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Journal of Virology, December 2000, p. 10882-10891, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

An In Vitro Rapid-Turnover Assay for Human Immunodeficiency Virus Type 1 Replication Selects for Cell-to-Cell Spread of Virus

Suryaram Gummuluru, C. Mathew Kinsey, and Michael Emerman*

Divisions of Human Biology and Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

Received 13 June 2000/Accepted 25 August 2000

We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in the vpu open reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the Delta vpu virus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the Delta vpu virus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.

* Corresponding author. Mailing address: Divisions of Human Biology and Basic Sciences, Mailstop C2-023, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Seattle, WA 98109. Phone: (206) 667-5058. Fax: (206) 667-6523. E-mail: memerman{at}fhcrc.org.

Journal of Virology, December 2000, p. 10882-10891, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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