Characterization of the Human Spuma Retrovirus Integrase by Site-directed Mutagenesis, by Complementation Analysis, and by Swapping the Zinc Finger Domain of HIV-1

Abstract

The human spuma retrovirus or foamy virus integrase (HFV IN) is an enzymatically active protein consisting of domains similar to other retroviral integrases: an amino-terminal HH-CC finger, a centrally located region with the conserved D, D-35-E protein motif required for catalytic activity and oligomerization, and at least one DNA binding domain implicated in the 3' DNA processing activity and integrase. Recombinant, purified HFV IN protein carrying 10 histidine residues displays a site-specific endonuclease, an integrase, and a disintegrase activity with oligonucleotide substrates that mimic the viral long terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV IN residues of the catalytic domain had increased endonuclease and disintegrase activities. Deletion mutants at both ends of the HFV IN protein were generated, purified, and characterized. Unexpectedly, it was found that the HFV integrase and disintegrase activities require an intact NH2-terminal sequence and that COOH-terminal deletions led to an increase in disintegrase activity. The HH-CC finger of HFV IN was exchanged with that of the human immunodeficiency virus-1 (HIV-1) IN protein. The resulting chimeric IN had a 3' processing activity that utilized the HFV LTR instead of the HIV LTR, indicating that the central domain is crucial for substrate recognition. Functional complementation of the amino-terminal deletion mutant of HFV IN was achieved by a carboxyl-terminal deletion mutant of the chimeric IN, resulting in high levels of integrase activity.

An active foamy virus integrase is required for virus replication.

“…With respect to the M70 mutation (DYIG DYTG) our results indicate that this mutant, in contrast to what has been expected from in vitro integration assays (Pahl & Flu$ gel, 1995), behaves in a very similar way to unmutated virus. Except for FVs the threonine (in HIV-1 IN at position 66) is highly conserved in retroviral IN proteins (Engelman & Craigie, 1992).…”

“…IN mutants of the infectious plasmids were generated by recombinant PCR on a subgenomic 0n33 kb EcoRI-HincII fragment (Higuchi, 1990). In pHSRV2\M70 the DYIG motif was changed to DYTG, similar to a previous report (Pahl & Flu$ gel, 1995), and in pHSRV2\M73 and pcHSRV2\M73 the DD35E motif was changed to DA35E. The mutations were verified by automated DNA sequence analysis of the complete PCR fragment using AmpliTaqFS and the ABI 310 sequence analysis system (Perkin Elmer).…”

“…The mutant M70 contains an isoleucine to threonine substitution (DYIG DYTG) around the first aspartic acid of the DD35E motif. This mutant was described previously as having a reduced endonuclease, a slightly elevated disintegrase and a largely impaired IN activity (reduction to less than 3 % of wild-type activity) in in vitro assays (Pahl & Flu$ gel, 1995). The mutant M73 contains an aspartic acid to alanine substitution (DD35E DA35E) in the highly conserved catalytic centre present in all known IN proteins (Brown, 1997).…”

Retroviral Integrase Proteins and HIV-1 DNA Integration

“…15). Consistent with results of in vitro integration assays with recombinant IN protein (27)(28)(29)(30) and crystal structures of a lentiviral IN two-domain NTD-CCD construct (31), the NTD of each inner monomer engages the CCD of the opposed inner monomer with its bound viral DNA in trans ( Fig. 2A, right) (18).…”

Minimal size of prototype foamy virus integrase for nuclear localization

“…First, it catalyzes covalent insertion of the DNA copy of viral genome into the host genomic DNA, a process called integration (Engelman et al, 1991;Pahl and Flugel, 1995). Second, it mediates the transport of preintegration complex (PIC) from cytoplasm to the nucleus of infected cell (Craigie, 2001).…”