A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse.

Lili Wang,Alice Sandmeyer,Hongru Li,Benjamin K Chen,Thomas Huser,Wolfgang Hübner

doi:10.3390/v14010038

Lili Wang, Alice Sandmeyer + Show 4 more

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https://doi.org/10.3390/v14010038

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Abstract

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.

Highlights

When conducting fluorescence recovery after photobleaching (FRAP) studies we found that the envelope glycoprotein (Env) recovery was dramatically decreased in the HIV-1 Env V4.2-Y712A-Superfolder green fluorescent protein (sfGFP) when compared to the non-mutated clone (Figure 6D-1, Supplemental Movie S7)
Env-green fluorescent protein (GFP) fusion protein resembles wild type Env in its subcellular distribution and is still able to participate in virological synapses (VS) formation and cell-to-cell infection
By utilizing a superfolder allele of GFP and employing favorable insertion sites described by Nakane et al into molecular clone NL4-3, we provide a description of an HIV-1 clone encoding a fluorescent

Summary

Introduction

Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS. The HIV-1 envelope glycoprotein (Env) plays crucial roles as the surface glycoprotein on the virus particle, mediating virus binding, fusion and entry, as well as in initiating the formation of cell–cell adhesions that facilitate viral transmission, called virological synapses (VS) [1–3].

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