This proposal will focus on the mechanism of human immunodeficiency virus (HIV) recombination as catalyzed by reverse transcriptase (RT). Recombination during first strand DNA synthesis (synthesis directed by the RNA genome of the retrovirus) is presumed to occur by strand transfer. This process, whereby DNA synthesized on one template (donor) is transferred to another template (acceptor) for additional elongation, occurs twice during the retroviral replication cycle. In addition to these required transfers which occur at the termini of viral templates, other transfers within internal regions of the genome can also occur. An in vitro system designed to examine internal strand transfers will be used to model recombination occurring within internal regions of the donor. Experiments will evaluate the kinetic parameters associated with this process and the influence of RT on specific steps (for example, the dissociation of the nascent DNA from the donor and its association with the acceptor) with the goal of formulating a model for recombination. Strand transfer on specific viral sequences with characteristics that may promote efficient transfer will be examined using a system that can measure the strand transfer efficiency of any region of RNA. The potential effect of base misincorporations on transfer efficiency and the frequency of misincorporation during transfer events will be evaluated. This is particularly relevant given the low fidelity of DNA synthesis by HIV-RT and the fact that misincorporation leads to DNA synthesis pausing which has been proposed to promote transfer. Nucleocapsid protein (NCp) is a nucleic acid binding protein found in the core of the retroviral virion. The properties of this protein suggest that it may influence the DNA synthetic and strand transfer catalysis properties of RT. These possibilities will be evaluated in the above systems. In addition to the studies on recombination, the RNase H cleavage specificity of HIV-RT will be examined, The experiments will use substrates that mimic structures present during replication to determine how the RT recognizes and cleaves such structures and to evaluate the spacial and topological arrangement of the polymerase and RNase H active sites of the RT. Overall, the studies will offer insight into one of the mechanisms by which retroviruses evolve and escape both drug therapy and the host immune response. An understanding of these processes is important for designing drug therapies or other strategies that target unique RT activities.
