The long term objective of the proposed research is to understand the mechanism by which RNA tumor viruses control the expression of their own genomes and influence the expression of genes in their host cells.
Specific aims i nclude studies of the biochemistry of retroviral DNA integration and identification and analysis of viral genomic sequences that control polyprotein and RNA processing. The avian sarcoma/leukosis viruses (ASLV) serve as the primary model systems. However, comparative studies with human immunodeficiency virus (HIV) are also planned. For studies of integration, experimental strategies include analysis of the relevant biochemical activities of proteins isolated from viral particles and produced in heterologous systems through recombinant DNA techniques. The heterologous systems will be exploited for their ability to provide large amounts of mature, active proteins and functional gene fragments that can be subjected to site-directed mutagenesis. A variety of DNA substrates containing putative viral integration sites will be tested in vivo using sensitive genetic assays to detect recombination events and to evaluate the investigated in vitro systems, which include cell extracts and virus- infected and/or purified protein and nucleic components. The protein processing studies will benefit from our growing knowledge of the biochemistry and three-dimensional structure of the retroviral protease. Mutants will be designed based on this knowledge and will be used to evaluate the contribution of protease activity to virion core morphogenesis. Genetic and biochemical approaches will also be used to identify and characterize viral RNA sequences involved in the control of RNA processing and to determine the mechanism of this control. For these studies, powerful genetic selection made possible by the availability of unique viral mutants will provide a special advantage. Results from this work will contribute to an understanding of the viral and cellular biochemical reactions that underlie retrovirus-induced cancer. In addition, the retroviruses represent a promising tool with which to enlarge our understanding of the general mechanisms involved in other instances of oncogenesis where genetic damage, gene amplifications or abnormal gene rearrangements and controls can trigger malignancy.
|Lubkowski, J; Dauter, Z; Yang, F et al. (1999) Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant. Biochemistry 38:13512-22|
|Bouck, J; Fu, X D; Skalka, A M et al. (1998) Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants. J Biol Chem 273:15169-76|
|Lubkowski, J; Yang, F; Alexandratos, J et al. (1998) Structural basis for inactivating mutations and pH-dependent activity of avian sarcoma virus integrase. J Biol Chem 273:32685-9|
|Asante-Appiah, E; Merkel, G; Skalka, A M (1998) Purification of untagged retroviral integrases by immobilized metal ion affinity chromatography. Protein Expr Purif 12:105-10|
|Kukolj, G; Jones, K S; Skalka, A M (1997) Subcellular localization of avian sarcoma virus and human immunodeficiency virus type 1 integrases. J Virol 71:843-7|
|Bujacz, G; Alexandratos, J; Wlodawer, A et al. (1997) Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity. J Biol Chem 272:18161-8|
|Asante-Appiah, E; Skalka, A M (1997) A metal-induced conformational change and activation of HIV-1 integrase. J Biol Chem 272:16196-205|
|Asante-Appiah, E; Skalka, A M (1997) Molecular mechanisms in retrovirus DNA integration. Antiviral Res 36:139-56|
|Bujacz, G; Jaskolski, M; Alexandratos, J et al. (1996) The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations. Structure 4:89-96|
|Levy-Mintz, P; Duan, L; Zhang, H et al. (1996) Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase. J Virol 70:8821-32|
Showing the most recent 10 out of 34 publications