Host Cell Neddylation Facilitates Alphaherpesvirus Entry in a Virus-Specific and Cell-Dependent Manner

Herpes simplex virus 1 (HSV-1) commandeers the host cell proteasome at several steps of its replication cycle, including entry. Here we demonstrate that HSV-2, pseudorabies virus (PRV), and bovine herpesvirus 1 (BoHV-1) entry are blocked by bortezomib, a proteasome inhibitor that is an FDA-approved cancer drug. Proteasome-dependent entry of HSV-1 is thought to be ubiquitin-independent. To interrogate further the proteasomal mechanism of entry, we determined the involvement of the ubiquitin-like molecule NEDD8 and the neddylation cascade in alphaherpesvirus entry and infection. MLN4924 is a small-molecule inhibitor of neddylation that binds directly to the NEDD8-activating enzyme. Cell treatment with MLN4924 inhibited plaque formation and infectivity by HSV-1, PRV, and BoHV-1 at noncytotoxic concentrations. Thus, the neddylation pathway is broadly important for alphaherpesvirus infection. However, the neddylation inhibitor had little effect on entry of the veterinary viruses but had a significant inhibitory effect on entry of HSV-1 and HSV-2 into seven different cell types. Washout experiments indicated that MLN4924's effect on viral entry was reversible. A time-of-addition assay suggested that the drug was acting on an early step in the entry process. Small interfering RNA (siRNA) knockdown of NEDD8 significantly inhibited HSV entry. In probing the neddylation-dependent step in entry, we found that MLN4924 dramatically blocked endocytic uptake of HSV from the plasma membrane by >90%. In contrast, the rate of HSV entry into cells that support direct fusion of HSV with the cell surface was unaffected by MLN4924. Interestingly, proteasome activity was less important for the endocytic internalization of HSV from the cell surface. The results suggest that the NEDD8 cascade is critical for the internalization step of HSV entry. IMPORTANCE Alphaherpesviruses are ubiquitous pathogens of humans and veterinary species that cause lifelong latent infections and significant morbidity and mortality. Host cell neddylation is important for cell homeostasis and for the infection of many viruses, including HSV-1, HSV-2, PRV, and BoHV-1. Inhibition of neddylation by a pharmacologic inhibitor or siRNA blocked HSV infection at the entry step. Specifically, the NEDD8 pathway was critically important for HSV-1 internalization from the cell surface by an endocytosis mechanism. The results expand our limited understanding of cellular processes that mediate HSV internalization. To our knowledge, this is the first demonstration of a function for the neddylation cascade in virus entry.