Knowledge Bank

The HIV-1 nucleocapsid (NC) domain of Gag has both specific and more general nucleic acid (NA) binding properties. NC acts specifically to recognize and package unspliced viral RNA and as a general chaperone to facilitate reverse transcription by annealing/ aggregating NAs via its highly positive character and by destabilizing NA secondary structure via its two CCHC zinc finger (ZF) motifs. Interestingly, it has recently been reported that ZF deletion or mutation to CCCC results in production of virions containing DNA instead of RNA, rendering them noninfectious. Thus, an additional role of NC is to prevent premature reverse transcription from occurring before or during assembly. Here, we probe the in vitro NA binding and chaperone properties of Gag variants containing the same ZF mutations or deletions tested in the cell-based assays. Fluorescence anisotropy equilibrium binding measurements reveal that mutation or deletion of both ZFs results in a modest reduction in binding to the ψ SL3 stem-loop relative to WT Gag, whereas binding to nonspecific single-stranded NAs is largely unaffected. Similarly, Gag’s ability to aggregate NAs or facilitate tRNALys,3 annealing to the primer-binding site, two functions of Gag during viral assembly, was only moderately affected upon ZF mutation or deletion. A time-resolved fluorescence resonance energy transfer assay was used to monitor hairpin stem opening of a DNA hairpin construct. Surprisingly, single CCCC and ZF deletion variants appeared to be more effective at opening the TAR hairpin than WT Gag, and Gag variants in which both zinc fingers were mutated or deleted showed even greater duplex destabilization capability than the single ZF variants. Previous studies with the freestanding NC domain of Gag indicated that NC’s duplex destabilization activity depended on the ZF structures. Our new results suggest that in the context of Gag, disruption of the ZF leads to an increased ability to disrupt nucleic acid secondary structure and we explore possible mechanisms by which this increased capability may cause premature reverse transcription.