Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding

The study of early events in the human immunodeficiency virus type 1 (HIV-1) life cycle can be limited by the relatively low numbers of cells that can be infected synchronously in vitro. Although the efficiency of HIV-1 infection can be substantially improved by centrifugal inoculation (spinoculation or shell vial methods), the underlying mechanism of enhancement has not been defined. To understand spinoculation in greater detail, we have used real-time PCR to quantitate viral particles in suspension, virions that associate with cells, and the ability of those virions to give rise to reverse transcripts. We report that centrifugation of HIV-1(IIIB) virions at 1,200 x g for 2 h at 25 degrees C increases the number of particles that bind to CEM-SS T-cell targets by similar to 40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37 degrees C for 5 h to allow membrane fusion and uncoating to occur, the number of reverse transcripts per target cell was similarly enhanced. Indeed, by culturing spinoculated samples for 24 h, similar to 100% of the target tells were reproducibly shown to be productively infected, as judged by the expression of p24(gag). Because the modest g forces employed in this procedure were found to be capable of sedimenting viral particles and because CD4-specific antibodies were effective at blocking virus binding, we propose that spinoculation works by depositing virions on the surfaces of target cells and that diffusion is the major rate-limiting step for viral adsorption under routine in vitro pulsing conditions. Thus, techniques that accelerate the binding of viruses to target cells not only promise to facilitate the experimental investigation of postentry steps of HIV-1 infection but should also help to enhance the efficacy of virus-based genetic therapies.