Relative Comparison of Catalytic Characteristics of human Foamy Virus and hIV-1 Integrases

Аннотация

Due to their ability to integrate into the host cell’s genome, retroviruses represent an optimal basis for the creation of gene therapy vectors. The integration reaction is carried out by a viral enzyme integrase: thus, a detailed research of this enzyme is required. In this work, the catalytic properties of human foamy virus integrase were studied. This virus belongs to the Retroviridae family. The dissociation constant was determined, together with the kinetics of integrase catalytic activity. The data obtained were compared to those for the human immunodeficiency virus integrase and a considerable similarity in the activity of the two enzymes was observed.

Полный текст

Due to their ability to integrate into the genomes of non-dividing cells, retroviruses are widely used as a base for gene therapy vectors construction. A number of papers [1-6] report on systems employing human immunodeficiency virus type 1 integrase (HIV-1 IN) as a basis for the creation of constructs enabling integration of a certain vector into a given DNA sequence. However, directed integration vectors on the basis of HIV carry a potential danger to human health because of their high pathogenicity. In this regard, human foamy virus (HFV), which infects human cells efficiently, but is not pathogenic [7], seems attractive. HFV belongs to the Spumaviridae genus of the retrovirus family and carries an enzyme, integrase (HFV IN), which accomplishes the integration of the viral genome into the host cell’s genome. At present, the HFV IN catalytic properties are relatively little-studied. In this paper, an attempt has been made to explore the IN HFV catalytic properties and compare them with those of HIV-1 IN, so as to evaluate the potential for using HFV integrase for sitedirected integration. One of the factors hampering the study of the catalytic properties of retroviral integrases is their low activity: to accomplish 3’-processing, a very large excess of the enzyme over DNA is required (usually > 30:1). Therefore, in our study of the HFV IN properties we first explored the dependence of the 3’-processing efficiency on the enzyme concentration in the reaction mixture. To this end, synthetic DNA duplexes imitating the terminal sequence of the U5 domain of the viral DNA’s long terminal repeat were employed. Incubation of IN with such DNA-substrate resulted in dinucleotide removal from the 3’-end of the processed strand (U5B-strand). For both Ins, maximum reaction efficiency was achieved at an enzyme concentration of 100 nM (Figure 1). HIV IN’s low enzymatic activity is accounted for by the single-turnover mechanism of the catalytic process, the causes for which include the formation of a strong complex between the enzyme and the DNA sequence [8].