Poxviruses and γ2-herpesviruses encode K3-family immune evasion proteins that share structural features with a group of human membrane-associated RING-CH (MARCH) proteins. This study shows that MARCH proteins localize to intracellular membranes and functionally resemble viral K3 proteins by reducing surface expression of specific target proteins. To identify physiological substrates, a quantitative proteomics approach using stable isotope labeling and membrane fractionation was developed. This method revealed distinct sets of host proteins selectively downregulated by viral K5 and human MARCH-VIII. These findings identify MARCH proteins as a novel family of human ubiquitin ligases and establish a powerful strategy for analyzing cellular and viral immune modulators.
