Involvement of protein-DNA interaction in adeno-associated virus Rep78-mediated inhibition of HIV-1.
OBJECTIVE
It has been well documented by several laboratories that adeno-associated virus (AAV) is able to inhibit HIV-1 replication and gene expression. This effect has been mapped to the AAV-encoded Rep78 protein. However, the mechanism by which Rep78 is able to inhibit HIV-1 is unclear. As Rep78 is a DNA binding transcription factor, the objective of this study was to investigate where Rep78 might bind within the HIV-1 long terminal repeat (LTR) sequences and to judge the importance of this protein-DNA interaction.


METHODS
Rep78's binding to HIV-LTR DNA was analyzed by electrophoretic mobility shift assay (EMSA). The importance of this protein-DNA interaction was analyzed using a Rep78 mutant defective for binding HIV-LTR DNA in an assay for monitoring gene expression (chloramphenicol acetyltransferase [CAT] assay).


RESULTS
The preferred site for Rep78 binding was found to be adjacent to the HIV-LTR TATA box, within nt -54 to -34 relative to the site of transcription initiation. Furthermore, a Rep78 mutant with substitutions at amino acid residues 64 and 65 which was found defective for binding HIV-LTR DNA, was also found to be defective for inhibition of tat transactivated HIV-LTR gene expression.


CONCLUSIONS
These data strongly suggest that Rep78's DNA binding ability is important for its mechanism of inhibition. Furthermore, the TATA box region of the HIV-LTR, to which Rep78 preferentially binds, is a likely target through which the inhibition takes place.
Kokorina
N A
NA
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Santin
A D
AD
Li
C
C
Hermonat
P L
PL
D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D016325:HIV Long Terminal Repeat / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D016385:TATA Box / Q000502:physiology; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic / Q000187:drug effects*; D014762:Viral Interference; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2