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Retroviruses exhibit high levels of variation primarily due to high rates of mutation and recombination during viral replication. The high levels of variation present in retroviral populations affect the genetic diversity and evolution of the virus, and play a significant role in viral pathogenesis as well as resistance to antiviral drugs and vaccines. The virally encoded reverse transcriptase (RT) is responsible for the majority of mutations that are introduced into the viral genome. The low processivity of RT also contributes to the high frequency of recombination that is observed. We have developed a powerful in vivo system utilizing directly repeated sequences within the green fluorescent protein (GFP) gene to analyze properties of RT that are involved in template switching and recombination. Mutations were introduced into various domains of RT and analyzed for their effects on template switching. Based on these results, we proposed a dynamic copy-choice model for RT template switching in which the dynamic equilibrium between DNA polymerization and RNase H degradation determines the rate of template switching. In the second study, we determined whether polymerase-dependent and/or polymerase-independent RNase H activity was important for template switching. Using a trans-complementation system, we were able to determine that the dynamic equilibrium between DNA polymerization and polymerase-dependent RNase H activity is responsible for high rates of RT template switching. We also demonstrated for the first time, the existence of polymerase-dependent RNase H activity in vivo. Finally, we determined the frequency of direct repeat deletion for short direct repeats and identified an RNA secondary structure that formed a hotspot for RT template switching. Analysis of the template switching hotspot revealed that the majority of template switching events occurred within 19 base pairs 3′ of the predicted RNA structure. We also showed that base pairing between the newly synthesized DNA and RNA acceptor template is essential for efficient RT template switching.