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Journal of Virology, October 2006, p. 9497-9510, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00856-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Jennifer Puglia,1,{dagger} Tan Wang,2,{dagger} Christine Smith-Snyder,1 Marie Cote,1 Michael Scher,1 Joelle N. Pelletier,4 Sinu John,3 Colleen B. Jonsson,2 and Monica J. Roth1*

Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, New Jersey 08854,1 Department of Biochemistry and Molecular Biology, Southern Research Institute, 2000 9th Ave. S., Birmingham, Alabama 35205,2 Graduate Program in Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294,3 Département de Chimie, Faculté des Arts et Sciences, et Département de Biochimie, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada4

Received 26 April 2006/ Accepted 7 July 2006

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.

* Corresponding author. Mailing address: Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-5048. Fax: (732) 235-4783. E-mail: roth{at}rwja.umdnj.edu.

{dagger} These authors contributed equally to the manuscript.

Journal of Virology, October 2006, p. 9497-9510, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00856-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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