Analysis of conserved and non-conserved amino acids critical for ALSV (Avian leukemia and sarcoma viruses) integrase functions in vitro

Abstract

Retroviral integrase (IN) is the viral enzyme responsible for the integration of viral DNA into host cellular DNA. In vitro, recombinant IN protein is able to catalyze the 3'-processing, strand transfer and disintegration activities. In order to analyze the importance of specific residues of ALSV (Avian leukemia and sarcoma viruses) IN protein, we introduced 31 amino acid substitutions either in residues previously shown by others to be involved in IN oligomerization or in selected conserved and non-conserved residues through the IN sequence. We tested, in vitro, the three catalytic activities of these mutants as well as their capacity to bind DNA. We found that (i) 88% of the substitutions occurring on well-conserved residues have an effect on IN activities (ii) two mutants (S85T in the central catalytic domain and N197C in the C-terminal domain) present a reduced efficiency of DNA binding compared to the wild type protein. Moreover, all mutations made on the dimer interface of C-terminal domain present reduced activities, suggesting an important role of this part of the protein. Finally, for some mutations, we observed differences between the ALSV and HIV (Human immunodeficiency virus) IN corresponding residues.

Recombinant Human Immunodeficiency Virus Type 1 Integrase Exhibits a Capacity for Full-Site Integration In Vitro That Is Comparable to That of Purified Preintegration Complexes from Virus-Infected Cells

“…A third donor, termed "P2" (480 bp in length), containing terminal wt U3 and U5 att sites, was also isolated by NdeI digestion as previously described (46). The supercoiled target DNAs (2.7 kbp) used in the integration assays were plasmids pUC19 (with the EcoRI site destroyed) and pBSK⌬2-zeo (2.6 kbp), kindly provided by Karen Moreau (40).…”

Cross-Packaging of Human Immunodeficiency Virus Type 1 Vector RNA by Spleen Necrosis Virus Proteins: Construction of a New Generation of Spleen Necrosis Virus-Derived Retroviral Vectors

“…We have constructed a series of SNV-based HIV-1 IN chimeric packaging constructs by replacing the SNV IN sequence with HIV-1 IN, using computer-based alignment and modeling information. The construct pSHI-ZP 1 was generated by removing 733 bp (AgeI and SalI sites) from the 3Ј end of pol region of the SNV packaging construct, pZP 32 ( Fig. 1B and 4) and was substituted by HIV-1 IN in the translational orf of the SNV gag-pol (pRDM 7 ) (38).…”

“…To analyze the ability of SNV proteins to crosspackage heterologous genomic HIV-1 RNA, we utilized the SNV packaging construct pZP 32 (expressing gag-pol) to crosspackage HIV-1 RNA generated from the pHRЈCMV LacZ vector (34), as well as homologous (SNV) RNA generated from the pZP 36 transfer vector (control) in a triple plasmid transfection assay. The G protein of VSV (pMDG) (33,34) was utilized as the envelope of choice.…”

“…The central domain is the catalytic core domain, and studies with chimeric HIV-1 IN have shown that this domain is particularly responsible for the selection of nonviral target DNA sites (23,(26)(27)(28)(29)46). There are three amino acids in this domain that are highly conserved among retrotransposon and retroviral INs (15,16,21,32,49). The third domain is entitled the DNAbinding domain or minimal binding domain and is located at the CЈ-terminal end of most of the retroviral INs.…”

“…Furthermore, we were able to utilize these new constructs to define the IN, and indirectly the RNase H domains of SNV (computer-based domain analysis) by using three different regions of SNV gag-pol packaging constructs pZP 32 and pRDM 7 to fuse HIV-1 IN (37). It has been documented that RNase H is responsible for the hydrolysis of the RNA-DNA hybrid (44) and inactivation of RNase H leads to a noninfectious virus, making this enzymatic activity a target for therapy of HIV-1 infection (19).…”