Cytoplasmic factories, virus assembly, and DNA replication kinetics collectively constrain the formation of poxvirus recombinants

Autoři: Quinten Kieser aff001;  Ryan S. Noyce aff001;  Mira Shenouda aff001;  Y.-C. James Lin aff001;  David H. Evans aff001
Působiště autorů: The Dept. of Medical Microbiology & Immunology, University of Alberta, Edmonton, Alberta, Canada aff001;  Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0228028


Poxviruses replicate in cytoplasmic structures called factories and each factory begins as a single infecting particle. Sixty-years ago Cairns predicted that this might have effects on vaccinia virus (VACV) recombination because the factories would have to collide and mix their contents to permit recombination. We've since shown that factories collide irregularly and that even then the viroplasm mixes poorly. We’ve also observed that while intragenic recombination occurs frequently early in infection, intergenic recombination is less efficient and happens late in infection. Something inhibits factory fusion and viroplasm mixing but what is unclear. To study this, we’ve used optical and electron microscopy to track factory movement in co-infected cells and correlate these observations with virus development and recombinant formation. While the technical complexity of the experiments limited the number of cells that are amenable to extensive statistical analysis, these studies do show that intergenic recombination coincides with virion assembly and when VACV replication has declined to ≤10% of earlier levels. Along the boundaries between colliding factories, one sees ER membrane remnants and other cell constituents like mitochondria. These collisions don't always cause factory fusion, but when factories do fuse, they still entrain cell constituents like mitochondria and ER-wrapped microtubules. However, these materials wouldn’t seem to pose much of a further barrier to DNA mixing and so it’s likely that the viroplasm also presents an omnipresent impediment to DNA mixing. Late packaging reactions might help to disrupt the viroplasm, but packaging would sequester the DNA just as the replication and recombination machinery goes into decline and further reduce recombinant yields. Many factors thus appear to conspire to limit recombination between co-infecting poxviruses.

Klíčová slova:

DNA recombination – DNA replication – Mitochondria – Recombinant proteins – Recombination reactions – Transmission electron microscopy – Viral replication – Viral structure


1. Yang Z, Cao S, Martens CA, Porcella SF, Xie Z, Ma M, et al. Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling. J Virol. 2015;89(13):6874–86. doi: 10.1128/JVI.00528-15 25903347

2. Yang Z, Reynolds SE, Martens CA, Bruno DP, Porcella SF, Moss B. Expression profiling of the intermediate and late stages of poxvirus replication. J Virol. 2011;85(19):9899–908. doi: 10.1128/JVI.05446-11 21795349

3. Teale A, Campbell S, Van Buuren N, Magee WC, Watmough K, Couturier B, et al. Orthopoxviruses require a functional ubiquitin-proteasome system for productive replication. J Virol. 2009;83(5):2099–108. doi: 10.1128/JVI.01753-08 19109393

4. Mercer J, Snijder B, Sacher R, Burkard C, Bleck CK, Stahlberg H, et al. RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection. Cell Rep. 2012;2(4):1036–47. doi: 10.1016/j.celrep.2012.09.003 23084750

5. Liu B, Panda D, Mendez-Rios JD, Ganesan S, Wyatt LS, Moss B. Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles. J Virol. 2018;92(7).

6. Cairns J. The initiation of vaccinia infection. Virology. 1960;11:603–23. doi: 10.1016/0042-6822(60)90103-3 13806834

7. Dales S, Siminovitch L. The development of vaccinia virus in Earle's L strain cells as examined by electron microscopy. J Biophys Biochem Cytol. 1961;10:475–503. doi: 10.1083/jcb.10.4.475 13719413

8. Tolonen N, Doglio L, Schleich S, Krijnse Locker J. Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei. Mol Biol Cell. 2001;12(7):2031–46. doi: 10.1091/mbc.12.7.2031 11452001

9. Katsafanas GC, Moss B. Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe. 2007;2(4):221–8. doi: 10.1016/j.chom.2007.08.005 18005740

10. Weisberg AS, Maruri-Avidal L, Bisht H, Hansen BT, Schwartz CL, Fischer ER, et al. Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants. Proc Natl Acad Sci U S A. 2017;114(51):E11001–E9. doi: 10.1073/pnas.1716255114 29203656

11. Harrison K, Haga IR, Pechenick Jowers T, Jasim S, Cintrat JC, Gillet D, et al. Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis. J Virol. 2016;90(22):10120–32. doi: 10.1128/JVI.01464-16 27581988

12. Czarnecki MW, Traktman P. The vaccinia virus DNA polymerase and its processivity factor. Virus Res. 2017;234:193–206. doi: 10.1016/j.virusres.2017.01.027 28159613

13. Rochester SC, Traktman P. Characterization of the single-stranded DNA binding protein encoded by the vaccinia virus I3 gene. J Virol. 1998;72(4):2917–26. 9525612

14. Welsch S, Doglio L, Schleich S, Krijnse Locker J. The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA. J Virol. 2003;77(10):6014–28. doi: 10.1128/JVI.77.10.6014-6028.2003 12719593

15. Magee WC, Shahhosseini S, Lin YC, Suresh MR, Evans DH. Production and characterization of antibodies against vaccinia virus DNA polymerase. J Virol Methods. 2009;161(1):44–51. doi: 10.1016/j.jviromet.2009.05.012 19477201

