Deep sequencing after alcelaphine gammaherpesvirus 1 infection reveals the nature of CD8+ T cell expansion and identify an essential viral protein for fatal bovine malignant catarrhal fever [ATAC-seq]

Alcelaphine gammaherpesvirus 1 (AlHV-1) is a member of the Gammaherpesvirinae subfamily and establishes asymptomatic latent infection in its natural host species, the wildebeest. Cross-species transmission to various ruminant species including cattle can occur, resulting in the induction of malignant catarrhal fever (MCF), a deadly peripheral T cell lymphoproliferative disease. Here, we experimentally infected calves to confirm that AlHV-1 latency-associated gene expression is essential for persistent infection of CD8+ T cells and MCF development. Then, deep sequencing of the T cell receptor repertoire revealed an oligoclonal expansion of peripheral CD8+ T cells during bovine MCF, associated with transcriptomic and epigenetic changes identified by (sc)RNA-seq and ATAC-seq analyses which indicated a mixed effector/memory and exhaustion phenotype of infected cells in vivo. Analysis of the viral genome transcription identified viral genomic regions being expressed in infected CD8+ T cells, such as the region predicted to encode the gene A10. A10 encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. We could demonstrate that impaired expression of A10 did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF in the rabbit experimental model, and we showed that A10 is phosphorylated in T lymphocytes in vitro and affects T cell signaling. Finally, while AlHV-1 viruses expressing mutated forms of A10 devoid of ITAM and/or SH3 motifs could induce MCF, an A10 knock-in viral mutant unable to phosphorylate tyrosine residues resulted in the absence of MCF development. Overall, we identified AlHV-1-induced phenotypic changes in CD8+ T cells during MCF and demonstrated that A10 expression in infected CD8+ T lymphocytes results in the dysregulation of T cell signaling and MCF. Bovine PBMC were isolated from veinous blood collected from the jugular vein. PBMCs were subjected to a microbead-based negative selection protocol to isolate CD8 T cells.