TY - JOUR
T1 - Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection
AU - Gérard, Annabelle
AU - Soler, Nicolas
AU - Ségéral, Emmanuel
AU - Belshan, Michael
AU - Emiliani, Stéphane
N1 - Funding Information:
We are grateful to Fabrizzio Mammano (Inserm U941, Paris, France) for the generous gift of the pBru IN-HA plasmid, to Eric Poeschla (Mayo Clinic, Rochester, Minnesota, USA) for the generous gift of the TC3 and TL34 cell lines, to Clarisse Berlioz-Torrent, Sarah Gallois-Montbrun and Lisa Hayden for critical reading of the manuscript, and to Jean-Luc Darlix and all members of the Berlioz-Emiliani laboratory for helpful discussions. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1SF2 p24 Antiserum. We thank the Genome and sequencing research facility from Institut Cochin. This work was supported by grants from the Agence Nationale de la Recherche sur le Sida et les Hépatites (ANRS) and Agence Nationale de la Recherche (ANR) [ANR-06-RIB-021-02, ANR-09-BIOT-006-02] to SE.. A.G. was recipient of postdoctoral fellowships from ANRS and SIDACTION. N.S. was recipient of a postdoctoral fellowship from ANRS.
PY - 2013/2/1
Y1 - 2013/2/1
N2 - Background: HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood.Results: Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization.Conclusions: Our findings indicate that, shortly after viral entry, a significant portion of DNA-free IN that is distinct from active pre-integration complexes accumulates in the nucleus.
AB - Background: HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood.Results: Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization.Conclusions: Our findings indicate that, shortly after viral entry, a significant portion of DNA-free IN that is distinct from active pre-integration complexes accumulates in the nucleus.
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U2 - 10.1186/1742-4690-10-13
DO - 10.1186/1742-4690-10-13
M3 - Article
C2 - 23369367
AN - SCOPUS:84873019961
VL - 10
JO - Retrovirology
JF - Retrovirology
SN - 1742-4690
IS - 1
M1 - 13
ER -