Mutations in the C-terminal domain of ALSV (Avian Leukemia and Sarcoma Viruses) integrase alter the concerted DNA integration process in vitro.
Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.
Moreau
Karen
K
Université Claude Bernard, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Lyon, France.
Faure
Claudine
C
Violot
Sébastien
S
Verdier
Gérard
G
Ronfort
Corinne
C
D001355:Alpharetrovirus / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D002384:Catalysis; D004279:DNA, Viral / Q000378:metabolism*; D019426:Integrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D016662:Virus Integration
D004279:DNA, Viral; D019426:Integrases