Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid.

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.