Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection.
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.
Inserm, U1016, Institut Cochin, Paris, France.
Annabelle
A
Gérard
Nicolas
N
Soler
Emmanuel
E
Ségéral
Michael
M
Belshan
Stéphane
S
Emiliani
D002467:Cell Nucleus / Q000737:chemistry* / Q000821:virology*; D019427:HIV Integrase / Q000032:analysis*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008970:Molecular Weight; D009698:Nucleoproteins / Q000737:chemistry; D014779:Virus Replication
D009698:Nucleoproteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1