Moss, B. Understanding the biology of monkeypox virus to prevent future outbreaks. Nat. Microbiol. 9, 1408–1416 (2024).
Oliveira Silva, N. I., Silva de Oliveira, J., Kroon, E. G., de Souza Trindade, G. & Drumond, B. P. Here, there, and everywhere: the wide host range and geographic distribution of zoonotic orthopoxviruses. Viruses 13, 43 (2020).
Condit, R. C., Moussatche, N. & Traktman, P. In a nutshell: structure and assembly of the vaccinia virion. Adv. Virus Res. 66, 31–124 (2006).
Hernandez-Gonzalez, M., Calcraft, T., Nans, A., Rosenthal, P. B. & Way, M. A succession of 2 viral lattices drives vaccinia virus assembly. PLoS Biol. 21, e3002005 (2023).
Boyd, O., Strahl, A. L., Rodeffer, C., Condit, R. C. & Moussatche, N. Temperature-sensitive mutant in the vaccinia virus E6 protein produce virions that are transcriptionally inactive. Virology 399, 221–230 (2010).
Kato, S. E. M., Condit, R. C. & Moussatché, N. The vaccinia virus E8R gene product is required for formation of transcriptionally active virions. Virology 367, 398–412 (2007).
Resch, W. & Moss, B. The conserved poxvirus L3 virion protein is required for transcription of vaccinia virus early genes. J. Virol. 79, 14719–14729 (2005).
Resch, W., Weisberg, A. S. & Moss, B. Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis. Virology 386, 478–485 (2009).
Boyd, O., Turner, P. C., Moyer, R. W., Condit, R. C. & Moussatche, N. The E6 protein from vaccinia virus is required for the formation of immature virions. Virology 399, 201–211 (2010).
Condit, R. C. & Moussatche, N. The vaccinia virus E6 protein influences virion protein localization during virus assembly. Virology 482, 147–156 (2015).
Zhao, Y. et al. TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6. Nature 620, 873–880 (2023).
Kilcher, S. et al. siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor. Cell Host Microbe 15, 103–112 (2014).
Ngo, T., Mirzakhanyan, Y., Moussatche, N. & Gershon, P. D. Protein primary structure of the vaccinia virion at increased resolution. J. Virol. 90, 9905–9919 (2016).
Resch, W., Hixson, K. K., Moore, R. J., Lipton, M. S. & Moss, B. Protein composition of the vaccinia virus mature virion. Virology 358, 233–247 (2007).
Dubochet, J., Adrian, M., Richter, K., Garces, J. & Wittek, R. Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy. J. Virol. 68, 1935–1941 (1994).
Cyrklaff, M. et al. Cryo-electron tomography of vaccinia virus. Proc. Natl Acad. Sci. USA 102, 2772–2777 (2005).
Hernandez-Gonzalez, M., Calcraft, T., Nans, A., Rosenthal, P. B. & Way, M. Palisade structure in intact vaccinia virions. mBio 15, e0313423 (2024).
Moss, B. Membrane fusion during poxvirus entry. Semin. Cell Dev. Biol. 60, 89–96 (2016).
Moss, B. in Fields Virology Vol. 2 (eds Knipe, D. M. et al.) 2129–2159 (Lippincott Williams & Wilkins, 2013).
Kates, J. & Beeson, J. Ribonucleic acid synthesis in vaccinia virus. I. The mechanism of synthesis and release of RNA in vaccinia cores. J. Mol. Biol. 50, 1–18 (1970).
Yang, Z. et al. Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling. J. Virol. 89, 6874–6886 (2015).
Greseth, M. D. & Traktman, P. The life cycle of the vaccinia virus genome. Annu. Rev. Virol. 9, 239–259 (2022).
Datler, J. et al. Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores. Nat. Struct. Mol. Biol. 31, 1114–1123 (2024).
Hong, Y. et al. Molecular architecture of monkeypox mature virus. Cell Discov. 10, 108 (2024).
Liu, J. et al. The palisade layer of the poxvirus core is composed of flexible A10 trimers. Nat. Struct. Mol. Biol. 31, 1105–1113 (2024).
Moussatche, N. & Condit, R. C. Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization. Virology 475, 204–218 (2015).
Dedeo, C. L., Cingolani, G. & Teschke, C. M. Portal protein: the orchestrator of capsid assembly for the dsDNA tailed bacteriophages and herpesviruses. Annu. Rev. Virol. 6, 141–160 (2019).
Sun, M. et al. Ring-stacked capsids of white spot syndrome virus and structural transitions with genome ejection. Sci. Adv. 9, eadd2796 (2023).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://doi.org/10.1101/2021.10.04.463034 (2022).
Mirzakhanyan, Y. & Gershon, P. The vaccinia virion: filling the gap between atomic and ultrastructure. PLoS Pathog. 15, e1007508 (2019).
Bidgood, S. R. et al. Poxviruses package viral redox proteins in lateral bodies and modulate the host oxidative response. PLoS Pathog. 18, e1010614 (2022).
Tolonen, N., Doglio, L., Schleich, S. & Krijnse Locker, J. Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei. Mol. Biol. Cell 12, 2031–2046 (2001).
