MYXV binds to unstimulated human T lymphocytes but activation of human T lymphocytes is required for MYXV replication. (A) To investigate whether MYXV binds to unstimulated human T lymphocytes, T cells were isolated using an EasySep negative selection HLA T cell enrichment kit (up to >95% purity). Approximately 1 × 10⁶ of these negatively isolated T lymphocytes were incubated with recombinant vMyx-Venus/M093L at an MOI of 10 for 1 hour on ice to allow virus binding but not entry. After this, unbound virus was washed twice with cold 1× PBS + 5% FBS. T cells were then stained with anti-CD3 antibody, and the levels of Venus⁺ labeling in the CD3⁺ population were determined by flow cytometry (bottom panel). A representative experiment from 1 donor is shown. To investigate virus infection, ∼3-4 × 10⁶ isolated human T lymphocytes were incubated with recombinant vMyx-GFP at an MOI of 10 for 1 hour at room temperature to allow virus adsorption. After this, mock-treated and infected T cells were stimulated with anti-CD3/CD28 beads at a cell:bead ratio of 1:1. This was followed by incubation at 37°C for 72 hours or 96 hours. The unstimulated (ie, without adding beads) mock- and MYXV-treated T lymphocytes were subjected to the same culturing conditions. (B) Seventy-two and 96 hours after culturing, expression of virus-expressed GFP was monitored using fluorescence microscopy. (C-F) To quantify the levels of infection of different T populations, 72 hours after vMyx-GFP exposure, cells were stained with antibodies against CD3, CD4, and CD8, and the levels of GFP⁺ in each population were quantified by using flow cytometry. Likewise, in panel G, levels of infection of T lymphocyte activation proteins CD25 and CD69 were also quantified using flow cytometry. Data reported are representative of al least 6 independent experiments. Significance (ie, P < .05) was determined using the Student t test. To investigate whether MYXV launches productive virus replication in stimulated human T lymphocytes, we performed 1-step growth curves. (H) T cells were infected with vMyx-GFP at an MOI of 10. Infected/unstimulated T cells, and infected/stimulated T cells, were harvested, cells were lysed using repeated freeze-thaw, and the amount of infectious virus in each sample was quantified using foci formation on BSC40 cells. (I) Stimulated or unstimulated T cells were infected with recombinant vMyx-GFP/TrFP at an MOI of 10. Expression of GFP (expressed at both early and late times postinfection) and TrFP (expressed only at late stages of virus infection) was determined 72 hours after infection using fluorescence microscopy. FBS, fetal bovine serum; PBS, phosphate-buffered saline.