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, Tan Wang, Christine Smith-Snyder, Marie Cote, Michael Scher, Joelle N. Pelletier, Sinu John, Colleen Jonsson, Monica J. Roth

Research output: Contribution to journalArticle

21 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)9497-9510
Number of pages14
JournalJournal of Virology
Volume80
Issue number19
DOIs
StatePublished - Oct 1 2006

Fingerprint

Murine leukemia virus
Ribonuclease H
Integrases
Murine Leukemia Viruses
Human immunodeficiency virus 1
RNA-directed DNA polymerase
HIV-1
Moloney murine leukemia virus
RNA-Directed DNA Polymerase
Proteins
proteins
stop codon
mutation
Terminator Codon
gene therapy
pol Genes
Mutation
mutants

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

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. / Puglia, Jennifer; Wang, Tan; Smith-Snyder, Christine; Cote, Marie; Scher, Michael; Pelletier, Joelle N.; John, Sinu; Jonsson, Colleen; Roth, Monica J.

In: Journal of Virology, Vol. 80, No. 19, 01.10.2006, p. 9497-9510.

Research output: Contribution to journalArticle

Puglia, Jennifer ; Wang, Tan ; Smith-Snyder, Christine ; Cote, Marie ; Scher, Michael ; Pelletier, Joelle N. ; John, Sinu ; Jonsson, Colleen ; Roth, Monica J. / 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. In: Journal of Virology. 2006 ; Vol. 80, No. 19. pp. 9497-9510.
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AU - Scher, Michael

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AU - John, Sinu

AU - Jonsson, Colleen

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