In cells infected with herpesviruses, two capsid-associated, or inner tegument, proteins, UL37 and UL36, control cytosolic trafficking of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure of the N-terminal half of UL37 from pseudorabies virus, an alphaherpesvirus closely related to herpes simplex viruses and varicella-zoster virus. The structure--the first for any alphaherpesvirus inner tegument protein--reveals an elongated molecule of a complex architecture rich in helical bundles. To explore the function of the UL37 N terminus, we used the three-dimensional framework provided by the structure in combination with evolutionary trace analysis to pinpoint several surface-exposed regions of potential functional importance and test their importance using mutagenesis. This approach identified a novel functional region important for cell-cell spread. These results suggest a novel role for UL37 in intracellular virus trafficking that promotes spread of viral infection, a finding that expands the repertoire of UL37 functions. Supporting this, the N terminus of UL37 shares structural similarity with cellular multisubunit tethering complexes (MTCs), which control vesicular trafficking in eukaryotic cells by tethering transport vesicles to their destination membranes. Our results suggest that UL37 could be the first viral MTC mimic and provide a structural rationale for the importance of UL37 for viral trafficking. We propose that herpesviruses may have co-opted the MTC functionality of UL37 to bring capsids to cytoplasmic budding destinations and further on to cell junctions for spread to nearby cells.
Importance: To move within an infected cell, viruses encode genes for proteins that interact with host trafficking machinery. In cells infected with herpesviruses, two capsid-associated proteins control the cytosolic movement of capsids by as yet poorly understood mechanisms. Here, we report the crystal structure for the N-terminal half of one of these proteins, UL37. Structure-based mutagenesis revealed a novel function for UL37 in virus trafficking to cell junctions for cell-cell spread. The unexpected structural similarity to components of cellular multisubunit tethering complexes, which control vesicular traffic, suggests that UL37 could be the first viral MTC mimic and provides a structural basis for the importance of UL37 for virus trafficking.