INTERCELLULAR TRANSFER OF HIV-1 NEF AMONG CD4 T CELLS IS MEDIATED BY DIRECT CELL-CELL CONTACT

Date

2013-04-12

Authors

Luo, Xiaoyu

ORCID

Journal Title

Journal ISSN

Volume Title

Publisher

Abstract

Purpose: HIV-1 Nef is an essential pathogenic factor for acquired immunodeficiency syndrome disease progression. It binds to over 30 putative cellular components and initiates a great variety of functions. Recent studies have demonstrated that Nef can be transferred from infected cells to uninfected cells and leads to significant changes on the bystander cells. This intercellular Nef transfer is possibly linked to the massive depletion of CD4 T cells in disease progression. However, the underlying mechanism for the intercellular Nef transfer is still not fully understood. In this study, by using advanced tools, we tested two potential mechanisms for intercellular Nef transfer among CD4 T cells including direct Nef transfer through cell-cell contact and Nef secretion in the form of exosomes. Methods: Nef-GFP Jurkat cells were co-cultured with red dye-labeled uninfected Jurkat cells for 8 hours. Cell mixture was analyzed under florescence microscope. To determine the percentage of Nef transfer during HIV infection, HIV and HIV Nef-deleted virus-infected jurkat cells were collected when cells reached 50% infected. The percentage of Nef+ cells in uninfected population and how it affected by cell density was analyzed by Immunostaining and flow-cytometry. In order to test if exosomes are involved in intercellular Nef transfer among CD4 T cells, we utilized optiprep gradient centrifugation to separate exosomes from viral particles and then detect Nef expression in exosome fractions. Results: Nef was detected in the bystander Jurkat cells after co-cultured with Nef-expressing /infected Jurkat cells. Nef was transferred through nanotubes and filopodia bridges by forming connections between infected cells and uninfected cells. There was about 3-10% of uninfected cells that were found to be Nef+ in infected Jurkat cells (50% infection). The intercellular Nef transfer was enhanced by increasing the cell density. Using optiprep gradient centrifugation, virus was successfully separated with exosomes and Nef was only detected in the virus fractions but not exosome fractions. Conclusions: Taken together, our studies showed that Nef can be transferred to uninfected bystander CD4+ T cells either from Nef-expressing cells or from HIV-infected cells. This intercellular Nef transfer is mediated by direct cell-cell contact possibly through synapse or nanotube like structures. Exosome is not involved in the intercellular Nef transfer.

Description

Keywords

Citation