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Journal of Virology, February 1999, p. 1460-1467, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Highly Purified Human Immunodeficiency Virus Type 1 Reveals a Virtual Absence of Vif in Virions

Markus Dettenhofer and Xiao-Fang Yu*

Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205

Received 23 July 1998/Accepted 4 November 1998

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.

* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3768. Fax: (410) 614-8263. E-mail: xfyu{at}jhsph.edu.

Journal of Virology, February 1999, p. 1460-1467, Vol. 73, No. 2
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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