title: Revealing domain structure through linker-scanning analysis of the murine leukemia virus (MuLV) RNase H and MuLV and human immunodeficiency virus type 1 integrase proteins.
abstract: Linker-scanning libraries were generated within the 3' terminus of the Moloney murine leukemia virus (M-MuLV) pol gene encoding the connection-RNase H domains of reverse transcriptase (RT) as well as the structurally related M-MuLV and human immunodeficiency virus type 1 (HIV-1) integrase (IN) proteins. Mutations within the M-MuLV proviral vectors were Tn7 based and resulted in 15-bp insertions. Mutations within an HIV-1 IN bacterial expression vector were based on Tn5 and resulted in 57-bp insertions. The effects of the insertions were examined in vivo (M-MuLV) and in vitro (HIV-1). A total of 178 individual M-MuLV constructs were analyzed; 40 in-frame insertions within RT connection-RNase H, 108 in-frame insertions within IN, 13 insertions encoding stop codons within RNase H, and 17 insertions encoding stop codons within IN. For HIV-1 IN, 56 mutants were analyzed. In both M-MuLV and HIV-1 IN, regions are identified which functionally tolerate multiple-linker insertions. For MuLV, these correspond to the RT-IN proteolytic junction, the junction between the IN core and C terminus, and the C terminus of IN. For HIV-1 IN, in addition to the junction between the IN core and C terminus and the C terminus of IN, insertions between the N terminus and core domains maintained integration and disintegration activity. Of the 40 in-frame insertions within the M-MuLV RT connection-RNase H domains, only the three C-terminal insertions mapping to the RT-IN proteolytic junction were viable. These results correlate with deletion studies mapping the domain and subdomain boundaries of RT and IN. Importantly, these genetic footprints provide a means to identify nonessential regions within RT and IN for targeted gene therapy applications.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Puglia
authors.forename: Tan
authors.initials: T
authors.lastname: Wang
authors.forename: Christine
authors.initials: C
authors.lastname: Smith-Snyder
authors.forename: Marie
authors.initials: M
authors.lastname: Cote
authors.forename: Michael
authors.initials: M
authors.lastname: Scher
authors.forename: Joelle N
authors.initials: JN
authors.lastname: Pelletier
authors.forename: Sinu
authors.initials: S
authors.lastname: John
authors.forename: Colleen B
authors.initials: CB
authors.lastname: Jonsson
authors.forename: Monica J
authors.initials: MJ
authors.lastname: Roth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D015870:Gene Expression; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009052:Leukemia Virus, Murine / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000378:metabolism*
chemical_list: D019426:Integrases; D016914:Ribonuclease H