Background: HIV infection is a multistep process that occurs by membrane fusion of virus and target cells.  Through interactions between gp120 and coreceptors, a 6 helix bundle is formed leading on the formation of fusion pores and release of the viral genome into target cells.  Although many of the factors required for HIV entry are known, movement and recruitment of these molecules to the cell surface is unclear.  To investigate the dynamics of components required for fusion, it is essential to examine fusion kinetics.  Cytoskeletal components may also play a role in membrane dynamics.  For example, ezrin a member of ERM family of cytoskeletal proteins may contribute to the recruitment of receptor complexes necessary for fusion.  We hypothesize that ezrin signalling may play a role in recruitment of receptor complexes.  We also believe that temperature sensitivity of fusion components will allow us to create a temperature arrested state (TAS) at which we can further examine events related to fusion.

Methods: Fusion kinetics were examined by incubating virus with MAGI reporter cells for 1 hour at 10º  to allow binding.  The cells were then either shifted to 20º for 2 hours (and finally shifted to 37º to promote fusion) or directly shifted to 37º.  Fusion inhibitor AMD 3100 was added at various timepoints.  These studies address the involvement of receptor dynamics and cytoskeletal reorganization in the formation of fusion complexes. In separate experiments, MAGI cells were transfected with dominant negative forms of ezrin and infected with virus.  Levels of infection were determined by measuring ?-gal activity.

Results: In our studies, we found that when cells were directly shifted to 37º, there was a gradual increase in the rate of fusion.  However, when cells were incubated at 20º there was a rapid increase.  These results suggest that like cell-cell fusion, a TAS can be induced for virus-cell fusion. Cells that were transfected with dominant negative ezrin constructs displayed a decrease in infection levels.

Conclusions: The absence of a lag period in fusion kinetics at 20º suggests that components necessary for fusion are being recruited to the sites of entry.  Therefore, by creating a TAS, these studies will allow us to observe the fusion process microscopically and examine other events related to fusion and membrane dynamics. Also, ezrin may be a potential factor involved in the movement and recruitment of molecules to the cell surface.

Keywords: entry; ezrin