16. DeLange AM, McFadden G. Sequence-nonspecific replication of transfected plasmid DNA in poxvirus- infected cells. Proc Natl Acad Sci U S A. 1986;83(3):614–8. doi: 10.1073/pnas.83.3.614 3003742

17. De Silva FS, Moss B. Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors. Virol J. 2005;2:23. doi: 10.1186/1743-422X-2-23 15784143

18. Parks RJ, Evans DH. Effect of marker distance and orientation on recombinant formation in poxvirus-infected cells. J Virol. 1991;65(3):1263–72. 1847453

19. Evans DH, Stuart D, McFadden G. High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells. J Virol. 1988;62(2):367–75. 2826801

20. Willer DO, Yao XD, Mann MJ, Evans DH. In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase. Virology. 2000;278(2):562–9. doi: 10.1006/viro.2000.0686 11118378

21. Gammon DB, Evans DH. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination. J Virol. 2009;83(9):4236–50. doi: 10.1128/JVI.02255-08 19224992

22. Yao XD, Evans DH. Characterization of the recombinant joints formed by single-strand annealing reactions in vaccinia virus-infected cells. Virology. 2003;308(1):147–56. doi: 10.1016/s0042-6822(02)00089-2 12706098

23. Wright WD, Shah SS, Heyer WD. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 2018;293(27):10524–35. doi: 10.1074/jbc.TM118.000372 29599286

24. Esposito JJ, Sammons SA, Frace AM, Osborne JD, Olsen-Rasmussen M, Zhang M, et al. Genome sequence diversity and clues to the evolution of variola (smallpox) virus. Science. 2006;313(5788):807–12. doi: 10.1126/science.1125134 16873609

25. Elde NC, Child SJ, Eickbush MT, Kitzman JO, Rogers KS, Shendure J, et al. Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses. Cell. 2012;150(4):831–41. doi: 10.1016/j.cell.2012.05.049 22901812

26. Qin L, Evans DH. Genome scale patterns of recombination between coinfecting vaccinia viruses. J Virol. 2014;88(10):5277–86. doi: 10.1128/JVI.00022-14 24574414

27. Paszkowski P, Noyce RS, Evans DH. Live-Cell Imaging of Vaccinia Virus Recombination. PLoS Pathog. 2016;12(8):e1005824. doi: 10.1371/journal.ppat.1005824 27525721

28. Lin YC, Evans DH. Vaccinia virus particles mix inefficiently, and in a way that would restrict viral recombination, in coinfected cells. J Virol. 2010;84(5):2432–43. doi: 10.1128/JVI.01998-09 20032178

29. Moss B. Poxvirus membrane biogenesis. Virology. 2015;479–480:619–26.

30. Roberts KL, Smith GL. Vaccinia virus morphogenesis and dissemination. Trends Microbiol. 2008;16(10):472–9. doi: 10.1016/j.tim.2008.07.009 18789694

31. Cepeda V, Esteban M. Novel insights on the progression of intermediate viral forms in the morphogenesis of vaccinia virus. Virus Res. 2014;183:23–9. doi: 10.1016/j.virusres.2014.01.016 24468494

32. McDonald WF, Crozel-Goudot V, Traktman P. Transient expression of the vaccinia virus DNA polymerase is an intrinsic feature of the early phase of infection and is unlinked to DNA replication and late gene expression. J Virol. 1992;66(1):534–47. 1727498

33. Fliegel L, Burns K, MacLennan DH, Reithmeier RA, Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1989;264(36):21522–8. 2600080

34. Kobiler O, Weitzman MD. Herpes simplex virus replication compartments: From naked release to recombining together. PLoS Pathog. 2019;15(6):e1007714. doi: 10.1371/journal.ppat.1007714 31158262

35. Willer DO, Mann MJ, Zhang W, Evans DH. Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions. Virology. 1999;257(2):511–23. doi: 10.1006/viro.1999.9705 10329561

36. Lee C, Ferguson M, Chen LB. Construction of the endoplasmic reticulum. J Cell Biol. 1989;109(5):2045–55. doi: 10.1083/jcb.109.5.2045 2478561

37. Hollinshead M, Rodger G, Van Eijl H, Law M, Hollinshead R, Vaux DJ, et al. Vaccinia virus utilizes microtubules for movement to the cell surface. J Cell Biol. 2001;154(2):389–402. doi: 10.1083/jcb.200104124 11470826

38. Schepis A, Schramm B, de Haan CA, Locker JK. Vaccinia virus-induced microtubule-dependent cellular rearrangements. Traffic. 2006;7(3):308–23. doi: 10.1111/j.1600-0854.2005.00381.x 16497225

39. Kieser Q, Paszkowski P, Lin J, Evans D, Noyce R. Visualizing Poxvirus Replication and Recombination Using Live-Cell Imaging. Methods Mol Biol. 2019;2023:221–35. doi: 10.1007/978-1-4939-9593-6_14 31240681

40. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. doi: 10.1038/nmeth.2019 22743772

41. Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. doi: 10.1006/jsbi.1996.0013 8742726

Článek vyšel v časopise


2020 Číslo 1