Doug's co-first author paper in collaboration with Juan Perilla (University of Delaware), Itay Rousso (Ben-Gurion University of Negev, Israel), and others was published in PLoS Biology this week. This collaborative study provides a novel mechanism for metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.
HIV-1 capsid has been described as semipermeable and regulates the passage of ions, small molecules, and dNTPs from the cytoplasm to its interior through pores in CA hexamers (Jacques et al., 2016; Perilla and Schulten, 2017). Inositol hexakisphosphate (IP6) has been previously reported to bind to HIV-1 Gag and CA hexamers, affecting virion assembly and reverse transcription. It has been proposed that IP6 stabilizes HIV-1 capsid by interacting with positively charged residues located near the central axis of a CA hexamer.
Reverse transcription has been reported to initiate within HIV-1 capsid and to induce changes in capsid morphology, triggering capsid uncoating. To support vDNA synthesis, HIV-1 reverse transcriptase (RT) requires dNTPs within the lumen of the capsid.
Using computer simulations, Juan's lab showed that the central HIV-1 capsid hexamer cavity could accommodate two small molecules simultaneously. His modeling suggested that addition of IP6 or a second dNTP lowers the free energy barrier to permit dNTP entry into the capsid interior.
Juan's simulations also suggested that a lysine within the pore (K25) is important in this process, such that amino acid changes (e.g. K25N) could inhibit dNTP translocation. Doug wanted to test whether this mutant would have decreased reverse transcription. He made K25N HIV-1 and showed with confocal imaging that despite normal looking capsids (thanks to the Engelman lab for electron microscopy), these capsids were unstable and reverse transcription and infectivity were abolished.
Because this effect could be due to just unstable capsids rather than dNTP binding, we had to figure out how to keep K25N and prevent the capsid from destabilizing. Thus, we made a second amino acid substitution, E45A, that has previously been shown to hyperstabilize HIV-1 capsid. The K25N/E45A double mutant had stable capsids but still had significantly reduced reverse transcription and, thus, infectivity, supporting Juan's model.
Likewise, Doug used confocal imaging to show that K25N (alone or with E45A) in HIV-1 CA reverses the effect of IP6 stabilization on HIV-1 capsid.
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