Upton, C., Slack, S., Hunter, A. L., Ehlers, A. & Roper, R. L. Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome. J. Virol. 77, 7590–7600 (2003).
Mallardo, M., Schleich, S. & Krijnse Locker, J. Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures. Mol. Biol. Cell 12, 3875–3891 (2001).
Grossegesse, M., Doellinger, J., Haldemann, B., Schaade, L. & Nitsche, A. A next-generation sequencing approach uncovers viral transcripts incorporated in poxvirus virions. Viruses 9, 296 (2017).
Ding, K. et al. In situ structures of rotavirus polymerase in action and mechanism of mRNA transcription and release. Nat. Commun. 10, 2216 (2019).
Zhang, X. et al. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus. Nature 527, 531–534 (2015).
Li, Y., Zhu, J., Guo, Y. & Yan, R. Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus. Nat. Struct. Mol. Biol. 31, 68–81 (2024).
Hutin, S. et al. The vaccinia virus DNA helicase structure from combined single-particle cryo-electron microscopy and AlphaFold2 prediction. Viruses 14, 2206 (2022).
Shahid, T. et al. Structural dynamics of DNA unwinding by a replicative helicase. Nature 641, 240–249 (2025).
Xu, Y. et al. Essential and multifunctional mpox virus E5 helicase-primase in double and single hexamer. Sci. Adv. 10, eadl1150 (2024).
Cheng, Y. et al. Assembly and breakage of head-to-head double hexamer reveals mpox virus E5-catalyzed DNA unwinding initiation. Nat. Commun. 16, 5176 (2025).
Szajner, P., Jaffe, H., Weisberg, A. S. & Moss, B. A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions. Virology 330, 447–459 (2004).
Locker, J. K. & Griffiths, G. An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins. J. Cell Biol. 144, 267–279 (1999).
Senkevich, T. G., White, C. L., Koonin, E. V. & Moss, B. Complete pathway for protein disulfide bond formation encoded by poxviruses. Proc. Natl Acad. Sci. USA 99, 6667–6672 (2002).
Schmidt, F. I. et al. Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies. Cell Rep. 4, 464–476 (2013).
Cyrklaff, M. et al. Whole cell cryo-electron tomography reveals distinct disassembly intermediates of vaccinia virus. PLoS ONE 2, e420 (2007).
Skorupka, K. A. et al. Hierarchical assembly governs TRIM5α recognition of HIV-1 and retroviral capsids. Sci. Adv. 5, eaaw3631 (2019).
Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem. J. 478, 4169–4185 (2021).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113 (2017).
Bepler, T., Kelley, K., Noble, A. J. & Berger, B. Topaz-Denoise: general deep denoising models for cryoEM and cryoET. Nat. Commun. 11, 5208 (2020).
Zheng, S. et al. AreTomo: an integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction. J. Struct. Biol. X 6, 100068 (2022).
Chaillet, M. L. et al. Extensive angular sampling enables the sensitive localization of macromolecules in electron tomograms. Int. J. Mol. Sci. 24, 13375 (2023).
Chen, M. et al. A complete data processing workflow for cryo-ET and subtomogram averaging. Nat. Methods 16, 1161–1168 (2019).
Ermel, U. H., Arghittu, S. M. & Frangakis, A. S. ArtiaX: an electron tomography toolbox for the interactive handling of sub-tomograms in UCSF ChimeraX. Protein Sci. 31, e4472 (2022).
Zivanov, J. et al. A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0. eLife 11, e83724 (2022).
Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).
Aiyer, S., Zhang, C., Baldwin, P. R. & Lyumkis, D. Evaluating local and directional resolution of cryo-EM density maps. Methods Mol. Biol. 2215, 161–187 (2021).
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
Gao, J. et al. DomainFit: identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models. Structure 32, 1248–1259 (2024).
Lu, Y., Chen, G., Sun, F., Zhu, Y. & Zhang, Z. De novo identification of protein domains in cryo-electron tomography maps from AlphaFold2 models. Preprint at bioRxiv https://doi.org/10.1101/2024.11.21.623534 (2025).
Jensen, R. K. et al. In-cell discovery and characterization of a non-canonical bacterial protein translocation-folding complex. Preprint at bioRxiv https://doi.org/10.1101/2025.04.25.650208 (2025).
Oeffner, R. D. et al. Putting AlphaFold models to work with phenix.process_predicted_model and ISOLDE. Acta Crystallogr. D 78, 1303–1314 (2022).
GraphPad Software. GraphPad Prism version 10.4.1 for MacOS (2024).
Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. D 74, 519–530 (2018).
Croll, T. I. & Read, R. J. Adaptive Cartesian and torsional restraints for interactive model rebuilding. Acta Crystallogr. D 77, 438–446 (2021).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. 66, 486–501 (2010).
Arnold, M. J. AlphaPickle. Zenodo https://doi.org/10.5281/zenodo.5708709 (2021).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018).
Yamashita, K., Palmer, C. M., Burnley, T. & Murshudov, G. N. Cryo-EM single-particle structure refinement and map calculation using Servalcat. Acta Crystallogr. D 77, 1282–1291 (2021).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Smart, O. S., Neduvelil, J. G., Wang, X., Wallace, B. A. & Sansom, M. S. P. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graphics 14, 354–360 (1996).
Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).

