.…”