Article

Genetic analysis of the human immunodeficiency virus type 1 integrase protein

Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Journal of Virology (Impact Factor: 4.65). 04/1994; 68(3):1633-42.
Source: PubMed

ABSTRACT Single-amino-acid changes in a highly conserved central region of the human immunodeficiency virus type 1 (HIV-1) integrase protein were analyzed for their effects on viral protein synthesis, virion morphogenesis, and viral replication. Alteration of two amino acids that are invariant among retroviral integrases, D116 and E152 of HIV-1, as well as a mutation of the highly conserved amino acid S147 blocked viral replication in two CD4+ human T-cell lines. Mutations of four other highly conserved amino acids in the region had no detectable effect on viral replication, whereas mutations at two positions, N117 and Y143, resulted in viruses with a delayed-replication phenotype. Defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis were observed for all of the replication-defective mutants, indicating that changes in integrase can have pleiotropic effects on viral replication.

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Available from: Chris M. Farnet, Aug 27, 2014
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    • "Although the enzymatic activities of HIV-1 IN in the integration reaction have been clearly defined by in vitro biochemical studies, numerous genetic analysis of HIV-1 DNA have demonstrated that mutations in the IN gene, including deletion mutants, influence many other stages of viral replication in addition to integration. This pleiotropic effect of IN is characterized by defects in uncoating, reverse transcription, nuclear import, viral gene expression, virion precursor protein processing, and virion morphology (Shin et al., 1994; Engelman et al., 1995; Masuda et al., 1995; Bukovsky & Gottlinger, 1996; Leavitt et al., 1996; Nakamura et al., 1997; Engelman, 1999; Tsurutani et al., 2000; Lu et al., 2004; Dar et al., 2009; Briones et al., 2010). However, the mechanisms for these pleiotropic effects of the IN gene are still poorly understood. "
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    • "Since IN is expressed as part of Gag-Pol, agents that bind to the IN domain in this polyprotein are likely to impact on the late stages of replication. Consistent with this notion, mutations in IN have been reported to effect virion formation (Shin et al. 1994). Truncations of IN at the C-terminus of Gag-Pol result in aberrant virion core structures, with a reduction in the overall levels of cell-associated viral Gag, suggesting a defect in Gag-Pol processing (Engelman et al. 1995; Bukovsky and Gottlinger, 1996). "
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    • "As these two mutations have the same effect in increasing the two ended-integration events, changes in plasticity of the protein do not explain the increased ability for concerted integration. It is well known that IN protein has other functions in addition to integration in the replicative cycle of retroviruses, such as precursor polyprotein processing, particle assembly and release, DNA synthesis, and nuclear import (Bouyac-Bertoia et al., 2001; Engelman et al., 1995; Shin et al., 1994). Therefore, it may be possible that these two residues in the a1 helix, whose mutations induce a more active protein for integration, are essential for other functions of IN in the replicative cycle and are not the best one for integration. "
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    ABSTRACT: During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.
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