| Human Immunodeficiency Virus Type I (HIV-1) is the cause of Acquired Immunodeficiency Syndrome (AIDS). Currently, there are many different types of therapies available. However, the development of drug resistance demonstrates the need for new therapeutic targets.HIV-1 nucleocapsid protein (NCp) is necessary for viral replication. This nucleic acid binding protein has been shown to enhance various steps of viral replication. NCp7 helps to facilitate primer-template annealing and demonstrates a direct protein-protein interaction with HIV-1 reverse transcriptase (RT). Using pre-steady state kinetics, we observed that the NCp7 facilitated annealing enhances reverse transcription by increasing the maximum rate of single nucleotide incorporation, as well as during extension. This enhancement in rate was observed with the NCp7 facilitated annealing and not due to presence of the protein with a heat annealed primer-template.Human apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) is a part of the innate immune system thought to protect the host from viral infections. APOBEC3G's cytidine deaminase activity was initially thought to be responsible for antiviral activity. However, recent work has revealed a new mechanism of protection against HIV---through inhibition of viral processes, such as reverse transcription. Using fluorescence spectroscopy, we revealed that there is a direct interaction between APOBEC3G and HIV-1 RT. We have shown that APOBEC3G inhibits various processes of reverse transcription by slowing the rate of extension, as well as the rate of RNase H cleavage by HIV-1 RT.There is an interesting interplay between many viral and cellular proteins that modulate the efficiency of viral replication. NCp7 enhances the rate of extension by HIV-1 RT while APOBEC3G hinders the same process. Using NCp7 facilitated annealed primer-template, we observed that the presence of APOBEC3G eliminates the enhancement observed by the viral protein. This suggests an interplay and perhaps competition between these two proteins.Through pre-steady state kinetic analysis, this thesis demonstrates the positive role of the viral protein, NCp7, and the inhibitory role of human protein, APOBEC3G, in the process of reverse transcription. These mechanistic studies examining these proteins and their effects on viral replication contribute to the development of effective anti-HIV therapy. |
