Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro.

INTRODUCTION Reverse transcription and integration are the signature events of retrovirus replication. Reverse transcription creates a double-stranded DNA (dsDNA) copy of the positive-sense viral RNA genome, and integration archives that copy within the genome of the infected cell. Both processes are targets of successful HIV-1 antiretroviral therapies, and the associated enzymatic activities have been characterized by elegant structural, biochemical, and molecular virological analyses. Nevertheless, mechanistic studies of these processes remain challenging because they are performed by viral core particles deep within the infected cell cytoplasm and nucleus. Of particular interest is defining whether the conical capsid that surrounds the viral RNA genome participates in the process of viral replication. RATIONALE In principle, informative mechanistic and imaging analyses of HIV-1 replication could be performed in vitro, but the coupled processes of reverse transcription and concerted integration have not yet been recapitulated outside of the cell. To address this limitation, we reconstituted these processes in a cell-free system, using purified HIV-1 virions as the source of viral genomes and enzymes. RESULTS We recapitulated the sequential processes of endogenous reverse transcription (ERT) and integration from viral core particles, which were released from purified HIV-1 virions by gently permeabilizing the viral membrane using a pore-forming peptide. ERT was initiated through addition of deoxynucleotide triphosphates (dNTPs), and the DNA products—early, intermediate, and late transcripts—appeared in high yields and in the expected temporal order, with late dsDNA products accumulating maximally after 8 to 10 hours. Integration of the resulting viral DNA into an exogenous target DNA was also recapitulated, provided that cell extract was added to the reaction mix. Deep sequencing and cloning confirmed that the 3′ and 5′ termini of the viral DNA were integrated in a concerted fashion, with the expected 5–base pair target site duplications in the target DNA, and with target site sequence preferences that resembled those reported for HIV-infected cells. HIV-1 capsids are composed of hexagonal lattices of the viral CA protein, arrayed in a “fullerene cone” structure. Analyses of the requirements for efficient ERT and integration in our cell-free system revealed that these processes require the presence of capsid lattices of appropriate stability and geometry, as determined by using site-directed CA mutations, capsid-stabilizing factors, and potent new capsid inhibitors. Imaging with electron cryotomography revealed that many viral capsids remained largely intact during the ERT reaction. Capsid uncoating, when observed, did not occur in an all-or-none fashion, as might have been anticipated for a highly cooperative structure. Rather, uncoating proceeded through a continuum of disassembly intermediates in which portions of the capsid wall appeared lost in patches, as revealed through subunit-level lattice mapping. Largely intact capsids in which viral nucleic acid strands extruded through lattice openings were observed after 8 to 10 hours, which was coincident with the maximal accumulation of late ERT products and integration events. CONCLUSIONS We have reconstituted efficient reverse transcription and integration—the major early steps of the HIV-1 life cycle—in a cell-free system. Our data indicate that the viral capsid plays an active and indispensable role in supporting efficient reverse transcription. Thus, we consider the entire core particle, including the outer capsid shell, to be the true viral “replication complex.” We further found that complete capsid uncoating may be a prerequisite for integration to occur. Thus, the capsid plays essential roles in the reactions that duplicate and archive the viral genome, in addition to previously established roles in protecting the viral genome from innate immune sensor surveillance and in helping the core to traverse the cytoplasm, enter the nucleus, and traffic to integration sites. We anticipate that our cell-free system will enable systematic analyses of the key steps in viral replication and integration and thereby elucidate the transformations that occur as HIV-1 proceeds through the first half of the viral life cycle.