Pathogenic retroviral infections occur in a wide variety of metazoans often with devastating consequences. Stable integration of the retroviral cDNA genome into the host chromosome is absolutely required for a productive infection. Integration is catalyzed by the retrovirus-encoded integrase (IN), which forms a multimeric complex with the two viral cDNA long terminal repeat (LTR) ends (termed an intasome). The IN protein removes two 3'-nucleotides and catalyzes end joining (strand transfer) of the resulting recessed 3'- hydroxyls across the major groove of the target DNA separated by 4-6 bp. While decades of biochemical and cellular studies have catalogued many of the details associated with retroviral integration, the dynamic processes associated with genomic target identification, integration and intasome disassembly are largely unknown. The non-pathogenic prototype foamy virus (PFV) is a member of the Spumaretrovirinae; one of the two subfamilies of the Retroviridae family. It forms a tetramer IN complex with similar structure and drug sensitivity to the pathogenic human lentivirus HIV-1, an Orthoretrovirinae subfamily member. Unlike HIV-1 IN, the PFV IN may be easily purified and reconstituted with viral cDNA into an extremely active intasome in vitro that appears to retain comparable integration properties to cellular integration events. Moreover, the PFV IN inner and outer subunits of the tetramer complex may be manipulated separately to examine IN mutations and domain functions that are impossible with other retroviruses. For these reasons PFV IN is an unsurpassed biophysical model for retrovirus integration analysis. Using single molecule imaging analysis we recently demonstrated that the PFV intasome catalyzes the two strand transfer events near simultaneously (470 msec). Visualizing PFV intasomes on a linear target DNA suggested that the vast majority of IN-mediated target search events were nonproductive. Together these observations suggested that target site-selection limited retroviral integration. Preliminary studies presented in this application suggest that integration into model chromatin substrates is substantially more efficient while the search process appears significantly different than on naked DNA. Moreover, the strategic deletion of PFV IN domains appears to enhance its catalytic efficiency. These observations prompt several key questions: How does PFV intasome structure collaborate in target identification and integration? How does the PFV intasome disassemble following integration? How does the PFV intasome target chromatin? We propose to utilize innovative single molecule-imaging techniques to visualize the PFV integration process in real time with the following Specific Aims: 1.) detail the target site selection process of the PFV Intasome, 2.) examine the dynamics of PFV IN protein and viral cDNA following integration, and 3.) determine the influence of defined chromatin on PFV intasome dynamics and target site selection. These studies are designed to interrogate the animated processes associated with PFV integration to provide a quantitative biophysical foundation for retroviral intasome progressions.
While integration of a retroviral genome into the host chromosome is essential to establishing a productive infection, the mechanical processes of integration remain poorly understood. The prototype foamy virus (PFV) integrase is an unparalleled model for retroviral integration that includes detailed structures with the viral and host target DNAs. We will use PFV IN to examine integration mechanics in real time using innovative single molecule analysis and physiologically relevant chromatin target DNA, with the goal of advancing our quantitative understanding of this critical step in the retroviral life cycle.
|Mackler, R M; Lopez, M A; Osterhage, M J et al. (2018) Prototype foamy virus integrase is promiscuous for target choice. Biochem Biophys Res Commun 503:1241-1246|
|Senavirathne, Gayan; Lopez Jr, Miguel A; Messer, Ryan et al. (2018) Expression and purification of nuclease-free protocatechuate 3,4-dioxygenase for prolonged single-molecule fluorescence imaging. Anal Biochem 556:78-84|
|Mackler, Randi M; Lopez Jr, Miguel A; Yoder, Kristine E (2018) Assembly and Purification of Prototype Foamy Virus Intasomes. J Vis Exp :|
|Lopez Jr, Miguel A; Mackler, Randi M; Altman, Matthew P et al. (2017) Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J Vis Exp :|