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Journal of Virology, November 2000, p. 10074-10080, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Human Immunodeficiency Virus Type 1 Spinoculation Enhances Infection through Virus Binding

Una O'Doherty,1,2 William J. Swiggard,1,3 and Michael H. Malim1,3,*

Departments of Microbiology,1 Pathology and Laboratory Medicine,2 and Medicine,3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148

Received 23 June 2000/Accepted 6 August 2000

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-1IIIB virions at 1,200 × g for 2 h at 25°C increases the number of particles that bind to CEM-SS T-cell targets by ~40-fold relative to inoculation by simple virus-cell mixing. Following subsequent incubation at 37°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, ~100% of the target cells were reproducibly shown to be productively infected, as judged by the expression of p24gag. 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.

* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, 347B Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6148. Phone: (215) 573-3493. Fax: (215) 573-2172. E-mail: malim{at}mail.med.upenn.edu.

Journal of Virology, November 2000, p. 10074-10080, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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