HIV-1 cores retain their integrity until minutes before uncoating in the nucleus

Abstract

Significance Here, we used a fluorescent protein that is free in solution and is trapped in nuclear HIV-1 capsids to demonstrate that the capsids retain integrity and prevent mixing of macromolecules within the viral core and the cellular environment until just before integration. We also found that capsid integrity is maintained until just minutes before disassembly in the nucleus, revealing that uncoating proceeds rapidly after integrity loss. These valuable insights into the early stage of HIV-1 replication indicate that intact HIV-1 capsids are imported through nuclear pores, that reverse transcription is mostly completed within intact capsids, and that preintegration complex-host interactions facilitating integration and target site selection must occur within a short time frame between capsid disassembly and integration. We recently reported that HIV-1 cores that retained >94% of their capsid (CA) protein entered the nucleus and disassembled (uncoated) near their integration site <1.5 h before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear. Here, we utilized a previously reported vector in which green fluorescent protein is inserted in HIV-1 Gag (iGFP); proteolytic processing efficiently releases GFP, some of which remains trapped inside capsids and serves as a fluid phase content marker that is released when the capsids lose their integrity. We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ∼1 to 3 min before loss of capsid-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6). In contrast, loss of GFP fused to CA and mRuby-CPSF6 occurred simultaneously, indicating that viral cores retain their integrity until just minutes before uncoating. Our results indicate that HIV-1 evolved to retain its capsid integrity and maintain a separation between macromolecules in the viral core and the nuclear environment until uncoating occurs just before integration. These observations imply that intact HIV-1 capsids are imported through nuclear pores; that reverse transcription occurs in an intact capsid; and that interactions between the preintegration complex and LEDGF/p75, and possibly other host factors that facilitate integration, must occur during the short time period between loss of capsid integrity and integration.