CHARACTERIZATION OF THE HUMAN SPUMA RETROVIRUS INTEGRASE BY SITE-DIRECTED MUTAGENESIS, BY COMPLEMENTATION ANALYSIS, AND BY SWAPPING THE ZINC-FINGER DOMAIN OF HIV-1

被引:39
作者
PAHL, A [1 ]
FLUGEL, RM [1 ]
机构
[1] DEUTSCH KREBSFORSCHUNGSZENTRUM, FORSCHUNGSSCHWERPUNKT ANGEW TUMORVIROL, RETROVIRALE GENEXPRESS ABT, D-69009 HEIDELBERG, GERMANY
关键词
D O I
10.1074/jbc.270.7.2957
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 NE(2)-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.
引用
收藏
页码:2957 / 2966
页数:10
相关论文
共 33 条
[1]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[2]  
BROWN PO, 1990, CURR TOP MICROBIOL, V157, P19
[3]  
BURKE CJ, 1992, J BIOL CHEM, V267, P9639
[4]   DOMAINS OF THE INTEGRASE PROTEIN OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RESPONSIBLE FOR POLYNUCLEOTIDYL TRANSFER AND ZINC-BINDING [J].
BUSHMAN, FD ;
ENGELMAN, A ;
PALMER, I ;
WINGFIELD, P ;
CRAIGIE, R .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (08) :3428-3432
[5]   ROUS-SARCOMA VIRUS INTEGRASE PROTEIN - MAPPING FUNCTIONS FOR CATALYSIS AND SUBSTRATE-BINDING [J].
BUSHMAN, FD ;
WANG, BB .
JOURNAL OF VIROLOGY, 1994, 68 (04) :2215-2223
[6]   TRANSCRIPTIONAL CONTROL OF HUMAN PAPILLOMAVIRUS (HPV) ONCOGENE EXPRESSION - COMPOSITION OF THE HPV TYPE-18 UPSTREAM REGULATORY REGION [J].
BUTZ, K ;
HOPPESEYLER, F .
JOURNAL OF VIROLOGY, 1993, 67 (11) :6476-6486
[7]   REVERSAL OF INTEGRATION AND DNA SPLICING MEDIATED BY INTEGRASE OF HUMAN-IMMUNODEFICIENCY-VIRUS [J].
CHOW, SA ;
VINCENT, KA ;
ELLISON, V ;
BROWN, PO .
SCIENCE, 1992, 255 (5045) :723-726
[8]   HOTSPOTS AND WARM SPOTS - INTEGRATION SPECIFICITY OF RETROELEMENTS [J].
CRAIGIE, R .
TRENDS IN GENETICS, 1992, 8 (06) :187-190
[9]   INFLUENCE OF SUBSTRATE STRUCTURE ON DISINTEGRATION ACTIVITY OF MOLONEY MURINE LEUKEMIA-VIRUS INTEGRASE [J].
DONZELLA, GA ;
JONSSON, CB ;
ROTH, MJ .
JOURNAL OF VIROLOGY, 1993, 67 (12) :7077-7087
[10]   IDENTIFICATION OF AMINO-ACID-RESIDUES CRITICAL FOR ENDONUCLEASE AND INTEGRATION ACTIVITIES OF HIV-1 IN PROTEIN INVITRO [J].
DRELICH, M ;
WILHELM, R ;
MOUS, J .
VIROLOGY, 1992, 188 (02) :459-468