A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities

Wesley M. Konsavage, Stephen Burkholder, Malgorzata Sudol, Amy L. Harper, Michael Katzman

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

Original languageEnglish (US)
Pages (from-to)4691-4699
Number of pages9
JournalJournal of virology
Volume79
Issue number8
DOIs
StatePublished - Apr 1 2005

Fingerprint

Rous sarcoma virus
Integrases
Viral DNA
DNA
Proteins
proteins
Nucleotides
nucleotides
aspartic acid
Aspartic Acid
serine
Serine
Virus Integration
Human immunodeficiency virus
Valine
enzymatic reactions
philopatry
valine
virion
glycine (amino acid)

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Konsavage, Wesley M. ; Burkholder, Stephen ; Sudol, Malgorzata ; Harper, Amy L. ; Katzman, Michael. / A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities. In: Journal of virology. 2005 ; Vol. 79, No. 8. pp. 4691-4699.
@article{b40dfdaab0a7495099a5e918c82c7e30,
title = "A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities",
abstract = "Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant {"}-2{"} site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.",
author = "Konsavage, {Wesley M.} and Stephen Burkholder and Malgorzata Sudol and Harper, {Amy L.} and Michael Katzman",
year = "2005",
month = "4",
day = "1",
doi = "10.1128/JVI.79.8.4691-4699.2005",
language = "English (US)",
volume = "79",
pages = "4691--4699",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "8",

}

A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities. / Konsavage, Wesley M.; Burkholder, Stephen; Sudol, Malgorzata; Harper, Amy L.; Katzman, Michael.

In: Journal of virology, Vol. 79, No. 8, 01.04.2005, p. 4691-4699.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities

AU - Konsavage, Wesley M.

AU - Burkholder, Stephen

AU - Sudol, Malgorzata

AU - Harper, Amy L.

AU - Katzman, Michael

PY - 2005/4/1

Y1 - 2005/4/1

N2 - Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

AB - Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

UR - http://www.scopus.com/inward/record.url?scp=16244383537&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=16244383537&partnerID=8YFLogxK

U2 - 10.1128/JVI.79.8.4691-4699.2005

DO - 10.1128/JVI.79.8.4691-4699.2005

M3 - Article

C2 - 15795255

AN - SCOPUS:16244383537

VL - 79

SP - 4691

EP - 4699

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 8

ER -