7th Conference - Abstract 649

HIV-1 Core Particles Travel on Microtubules to Reach the Nucleus of Infected Cells

D. MCDONALD*¹, M. VODICKA², M. EMMERMAN², and T. HOPE¹. ¹The Salk Inst. for Biological Studies, La Jolla, CA; and ²The Fred Hutchinson Cancer Res. Ctr., Seattle, WA

Despite the recent progress made in understanding receptor-mediated entry of the Human Immunodeficiency Virus (HIV), there is little known about HIV-cell interactions that occur after entry but early in infection. One reason for this paucity of understanding is the difficulty in studying early post-entry events. To study these early events we have labeled the core of HIV by incorporation of a Gfp-Vpr fusion protein. Co-localization of viral proteins and cellular membranes with the Gfp-labeled virions demonstrates that authentic viral particles are visualized. Observation of living cells infected with labeled HIV reveals that the particles move in linear paths, suggesting a possible interaction with cytoskeletal components. Co-localization analysis and video microscopy confirms that the labeled particles are associated with and move along microtubules. Detergent extraction of infected cells demonstrates that when microtubules are stabilized by taxol treatment, the labeled particles remain attached to the cytoskeleton. In contrast, when cells are extracted without taxol treatment, the particles are found in the soluble fraction. Extracted particles bind to the microtubules of taxol-treated permeabilized target cells, and their movement can be reconstituted by the addition of ATP. We will also present evidence that we can detect membrane fusion and subsequent microtubule traficking of incoming viral particles in living cells when viral entry is routed through the endocytic pathway by Vesicular Stomatitis Virus glycoprotein pseudotyping. We propose that like herpesvirus and adenovirus, HIV uses microtubules to move to the nucleus as a normal part of its life cycle.

Key Words: GFP, microtubules, video microscopy