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.
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.
Puglia
Jennifer
J
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Wang
Tan
T
Smith-Snyder
Christine
C
Cote
Marie
M
Scher
Michael
M
Pelletier
Joelle N
JN
John
Sinu
S
Jonsson
Colleen B
CB
Roth
Monica J
MJ
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*
D019426:Integrases; D016914:Ribonuclease H