Background:  Fusion induced by HIV is a multi-step process. The gp120 subunit undergoes sequential conformational changes upon interactions with CD4 and principle co-receptors CXCR4 and CCR5. Previously, studies using cell–cell fusion assays found that a temperature-arrested stage can be created by pre-incubating cells at 23ºC prior to fusing with target cells. However, HIV is an enveloped virus containing distinct differences from that of the host cell plasma membrane; therefore we extend these studies using virus–cell fusion assays to fully understand all 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 a temperature intermediate. Cells were directly shifted to 37ºC to promote fusion. Fusion inhibitors were added at various times to assess fusion kinetics. Fusion was determined by measuring levels of β-gal activity.

Results:  These data reveal that a temperature arrested stage can be created for envelope-mediated fusion of both CXCR4- and CCR5-tropic HIV-1 strains. We found that preincubating cells at 23ºC allowed cells to proceed toward the fusion process much faster than cells preincubated at 4ºC. We also show that cells are resistant to inhibition by the c34 peptide before TAS compared with their sensitivity to inhibition by sCD4 before TAS and vice versa.

Conclusions:  This kinetic analysis can be used as a tool to synchronize entry and study HIV fusion. Our data suggest that a temperature-arrested intermediate contributes to the exposure of heptad repeats within gp41 that are necessary for 6-helix bundle formation. Further, the time required to become resistant to TAK779 is less after the temperature-arrested stage than before. These data imply that a temperature-arrested stage provides a kinetic predisposition for engagement of CCR5 by virion-associated gp120.