Molecular Architecture of the Vaccinia virus factory - The poxviruses comprise a major virus family of medical, ecological and agricultural importance. Although the key family member, smallpox, was eradicated some 45 years ago the risk of reintroduction or reconstruction remains real. The possibility of (enhanced) smallpox re- appearance at a future time has increased substantially with the recent demonstration of the facile recreation, de novo in the laboratory, of a poxvirus genetically very similar to smallpox. Moreover, the cessation of smallpox vaccination has coincided with the progressively more widespread (global) appearance of human Monkeypox (Mpox) with a minor 2003 outbreak reaching the USA and more recently the 2022 clade IIb global outbreak (99,000+ cases, 0.2% cfr). There have been continuing smaller outbreaks since, including a clade Ib outbreak in Central Africa (2023-2024, 29,000+ cases, 43% cfr with cases reported in the US and Canada). Two FDA-approved anti- orthopoxviral drug are TPOXX (stockpiled in the US and with a relatively low barrier to resistance) and Brincidofovir (with a black box warning). Emergency interventions include Vaccinia Immune Globulin. Additional drugs would be valuable for combinatorial or sequential use. Perhaps the richest source of potential viral drug targets is provided by the protein interfaces forming during virion morphogenesis in which the relative complexity of the vaccinia virion presents a therapeutic Achilles heel. This may be an under-exploited environment for rational drug design. Although the ultrastructural transformations occurring during Vaccinia virion morphogenesis are relatively well understood, protein interactions at the molecular level are not. The P.I. has achieved a biochemical affinity purification of the Vaccinia virus nucleus-factory (NF) complex, that now can be characterized at the molecular level. In Aim 1 of this proposal, the P.I will characterize its quantitative proteome to define the proteins present and their relative amounts. In Aim 2, via protein-protein chemical crosslinking mass spectrometry (XLMS), the P.I. will aim to identify protein-protein interfaces in the NF-complex while distinguishing proteins present in the factory from those in the associated nucleus. The P.I. already has successfully applied an XLMS approach to discover domain-level protein interfaces in situ within the vaccinia virion. In Aim 3, the P.I. will attempt to associate specific protein interfaces with specific morphogenic forms in the virus’s developmental pathway, by applying XLMS to the factories of mutant viruses, with repressible genes, that cease morphogenesis while yielding relatively homogeneous populations of morphogenic intermediates.
