Mechanism of Tetherin Inhibition of Alphavirus Release

Abstract

Tetherin is an interferon-inducible, antiviral host factor that broadly restricts enveloped virus release by tethering budded viral particles to the plasma membrane. In response, many viruses have evolved tetherin antagonists. The human tetherin gene can express two isoforms, long and short, due to alternative translation initiation sites in the N-terminal cytoplasmic tail. The long isoform (L-tetherin) contains 12 extra amino acids in its N terminus, including a dual tyrosine motif (YDYCRV) that is an internalization signal for clathrin-mediated endocytosis and a determinant of NF-κB activation. Tetherin restricts alphaviruses, which are highly organized enveloped RNA viruses that bud from the plasma membrane. L-tetherin is more efficient than S-tetherin in inhibiting alphavirus release in 293 cells. Here, we demonstrated that alphaviruses do not encode an antagonist for either of the tetherin isoforms. Instead, the isoform specificity reflected a requirement for tetherin endocytosis. The YXY motif in L-tetherin was necessary for alphavirus restriction in 293 cells but was not required for rhabdovirus restriction. L-tetherin’s inhibition of alphavirus release correlated with its internalization but did not involve NF-κB activation. In contrast, in U-2 OS cells, the YXY motif and the L-tetherin N-terminal domain were not required for either robust tetherin internalization or alphavirus inhibition. Tetherin forms that were negative for restriction accumulated at the surface of infected cells, while the levels of tetherin forms that restrict were decreased. Together, our results suggest that tetherin-mediated virus internalization plays an important role in the restriction of alphavirus release and that cell-type-specific cofactors may promote tetherin endocytosis. IMPORTANCE The mechanisms of tetherin’s antiviral activities and viral tetherin antagonism have been studied in detail for a number of different viruses. Although viral countermeasures against tetherin can differ significantly, overall, tetherin’s antiviral activity correlates with physical tethering of virus particles to prevent their release. While tetherin can mediate virus endocytic uptake and clearance, this has not been observed to be required for restriction. Here we show that efficient tetherin inhibition of alphavirus release requires efficient tetherin endocytosis. Our data suggest that this endocytic uptake can be mediated by tetherin itself or by a tetherin cofactor that promotes uptake of an endocytosis-deficient variant of tetherin.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes

“…(iii) One or more antiviral effectors acting during assembly or egress may have roles in this assay, but were missed in the single‐cycle assay of the original screen. Accordingly, we sought to determine whether some of the other previously described anti‐alphavirus effectors contribute to this residual IFN‐mediated restriction of VEEV (Zhang et al , 2007 ; Teng et al , 2012 ; Jones et al , 2013 ; Li et al , 2013 ; Mahauad‐Fernandez et al , 2014 ; Ooi et al , 2015 ; Poddar et al , 2016 ; Weston et al , 2016 ; Weiss et al , 2018 ; Wan et al , 2019 ). We used multiplexed CRISPR‐Cas9 to engineer U‐2 OS cells that have up to four antiviral effectors simultaneously targeted.…”

Interferon inhibits a model RNA virus via a limited set of inducible effector genes

“…It is also possible that other IFN-induced genes that were not identified by our screens may play a role in restricting VEEV infection. Accordingly, we sought to determine if some of the previously described anti-alphavirus effectors contribute to this residual IFN-mediated restriction of VEEV (Jones et al , 2013; Li et al , 2013; Mahauad-Fernandez et al , 2014; Ooi et al , 2015; Poddar et al , 2016; Teng et al , 2012; Wan et al , 2019; Weiss et al , 2018; Weston et al , 2016; Zhang et al ., 2007). We used multiplexed CRISPR-Cas9 to engineer U-2 OS cells that have up to 4 antiviral effectors simultaneously targeted.…”

Current and Promising Antivirals Against Chikungunya Virus

“…These findings gave rise to hope that nsP1 could be considered as a target for developing tetherin-mediated therapeutics against CHIKV (80). However, a more recent study on the subject could not confirm Jones' report since no evidence for tetherin-antagonists in alphaviruses was found (81).…”

“…Jones ( 80 ) postulated that CHIKV nsP1 is such a BST-2/tetherin antagonist. However, Wan et al ( 81 ) could not confirm Jones' findings and suggested that the sole physical tethering of virus particles to the PM is not sufficient to restrict alphaviruses and that the subsequent virus endocytosis is a requirement for efficient inhibition of alphavirus release.…”