Localization of DNA Binding Activity of HIV-1 Integrase to the C-Terminal Half of the Protein

Abstract

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex.

“…The conserved His and Cys residues within this domain may form part of a folded structure that includes a bound metal ion; a peptide comprising the N-terminal region folds stably in the presence of zinc (Burke et al, 1992) and the conserved His and Cys residues are important for zinc binding by the intact protein in vitro (Bushman et al, 1993). Sequences to the C-terminal side of the core may contribute to interactions with target DNA, as deletions of these regions of HIV-1 integrase affect the non-specific DNA binding activity of the protein (van Gent et al, 1991;Schauer and Billich, 1992;Woerner et al, 1992).…”

Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1

“…The carboxy terminus is the least conserved region among retroviral integrases. This domain was shown to bind both the viral DNA ends and non-specific DNA with similar affinity (Vink et al, 1991a;LaFemina et al, 1992;Schauer & Billich, 1992;Woerner et al, 1992). How the IN protein discriminates between viral DNA ends and other DNA is not yet known.…”

Uneven Genetic Robustness of HIV-1 Integrase

“…The CCD adopts an RNase H fold and contains a D,D(35)-E amino acid motif that binds divalent metal ions, specifically Mg 2ϩ (20,22). The C-terminal domain (CTD), which includes residues 213 to 288 (or in some schemes 223 to 270), adopts a beta barrel structure, resembling an Src homology 3 (SH3)-type fold, is involved in binding to host cell target DNA, and also contributes to multimerization (21,23,24). Interactions involving the three domains result in the formation of a tetramer comprised of a dimer of dimers, which, when bound to the vDNA, forms a complex known as the intasome (25).…”