Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions.
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.
Dettenhofer
M
M
Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205, USA.
Yu
X F
XF
D000818:Animals; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D016346:Gene Products, vif / Q000302:isolation & purification*; D015497:HIV-1; D006801:Humans; D014771:Virion; D054320:vif Gene Products, Human Immunodeficiency Virus
D016346:Gene Products, vif; D054320:vif Gene Products, Human Immunodeficiency Virus