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SUMMARY Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe poxvirus infections, but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal Abs from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.

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The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions. Viruses belonging to several different families utilize or modulate the system for their advantage. Here we showed that the proteasome inhibitors MG132 and epoxomicin blocked a postentry step in vaccinia virus (VACV) replication. When proteasome inhibitors were added after virus attachment, early gene expression was prolonged and the expression of intermediate and late genes was almost undetectable. By varying the time of the removal and addition of MG132, the adverse effect of the proteasome inhibitors was narrowly focused on events occurring 2 to 4 h after infection, the time of the onset of viral DNA synthesis. Further analyses confirmed that genome replication was inhibited by both MG132 and epoxomicin, which would account for the effect on intermediate and late gene expression. The virus-induced replication of a transfected plasmid was also inhibited, indicating that the block was not at the step of viral DNA uncoating. UBEI-41, an inhibitor of the ubiquitin-activating enzyme E1, also prevented late gene expression, supporting the role of the ubiquitin-proteasome system in VACV replication. Neither the overexpression of ubiquitin nor the addition of an autophagy inhibitor was able to counter the inhibitory effects of MG132. Further studies of the role of the ubiquitin-proteasome system for VACV replication may provide new insights into virus-host interactions and suggest potential antipoxviral drugs.The ubiquitin-proteasome system has a central role in the degradation of intracellular proteins and regulates a variety of functions (22). Proteins to be degraded are modified by the addition of multiple copies of the 76-amino-acid ubiquitin through the sequential activities of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) (4, 12). The degradation is mediated by the 26S proteasome, a large multiprotein complex containing trypsin-, chymotrypsin-, and postglutamyl peptidyl hydrolytic-like protease activities. In addition, ubiquitylation has nondegradative roles in DNA repair, transcriptional regulation, signal transduction, endocytosis, and intracellular trafficking (48). Viruses belonging to several families utilize or modulate the ubiquitin-proteasome system (2, 13). The inhibition of proteasomal degradation prevents the entry of influenza virus (23) and mouse hepatitis virus (54); the early postentry steps of minute virus of mice (44) and herpes simplex virus (7); and the genome replication or expression of human coxsackie 3B virus (27), adenovirus (5), cytomegalovirus (20), infectious bursal disease virus (26), and vesicular stomatitis virus (40). In some cases the effects may be secondary to the activation of a cellular stress response and signaling pathway (24, 40, 52). Proteasomal inhibitors have an indirect effect on retroviruses and rhabdoviruses by depleting free ubiquitin needed to modify proteins for budding (16).Vaccinia virus (VACV), the repre...