Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry

English version

Autoři: Remigiusz A. Serwa ^aff001; Eiki Sekine ^aff002; Jonathan Brown ^aff002; Su Hui Catherine Teo ^aff002; Edward W. Tate ^aff001; Peter O’Hare ^aff002
Působiště autorů: Department of Chemistry, Molecular Sciences Research Hub, White City Campus, London, United Kingdom ^aff001; Section of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom ^aff002
Vyšlo v časopise: Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry. PLoS Pathog 15(10): e32767. doi:10.1371/journal.ppat.1007956
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1007956

Souhrn

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSV^AHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSV^AHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSV^AHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we revealing novel aspects of entry pathways using HSV^AHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSV^AHA also enabled the discrimination or primary entering from secondary assembling, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, the work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.

Klíčová slova:

Amino acid analysis – Immunofluorescence – Ligation assay – Virions – Capsids – Methionine – Structural proteins – Viral packaging

Zdroje

1. Beatty KE, Liu JC, Xie F, Dieterich DC, Schuman EM, Wang Q, et al. Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew Chem Int Ed Engl. 2006;45(44):7364–7. Epub 2006/10/13. doi: 10.1002/anie.200602114 17036290.

2. Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat Protoc. 2007;2(3):532–40. Epub 2007/04/05. doi: 10.1038/nprot.2007.52 17406607.

3. Kolb HC, Finn MG, Sharpless KB. Click chemistry: Diverse chemical function from a few good reactions. Angewandte Chemie-International Edition. 2001;40(11):2004–+. doi: 10.1002/1521-3773(20010601)40:11<2004::aid-anie2004>3.3.co;2-x ISI:000169168100001. 11433435

4. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angewandte Chemie-International Edition. 2002;41(14):2596–+. doi: 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4 ISI:000176972000038. 12203546

5. tom Dieck S, Muller A, Nehring A, Hinz FI, Bartnik A, Schuman EM, et al. Metabolic labelling with noncanonical amino acids and visualisation by Chemoselective fluorescent tagging. Curr Protoc Cell Biol. 2012;Unit 7.11:1–37. doi: 10.1002/0471143030.cb0711s56 22968844

6. Hinz FI, Dieterich DC, Tirrell DA, Schuman EM. Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish. Acs Chemical Neuroscience. 2012;3(1):40–9. doi: 10.1021/cn2000876 ISI:000299353800007. 22347535

7. Tirrell DA. Cell-selective metabolic labeling of proteins. Abstracts of Papers of the American Chemical Society. 2010;239. ISI:000208189300599.

8. Dieterich DC, Hodas JJL, Gouzer G, Shadrin IY, Ngo JT, Triller A, et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience. 2010;13(7):897–U149. doi: 10.1038/nn.2580 ISI:000279173900024. 20543841

9. Beatty KE, Fisk JD, Smart BP, Lu YY, Szychowski J, Hangauer MJ, et al. Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide-Alkyne Cycloaddition. Chembiochem. 2010;11(15):2092–5. doi: 10.1002/cbic.201000419 ISI:000284050000005. 20836119

10. McClatchy DB, Ma Y, Liu C, Stein BD, Martinez-Bartolome S, Vasquez D, et al. Pulsed Azidohomoalanine Labeling in Mammals (PALM) Detects Changes in Liver-Specific LKB1 Knockout Mice. J Proteome Res. 2015;14(11):4815–22. Epub 2015/10/08. doi: 10.1021/acs.jproteome.5b00653 26445171; PubMed Central PMCID: PMC4642245.

11. Sekine E, Schmidt N, Gaboriau D, O'Hare P. Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy. PLoS Pathog. 2017;13(11):e1006721. doi: 10.1371/journal.ppat.1006721 29121649; PubMed Central PMCID: PMC5697887.

12. Su Hui Teo C, Serwa RA, O'Hare P. Spatial and Temporal Resolution of Global Protein Synthesis during HSV Infection Using Bioorthogonal Precursors and Click Chemistry. PLoS Pathog. 2016;12(10):e1005927. doi: 10.1371/journal.ppat.1005927 27706239; PubMed Central PMCID: PMC5051704.

13. Serwa RA, Abaitua F, Krause E, Tate EW, O'Hare P. Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics. Cell Chem Biol. 2015;22(8):1008–17. Epub 2015/08/11. doi: 10.1016/j.chembiol.2015.06.024 26256475; PubMed Central PMCID: PMC4543063.

14. Schmidt N, Hennig T, Serwa RA, Marchetti M, O'Hare P. Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission. J Virol. 2015;89(21):11107–15. Epub 2015/08/28. doi: 10.1128/JVI.01950-15 26311877; PubMed Central PMCID: PMC4621119.

15. Ouyang T, Liu X, Ouyang H, Ren L. Recent trends in click chemistry as a promising technology for virus-related research. Virus Res. 2018;256:21–8. doi: 10.1016/j.virusres.2018.08.003 30081058.

16. Sakin V, Paci G, Lemke EA, Muller B. Labeling of virus components for advanced, quantitative imaging analyses. FEBS Lett. 2016;590(13):1896–914. doi: 10.1002/1873-3468.12131 26987299.

17. Plass T, Milles S, Koehler C, Szymanski J, Mueller R, Wiessler M, et al. Amino acids for Diels-Alder reactions in living cells. Angew Chem Int Ed Engl. 2012;51(17):4166–70. doi: 10.1002/anie.201108231 22473599.

18. Hao J, Huang LL, Zhang R, Wang HZ, Xie HY. A mild and reliable method to label enveloped virus with quantum dots by copper-free click chemistry. Anal Chem. 2012;84(19):8364–70. doi: 10.1021/ac301918t 22946933.

