Viruses commandeer host cell 26S proteasome activity to promote viral entry, 
gene expression, replication, assembly, and egress. Proteasomal degradation 
activity is critical for herpes simplex virus (HSV) infection. The proteasome 
inhibitor bortezomib (also known as Velcade and PS-341) is a clinically 
effective antineoplastic drug that is FDA approved for treatment of hematologic 
malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar 
concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and 
acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. 
Bortezomib did not affect attachment of HSV to cells or inactivate the virus 
directly. Bortezomib acted early in HSV infection by perturbing two distinct 
proteasome-dependent steps that occur within the initial hours of infection: the 
transport of incoming viral nucleocapsids to the nucleus and the virus-induced 
disruption of host nuclear domain 10 (ND10) structures. The combination of 
bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV 
infection. Thus, bortezomib is a novel potential therapeutic for HSV with a 
defined mechanism of action.IMPORTANCE Viruses usurp host cell functions to 
advance their replicative agenda. HSV relies on cellular proteasome activity for 
successful infection. Proteasome inhibitors, such as MG132, block HSV infection 
at multiple stages of the infectious cycle. Targeting host cell processes for 
antiviral intervention is an unconventional approach that might limit antiviral 
resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which 
is a clinically effective cancer drug, has the in vitro features of a promising 
anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours 
of infection at nanomolar concentrations that were minimally cytotoxic. The 
mechanism of bortezomib's inhibition of early HSV infection was to halt 
nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense 
complex. Bortezomib and acyclovir acted synergistically to inhibit HSV 
infection. Overall, we present evidence for the repurposing of bortezomib as a 
novel antiherpesviral agent and describe specific mechanisms of action.