Unknown

Dataset Information

0

Biochemical characterization of novel retroviral integrase proteins.


ABSTRACT: Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3' processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

SUBMITTER: Ballandras-Colas A 

PROVIDER: S-EPMC3790719 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

altmetric image

Publications

Biochemical characterization of novel retroviral integrase proteins.

Ballandras-Colas Allison A   Naraharisetty Hema H   Li Xiang X   Serrao Erik E   Engelman Alan A  

PloS one 20131004 10


Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable cryst  ...[more]

Similar Datasets

| S-EPMC3510789 | biostudies-literature
| S-EPMC4810031 | biostudies-literature
| S-EPMC2637324 | biostudies-other
| S-EPMC7408472 | biostudies-literature
| S-EPMC4334468 | biostudies-literature
| S-EPMC7240061 | biostudies-literature
| S-EPMC5329712 | biostudies-literature
| S-EPMC3395085 | biostudies-literature
| S-EPMC2791572 | biostudies-literature
| S-EPMC3716682 | biostudies-literature