Modulation of activity of Moloney murine leukemia virus preintegration complexes by host factors in vitro.
We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCI), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.
Li
L
L
Gene Therapy Laboratories, Norris Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA.
Farnet
C M
CM
Anderson
W F
WF
Bushman
F D
FD
D016475:3T3 Cells; D000818:Animals; D002457:Cell Extracts; D005591:Chemical Fractionation; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D005816:Genetic Complementation Test; D025741:HMGA1a Protein; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011189:Potassium Chloride / Q000494:pharmacology; D016662:Virus Integration
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D025741:HMGA1a Protein; D011189:Potassium Chloride; D004247:DNA; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII