The genome of Moloney murine leukemia virus can be integrated by the integrase of human immunodeficiency virus type 1 expressed alone in vivo.
An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.
Peng
W J
WJ
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.
Pan
J T
JT
Lai
M C
MC
Chiu
C F
CF
Lin
T H
TH
D002460:Cell Line; D018014:Gene Transfer Techniques; D016679:Genome, Viral; D019427:HIV Integrase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009052:Leukemia Virus, Murine / Q000502:physiology*; D016662:Virus Integration / Q000235:genetics*
D019427:HIV Integrase