19. Banerjee PS, Ostapchuk P, Hearing P, Carrico IS. Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors. J Virol. 2011;85(15):7546–54. Epub 2011/05/27. doi: 10.1128/JVI.00118-11 21613404; PubMed Central PMCID: PMC3147895.

20. Oroskar AA, Read GS. Control of mRNA stability by the virion host shutoff function of herpes simplex virus. J Virol. 1989;63(5):1897–906. 2539493

21. Smiley JR. Herpes simplex virus virion host shutoff protein: immune evasion mediated by a viral RNase? J Virol. 2004;78(3):1063–8. doi: 10.1128/JVI.78.3.1063-1068.2004 14722261; PubMed Central PMCID: PMC321390.

22. Broncel M, Serwa RA, Ciepla P, Krause E, Dallman MJ, Magee AI, et al. Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development. Angewandte Chemie-International Edition. 2015. doi: 10.1002/anie.201500342 25807930

23. Dieterich DC, Link AJ, Tirrell DA, Graumann J, Schuman EM. Identification of newly synthesized proteins using bioorthogonal noncanonical amino acid tagging (BONCAT). Molecular & Cellular Proteomics. 2007;6(8):20–. ISI:000253299000017.

24. Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nature Protocols. 2007;2(3):532–40. doi: 10.1038/nprot.2007.52 ISI:000253138200010. 17406607

25. Bagert JD, Xie YJ, Sweredoski MJ, Qi Y, Hess S, Schuman EM, et al. Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics. 2014;13(5):1352–8. doi: 10.1074/mcp.M113.031914 24563536; PubMed Central PMCID: PMC4014290.

26. Bohannon KP, Sollars PJ, Pickard GE, Smith GA. Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection. J Gen Virol. 2012;93(Pt 1):124–9. Epub 2011/10/07. doi: 10.1099/vir.0.036145-0 21976610.

27. El Bilali N, Duron J, Gingras D, Lippe R. Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles. J Virol. 2017;91(10). doi: 10.1128/JVI.00320-17 28275191; PubMed Central PMCID: PMC5411592.

28. McLauchlan J, Rixon FJ. Characterization of enveloped tegument structures (L particles) produced by alphaherpesviruses: integrity of the tegument does not depend on the presence of capsid or envelope. J Gen Virol. 1992;73(Pt 2):269–76.

29. Szilagyi JF, Cunningham C. Identification and characterization of a novel non-infectious herpes simplex virus-related particle. J Gen Virol. 1991;72(Pt 3):661–8.

30. Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM. Principles of Virology. Washington DC: ASM Press; 2009.

31. Sodeik B, Ebersold MW, Helenius A. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol. 1997;136:1007–21. doi: 10.1083/jcb.136.5.1007 9060466

32. Whittaker GR, Kann M, Helenius A. Viral entry into the nucleus. Annu Rev Cell Dev Biol. 2000;16:627–51. Epub 2000/10/14. doi: 10.1146/annurev.cellbio.16.1.627 11031249.

33. Nicola AV, Aguilar HC, Mercer J, Ryckman B, Wiethoff CM. Virus entry by endocytosis. Adv Virol. 2013;2013:469538. doi: 10.1155/2013/469538 23710180; PubMed Central PMCID: PMC3654661.

34. Su Hui Teo C, O'Hare P. A Bimodal Switch in Global Protein Translation Coupled to eIF4H Relocalisation during Advancing Cell-Cell Transmission of Herpes Simplex Virus PLoS Pathog. 2018;14(7): e1007196 2018.

35. Rixon F. Stucture and assembly of herpesviruses. Seminars in Virology. 1993;4(3):135–44.

36. Rahn E, Petermann P, Hsu MJ, Rixon FJ, Knebel-Morsdorf D. Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent. PLoS One. 2011;6(10):e25464. doi: 10.1371/journal.pone.0025464 22022400; PubMed Central PMCID: PMC3192061.

37. Meckes DG Jr., Raab-Traub NMicrovesicles and viral infection. J Virol. 2011;85(24):12844–54. Epub 2011/10/07. doi: 10.1128/JVI.05853-11 21976651; PubMed Central PMCID: PMC3233125.

38. Oum YH, Carrico IS. Altering adenoviral tropism via click modification with ErbB specific ligands. Bioconjug Chem. 2012;23(7):1370–6. Epub 2012/06/12. doi: 10.1021/bc200477z 22681483.

39. Chu Y, Oum YH, Carrico IS. Surface modification via strain-promoted click reaction facilitates targeted lentiviral transduction. Virology. 2016;487:95–103. Epub 2015/10/27. doi: 10.1016/j.virol.2015.09.009 26499046.

40. Heal WP, Wright MH, Thinon E, Tate EW. Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry. Nature Protocols. 2012;7(1):105–17. doi: 10.1038/nprot.2011.425 ISI:000299108900010. 22193303

41. Freedman D, Diaconis P. On the Histogram as a Density Estimator: LZ Theory. Zeitschrgt her Wahrsheinlichkeitstheorie und Verwandte Gebeite. 1981;57:453–76.

42. Wand MP. Data-Based Choice of Histogram Bin Width. The American Statistician. 1997;51(1):59–64.

43. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology. 2008;26(12):1367–72. doi: 10.1038/nbt.1511 ISI:000261591300022. 19029910

44. Zhang J, Xin L, Shan B, Chen W, Xie M, Yuen D, et al. PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Mol Cell Proteomics. 2012;11(4):M111 010587. doi: 10.1074/mcp.M111.010587 22186715; PubMed Central PMCID: PMC3322562.

45. Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, et al. UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 2004;32(Database issue):D115–9. doi: 10.1093/nar/gkh131 14681372; PubMed Central PMCID: PMC308865.