A Single Nucleotide Polymorphism in Tetherin Promotes Retrovirus Restriction In Vivo

Tetherin is a membrane protein of unusual topology expressed from rodents to humans that accumulates enveloped virus particles on the surface of infected cells. However, whether this ‘tethering’ activity promotes or restricts retroviral spread during acute retrovirus infection in vivo is controversial. We report here the identification of a single nucleotide polymorphism in the Tetherin gene of NZW/LacJ (NZW) mice that mutated the canonical ATG start site to GTG. Translation of NZW Tetherin from downstream ATGs deleted a conserved dual-tyrosine endosomal sorting motif, resulting in higher cell surface expression and more potent inhibition of Friend retrovirus release compared to C57BL/6 (B6) Tetherin in vitro. Analysis of (B6×NZW)F1 hybrid mice revealed that increased Tetherin cell surface expression in NZW mice is a recessive trait in vivo. Using a classical genetic backcrossing approach, NZW Tetherin expression strongly correlated with decreased Friend retrovirus replication and pathogenesis. However, the protective effect of NZW Tetherin was not observed in the context of B6 Apobec3/Rfv3 resistance. These findings identify the first functional Tetherin polymorphism within a mammalian host, demonstrate that Tetherin cell surface expression is a key parameter for retroviral restriction, and suggest the existence of a restriction factor hierarchy to counteract pathogenic retrovirus infections in vivo.


Supporting Figure 2. Tetherin expression in 293T cells visualized by fluorescence microscopy.
Untagged Tetherin constructs (100 ng) were transfected into 293T cells and stained with an anti-Tetherin antibody directly conjugated to Alexa-488. Tetherin expression is shown in green, while nuclear staining with DAPI is shown in blue. A 40× panel showing several Tetherinexpressing cells are shown. Background staining is shown with vector control (leftmost panel). NZW Tetherin consistently stained brighter and more frequently localized to the plasma membrane compared to B6 Tetherin.

Supporting Figure 3. Intracellular expression of B6 Tetherin endocytosis mutants.
293T cells were transfected with 500 ng untagged Tetherin constructs and analyzed after 2 days. (A) Strategy to quantify Tetherin + 293T cells in an intact (upper) versus permeabilized (lower) state. Vector control was used for gating and B6 Tetherin frequencies are shown as an example. (B) Percentage of Tetherin + cells expressed in intact (surface) versus permeabilized (total) cells. Total Tetherin expression was consistently higher than cell surface expression, suggesting that a proportion of Tetherin is located intracellularly. (C) Intracellular Tetherin levels. The percentage of Tetherin expressed intracellularly was calculated by multiplying 1 minus the ratio of surface versus total Tetherin expression by 100. Data were based on triplicate measurements, error bars correspond to standard deviations and differences between means were calculated using a 2-tailed Student's t test.
(A) Representative histograms demonstrating that treatment of B6 Tetherin transfected 293T cells with the endocytosis inhibitor Dynasore resulted in increased MFI levels. (B) At 5 µM Dynasore dose, there was no significant difference in Tetherin MFI levels between treated B6 Tetherin and untreated NZW Tetherin. Data were based on triplicate measurements, error bars correspond to standard deviations and differences between means were calculated using a 2tailed Student's t test.

Supporting Figure 5. Blocking B6 Tetherin endocytosis with a dominant-negative dynamin mutant.
293T cells were co-transfected with untagged Tetherin (250 ng) and either wild-type or K44A Dynamin-eGFP (1 µg) constructs. After 2 days, the cells were subjected to flow cytometry. (A) Gating strategy and Tetherin cell surface expression analysis. Tetherin expression levels were analyzed from gated eGFP+ cells. Right panels correspond to representative histograms, showing increased B6 Tetherin surface levels following co-expression with the dominantnegative K44A dynamin mutant. (B) Increased B6 Tetherin MFI following blockade of endocytosis using a dominant-negative dynamin mutant. (C) Increased percentage of B6 Tetherin expression following blockade of endocytosis using a dominant-negative dynamin mutant. For panels B and C, data were based on triplicate measurements, error bars correspond to standard deviations and differences between means were calculated using a 2tailed Student's t test.

Supporting Figure 6. B6 Tetherin cell surface expression is dominant over NZW Tetherin in vitro.
Tetherin MFI levels in 293T cells following equimolar co-transfection of B6 and NZW Tetherin (250 ng each) were compared to 500 ng individual transfections with B6 or NZW Tetherin. (A) Distinct histogram profiles were observed between B6 and NZW Tetherin (left), but not when B6 and NZW Tetherin were co-transfected at equimolar levels (right). (B) Equimolar B6:NZW cotransfection resulted in decreased Tetherin MFI levels, consistent with the formation of a heterodimer that is endocytosed as efficiently as B6 Tetherin. Data were based on triplicate measurements, error bars correspond to standard deviations and differences between means were calculated using a 2-tailed Student's t test.

Supporting Figure 7. Baseline information on B6, NZW and F 1 hybrid mice.
Spleens from uninfected B6, NZW and F 1 hybrid mice were harvested. Tetherin MFI levels in (A) B cells and (B) erythroblasts. Each dot corresponds to a one mouse analyzed. (C) Baseline spleen mass for these three strains were equivalent. Differences between means were calculated using a 2-tailed Student's t test.