Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases.

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

Strategies for Targeting Retroviral Integration for Safer Gene Therapy: Advances and Challenges.

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for both close proximity to the target DNA and specificity of targeting. Each retrovirus appears to have distinct preferences for DNA sequence and chromatin features at the integration site. Tethering factors determine the preference for chromatin features, but do not affect the subtle sequence preference at the integration site. The sequence preference is likely intrinsic to the integrase protein. New developments may uncouple the requirement for a tethering factor and increase the ability to redirect retroviral integration.

Retroviral prototype foamy virus intasome binding to a nucleosome target does not determine integration efficiency.

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.