Influence of Viral Nucleocapsid Protein and Genomic RNA Structural Intricacies on the Mechanism of HIV Recombination

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Internal strand transfers involve template switching during retroviral replication, within internal regions of the viral genome. Such transfers are a major source of genetic variability in retroviruses like HIV-1. An in vitro strand transfer assay that mimicked recombinational events occurring during reverse transcription in HIV-1 was used to assess the role of nucleocapsid protein (NC) and structural intricacies of genomic RNA in strand transfer. Transfers in highly structured templates from the U3 3' LTR, gag-pol frameshift region, and Rev response element (RRE) were strongly enhanced by NC. In contrast, weakly structured templates from the env and pol-vif regions transferred well without NC and showed lower enhancement. Assays conducted using NC zinc finger mutants supported a differential role for the two fingers in strand transfer with finger one (N-terminal) being more important on highly structured RNAs. The lack of strong polymerase pause sites in the weakly structured templates from the env and pol-vif regions demonstrated that non-pause driven mechanisms could also promote transfer.

Mapping assays were conducted on high and low structured templates from the gag-pol and the env regions respectively (called GagPol and Env templates), to locate the point(s) of transfer in each case. The majority of transfers were located near a major pause site in the gag-pol region; in contrast, in the env region, most transfers were located towards the end of the homologous region between donor and acceptor templates. Various truncated/mutant GagPol acceptor templates were analyzed with wild type GagPol donor templates in strand transfer assays. Results indicated that destabilized acceptor templates enhance the level of transfer and cause a highly efficient 'chasing' of the pause site into transfer products. The outcome of these experiments also suggested that strand transfer in the GagPol templates is via a pause induced 'donor dissociation' method. In the Env template the mechanism of transfer was proposed to occur by a pause independent, 'acceptor invasion' method. This knowledge about the interplay of RNA structure and NC protein on recombination could help in designing antiviral vaccines and drug inhibitors.