Background:  The HIV fusion process occurs via membrane-mediated fusion of virus and target cells. Interactions between gp120 and co-receptors lead to the formation of fusion pores and release of the viral genome into target cells. Previous studies using cell–cell fusion assays demonstrate that a temperature arrested state can be created by pre-incubating cells at 23ºC prior to fusing with target cells. It is widely accepted that there are differences between viral and plasma membranes; therefore we further extend these studies using virus–cell fusion assays to fully understand all of the variables governing virus–cell fusion.

Methods:  MAGI reporter cells were incubated with virus for 2 hours at 4ºC. After washing unbound virus, cells were shifted to 23ºC for 2 hours to create temperature arrested state. Cells were directly shifted to 37ºC to promote fusion. Fusion inhibitors AMD 3100 (CXCR4 antagonist) and sCD4, were added at various time points to assess fusion kinetics. In separate experiments, both sCD4 and AMD 3100 were added either before or after temperature arrested state. 48 hours later, fusion was determined by measuring levels of b-gal activity.

Results:  Our studies reveal that a temperature arrested state allowed cells to proceed toward the fusion process more rapidly compared to control cells. We able differentiated which steps of the fusion process had already traversed. sCD4 achieved inhibition before, but not after temperature arrested state, unlike AMD 3100, which did not inhibit fusion both before and after temperature arrested state. Pre-incubation at 23ºC generated a state that was sensitive to inhibition by AMD 3100 but not sCD4.

Conclusions:  These studies demonstrate that a temperature arrested state can be created for virus–cell fusion, which can be used as a valuable tool to synchronize entry and study HIV fusion. The use of various inhibitors gives insight to how far virus-cell fusion has proceeded during temperature arrested state. Successful inhibition by sCD4 before, but not after temperature arrested state implies that engagement between gp120 and cell surface receptor, CD4 had already taken place during temperature arrested state. Further, the time required to become resistant to AMD 3100 is less after temperature arrested state than before. However, complete inhibition by AMD 3100 suggests that CXCR4 involving steps in the fusion process have not yet been fulfilled. These data imply that temperature arrested state provides a kinetic predisposition for engagement of CXCR4 by virion-associated gp120.

Keywords: Fusion; CD4; CXCR4