PROJECT SUMMARY/ABSTRACT Herpesviruses cause a substantial worldwide burden on human health. In response to infections by these and other pathogens, host cells express thousands of interferon-stimulated genes. One such gene encodes the myxovirus resistance protein B (MxB) that potently restricts several herpesviruses, including herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), and cytomegalovirus (CMV). MxB has been undergoing rapid evolution in primates, as reflected in genetic signatures of positive selection, suggesting that MxB has been engaged in arms races with viruses over millions of years. Intriguingly, unlike human MxB, the MxB alleles from several non-human primates do not inhibit HSV-1 replication. In contrast, all tested orthologs retain antiviral activity against CMV. These results suggest that human MxB, but not non-human primate orthologs, including the closely related chimpanzee gene, has evolved to restrict HSV-1. This scenario appears to be a rare example of the host side “winning” during an evolutionary arms race with a virus. To dissect the mechanism MxB utilizes to restrict HSV-1, studies in Aim 1 will define the role of specific residues that are necessary and sufficient to explain differing restrictive phenotypes of human and chimpanzee MxB orthologs. Experiments aiming to identify viral factors that are the targets of MxB, as well as which host co-factors are potentially required, will employ proximity ligation assays coupled with mass spectrometry. Building on the observation that HSV-2 strains vary in their sensitivity to human and chimpanzee MxB, Aim 2 will identify which other primate MxB orthologs restrict which strains of HSV-2, which will help elucidate the evolutionary trajectory of the development of the phenotype. Additionally, because human MxB restricts only a subset of HSV-2 strains, analyses of sequence differences among the strains will aid in the identification of viral determinants of MxB sensitivity. Together, this proposal will reveal the critical factors mediating the arms races between MxB and herpes simplex viruses and will aid in elucidating the anti-herpesvirus mechanism of MxB.