Human Polycomb group EED protein negatively affects HIV-1 assembly and release.
BACKGROUND
The human EED protein, a member of the superfamily of Polycomb group (PcG) proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA), the integrase enzyme (IN) and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells.


RESULTS
During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefDelta57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefDelta57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm.


CONCLUSIONS
Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic RNA packaging and virus assembly, resulting possibly from a mistrafficking of viral genomic RNA (gRNA) or gRNA/Gag complex. Nef reversed the EED negative effect on virus production, a function which required the integrity of the Nef N-terminal domain, but not its N-myristoyl group. The antagonistic effect of Nef correlated with a cellular redistribution of both EED and Nef.
Rakotobe
Dina
D
Laboratoire de Virologie & Pathologie Humaine, Université Lyon I & CNRS FRE-3011, Faculté de Médecine Laennec, Lyon Cedex 08, France. dinaraktb@yahoo.fr
Tardy
Jean-Claude
JC
André
Patrice
P
Hong
Saw See
SS
Darlix
Jean-Luc
JL
Boulanger
Pierre
P
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D015683:Gene Products, gag / Q000378:metabolism; D015693:Gene Products, nef / Q000378:metabolism; D016679:Genome, Viral; D015488:HIV Antigens / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000032:analysis; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D063151:Polycomb Repressive Complex 2; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus; D054311:nef Gene Products, Human Immunodeficiency Virus
C491488:EED protein, human; D015683:Gene Products, gag; D015693:Gene Products, nef; D015488:HIV Antigens; D016655:HIV Core Protein p24; D020033:Protein Isoforms; D012097:Repressor Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D063151:Polycomb Repressive Complex 2