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A focused deep mutational scan on the Env transmembrane and proximal cytosolic regions reveals an absence of sequence features for MA association

ABSTRACT: The HIV-1 surface glycoprotein Env binds target receptors to mediate fusion of the viral and host cell membranes during infection. Env is incorporated with very low density into virions, and in some cell types, regions within the long cytosolic C-terminal tail may mediate direct or indirect associations with Gag during virus assembly and budding. Here, the mutational landscape is determined across the transmembrane and proximal cytosolic domains of Env (001428 isolate from clade C) interacting with the MA domain of Gag at cellular membranes. No evidence is found in a derivative line of HEK293 cells for specific motifs that mediate Env/MA associations.

ORGANISM(S): Human immunodeficiency virus 1

PROVIDER: GSE126136 | GEO | 2019/02/07

REPOSITORIES: GEO

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Project description:Human Endogenous Retroviruses (HERVs) are exogenous viral elements that integrated into the germline millions of years ago. They comprise 8% of the human genome and play a critical role in cellular differentiation during development. HERV-K(HML-2) is the most recently integrated and actively transcribed HERV. It is found to be highly expressed in pluripotent stem cells and strongly downregulated during cellular differentiation. Moreover, expression of HERV-K in terminally differentiated neurons results in cytotoxicity. Expression of HERVs has been implicated in carcinogenesis and is suggested to confer stem cell like features important for cell growth. Atypical Teratoid / Rhabdoid Tumor (AT/RT) is a rare pediatric brain cancer that results from incomplete neuronal differentiation during embryonic development. In this paper, we investigated the cytopathic effect of Ouabain, a cardiac glycoside that induces cytotoxicity in stem cells, on AT/RT cells. We characterized four AT/RT cell lines by immunostaining and observed variable levels of stem cell, Oct4 and Pax6, and neuronal differentiation markers, TUBB3 and MAP2, as well as HERV-K envelope (env), polymerase (pol), and gag transcripts. We stained formalin-fixed brain tissues from 20 AT/RT patients and 5 unaffected controls for HERV-K ENV protein expression. 40% of AT/RT patient tissues showed ENV expression. In contrast, no ENV immunostaining was observed in all 5 control brain tissues. We used Ouabain, known to be cytotoxic to stem cells, observed up to 50% toxicity at 24 hr and 80% toxicity at 48 hr after treatment. While AT/RT primarily exist as large cell aggregates in suspension, Ouabain treatment downregulated HERV-K ENV protein and caused disruption of cell-to-cell interactions. Similarly, when AT/RT cells were transfected with a CRISPR/Cas9 construct designed to repress HERV-K promotor activity, cell aggregates separated and cell viability decreased significantly. Using a luciferase reporter construct under the control of a HERV-K promotor, we observed a significant decrease in HERV-K promotor activity at 8 to 72 hr post treatment with 0.5 uM Ouabain. Given that downregulation of HERV-K alone was sufficient to cause cytotoxicity in the cells, HERV-K expression may be essential for AT/RT growth and survival. Ouabain and other compounds that modulate HERV expression may provide an alternative or adjunctive therapeutic option for treatment of embryonal tumors.

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A focused deep mutational scan on the Env transmembrane and proximal cytosolic regions reveals an absence of sequence features for MA association

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