The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

Daniel Ivanusic,Joachim Denner,Norbert Bannert,Kazimierz Madela

doi:10.1038/s41598-021-89523-7

Daniel Ivanusic, Joachim Denner + Show 2 more

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https://doi.org/10.1038/s41598-021-89523-7

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Abstract

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system. One of these strategies is the direct cell-to-cell spread of HIV-1. The formation of a virological synapse (VS) between donor and target cell is important for this process. Tetraspanins are cellular proteins that are actively involved in the formation of a VS. However, the molecular mechanisms of recruiting host proteins for the cell–cell transfer of particles to the VS remains unclear. Our study has mapped the binding site for the transmembrane envelope protein gp41 of HIV-1 within the large extracellular loop (LEL) of CD63 and showed that this interaction occurs predominantly at the VS between T cells where viral particles are transferred. Mutations within the highly conserved CCG motif of the tetraspanin superfamily abrogated recruiting of expressed HIV-1 GFP fused Gag core protein and CD63 to the VS. This demonstrates the biological significance of CD63 for enhanced formation of a VS. Since cell–cell spread of HIV-1 is a major route of persistent infection, these results highlight the central role of CD63 as a member of the tetraspanin superfamily during HIV-1 infection and pathogenesis.

Highlights

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system
Transformed yeast colonies containing the full-length CD63 sequence TM1-4, sequence transmembrane domain (TM) 3–4 and single membrane spanning mutant variant containing the TM4LEL showed a strong growth on selective plates SD W-L-H-A supplemented with 3-Amino-1,2,4-triazole (3-AT) (Fig. 1C). 3-AT competitively inhibits the HIS3 gene product, while 3-AT supplementation reduces the background growth due to HIS3 gene activity in the absence of a protein–protein interaction (PPI)[33,34]
Yeast-based split ubiquitin screen and pair-wise interaction analysis identified CD63 as an interaction partner of gp4129, and this finding was confirmed by the correlative Fluorescence resonance energy transfer (FRET)-Proximity ligation assays (PLAs) technique[40]

Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system One of these strategies is the direct cellto-cell spread of HIV-1. Mutations within the highly conserved CCG motif of the tetraspanin superfamily abrogated recruiting of expressed HIV-1 GFP fused Gag core protein and CD63 to the VS This demonstrates the biological significance of CD63 for enhanced formation of a VS. There are several lines of evidence supporting a role of tetraspanins in the replication process of HIV, mainly in very early or late events In this respect, it has been demonstrated that HIV-1 particles contain selectively incorporated CD63 as well as other tetraspanins[25,26,27]. The findings provide novel insights concerning the mechanism how CD63 might act as a regulatory protein for cell-to-cell transfer

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