| HIV-1 can directly spread between immune cells through the virological synapse, a cell-cell adhesion structure that greatly enhances the spread of HIV-1. The formation of the virological synapse occurs in a CD4-Env dependent manner but the process by which Env, a protein required for the assembly of infectious virions, is recruited to the cell membrane for the formation of virological synapses, remains poorly understood. In order to create a tool to elucidate Env's role, two novel fluorescent variants of Env were constructed by replacing the HIV-1 V1 region with a fluorescent probe, mCherry or super-folder GFP. The development of HIV-1 variants carrying fluorescent proteins within the context of the viral genome has facilitated the study of infection and has been particularly useful in monitoring the transfer of virus between cells. Transfection of the constructs into 293T, HeLa, and Jurkat cells showed that Env localizes and infects like wild-type HIV-1 but fails to admit as robust viral protein and virion production. Co-transfection with wild-type HIV-1 restores packaging of Env to wild-type levels and increases cell-cell transfer. These results demonstrate the feasibility of constructing a means to visualize HIV-1 Env. Understanding HIV-1 protein interaction during the viral life cycle, and the molecular mechanisms of cell-cell transfer will lead to a better understanding of HIV-1 pathogenesis and provide the basis for the development of novel anti-viral treatments for inhibiting HIV-1 transmission. |
