Involvement of protein-DNA interaction in adeno-associated virus Rep78-mediated inhibition of HIV-1

J Hum Virol. Nov-Dec 1998;1(7):441-50.

Abstract

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.

Study design/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.

Conclusion: 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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA, Viral / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dependovirus / genetics
  • Dependovirus / physiology*
  • Electrophoresis, Polyacrylamide Gel
  • HIV Long Terminal Repeat / physiology*
  • HIV-1 / genetics
  • HIV-1 / physiology*
  • Humans
  • Mutation
  • TATA Box / physiology
  • Transcription Factors / physiology
  • Transcription, Genetic / drug effects*
  • Viral Interference
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*
  • Virus Replication / physiology

Substances

  • DNA, Viral
  • DNA-Binding Proteins
  • Transcription Factors
  • Viral Proteins
  • rep proteins, Adeno-associated virus 2