HIV-1 capsid core disassembly (uncoating) is a prerequisite for viral DNA integration into the host genome and a promising target for antiviral therapy. We recently developed a method to directly label capsid protein (CA) with green fluorescent protein (GFP-CA) in infectious viral complexes and reported that HIV-1 cores that retained >94% of their CA entered the nucleus and uncoated near their integration site ?1.5 hours before integration. However, whether the nuclear capsids lost their integrity by rupturing or a small loss of CA before capsid disassembly was unclear.
We utilized a previously reported vector in which GFP 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 used live-cell imaging to track GFP and core-associated mRuby-tagged cleavage and polyadenylation specificity factor 6 (mRuby-CPSF6).
We found that nuclear capsids retained their integrity until shortly before integration and lost their GFP content marker ~1-3 minutes before loss of capsid-associated mRuby-CPSF6. In contrast, when CA was tagged with GFP, loss of GFP and mRuby-CPSF6 occurred simultaneously. Thus, capsids 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 within a short time frame between uncoating and integration.