Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Multi-virion infectious units arise from free viral particles in an enveloped virus

Nature Microbiology volume 2, Article number: 17078 (2017) Cite this article

Subjects

Abstract

Many animal viruses are enveloped in a lipid bilayer taken up from cellular membranes. Because viral surface proteins bind to these membranes to initiate infection, we hypothesized that free virions may also be capable of interacting with the envelopes of other virions extracellularly. Here, we demonstrate this hypothesis in the vesicular stomatitis virus (VSV), a prototypic negative-strand RNA virus composed of an internal ribonucleocapsid, a matrix protein and an external envelope1. Using microscopy, dynamic light scattering, differential centrifugation and flow cytometry, we show that free viral particles can spontaneously aggregate into multi-virion infectious units. We also show that, following establishment of these contacts, different viral genetic variants are co-transmitted to the same target cell. Furthermore, virion–virion binding can determine key aspects of viral fitness such as antibody escape. In purified virions, this process is driven by protein–lipid interactions probably involving the VSV surface glycoprotein and phosphatidylserine. Whereas we found that multi-virion complexes occurred unfrequently in standard cell cultures, they were abundant in other fluids such as saliva, a natural VSV shedding route2. Our findings contrast with the commonly accepted perception of virions as passive propagules and show the ability of enveloped viruses to establish collective infectious units, which could in turn facilitate the evolution of virus–virus interactions and of social-like traits3.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Spontaneous aggregation of purified VSV virions.
Figure 2: Functional implications of multi-virion infectious units.
Figure 3: Envelope protein–lipid interactions mediate virion–virion binding in VSV.
Figure 4: VSV virion–virion binding in cell culture media, plasma and saliva.

Similar content being viewed by others

References

  1. Ge, P. et al. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science 327, 689–693 (2010).

    Article  CAS  Google Scholar 

  2. Letchworth, G. J., Rodríguez, L. L. & Del Cbarrera, J. Vesicular stomatitis. Vet. J. 157, 239–260 (1999).

    Article  CAS  Google Scholar 

  3. Sanjuán, R. Collective infectious units in viruses. Trends Microbiol. 25, 402–412 (2017).

    Article  Google Scholar 

  4. Sanjuán, R., Moya, A. & Elena, S. F. The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc. Natl Acad. Sci. USA 101, 8396–8401 (2004).

    Article  Google Scholar 

  5. Kalvodova, L. et al. The lipidomes of vesicular stomatitis virus, Semliki Forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry. J. Virol. 83, 7996–8003 (2009).

    Article  CAS  Google Scholar 

  6. Coil, D. A. & Miller, A. D. Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus. J. Virol. 78, 10920–10926 (2004).

    Article  CAS  Google Scholar 

  7. Carneiro, F. A. et al. Probing the interaction between vesicular stomatitis virus and phosphatidylserine. Eur. Biophys. J. 35, 145–154 (2006).

    Article  CAS  Google Scholar 

  8. Hall, M. P., Burson, K. K. & Huestis, W. H. Interactions of a vesicular stomatitis virus G protein fragment with phosphatidylserine: NMR and fluorescence studies. Biochim. Biophys. Acta 1415, 101–113 (1998).

    Article  CAS  Google Scholar 

  9. MacFarlane, M. G. The biochemistry of bacterial toxins; the enzymic specificity of Clostridium welchii lecithinase. Biochem. J. 42, 587–590 (1948).

    Article  CAS  Google Scholar 

  10. Tait, J. F. & Gibson, D. Phospholipid binding of annexin V: effects of calcium and membrane phosphatidylserine content. Arch. Biochem. Biophys. 298, 187–191 (1992).

    Article  CAS  Google Scholar 

  11. Moerdyk-Schauwecker, M., Hwang, S. I. & Grdzelishvili, V. Z. Cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions. PLoS ONE 9, e104688 (2014).

    Article  Google Scholar 

  12. Finkelshtein, D., Werman, A., Novick, D., Barak, S. & Rubinstein, M. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc. Natl Acad. Sci. USA 110, 7306–7311 (2013).

    Article  CAS  Google Scholar 

  13. Prasad, J. M., Migliorini, M., Galisteo, R. & Strickland, D. K. Generation of a potent low density lipoprotein receptor-related protein 1 (LRP1) antagonist by engineering a stable form of the receptor-associated protein (RAP) D3 domain. J. Biol. Chem. 290, 17262–17268 (2015).

    Article  CAS  Google Scholar 

  14. Simon, K. O., Cardamone, J. J. Jr, Whitaker-Dowling, P. A., Youngner, J. S. & Widnell, C. C. Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus. Virology 177, 375–379 (1990).

    Article  CAS  Google Scholar 

  15. Bald, J. G. & Briggs, G. E. Aggregation of virus particles. Nature 140, 111 (1937).

    Article  Google Scholar 

  16. Galasso, G. J. & Sharp, D. G. Virus particle aggregation and the plaque-forming unit. J. Immunol. 88, 339–347 (1962).

    CAS  PubMed  Google Scholar 

  17. Wallis, C. & Melnick, J. L. Virus aggregation as the cause of the non-neutralizable persistent fraction. J. Virol. 1, 478–488 (1967).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Galasso, G. J. Quantitative studies on the quality, effects of aggregation and thermal inactivation of vesicular stomatitis virus. Arch. Gesamte Virusforsch. 21, 437–446 (1967).

    Article  CAS  Google Scholar 

  19. Floyd, R. Viral aggregation: mixed suspensions of poliovirus and reovirus. Appl. Environ. Microbiol. 38, 980–986 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Combe, M., Garijo, R., Geller, R., Cuevas, J. M. & Sanjuán, R. Single-cell analysis of RNA virus infection identifies multiple genetically diverse viral genomes within single infectious units. Cell Host Microbe 18, 424–432 (2015).

    Article  CAS  Google Scholar 

  21. Aguilera, E. R., Erickson, A. K., Jesudhasan, P. R., Robinson, C. M. & Pfeiffer, J. K. Plaques formed by mutagenized viral populations have elevated coinfection frequencies. mBio 8, e02020–16 (2017).

    Article  CAS  Google Scholar 

  22. Marcus, P. I., Rodríguez, L. L. & Sekellick, M. J. Interferon induction as a quasispecies marker of vesicular stomatitis virus populations. J. Virol. 72, 542–549 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Nunamaker, R. A. et al. Grasshoppers (Orthoptera: Acrididae) could serve as reservoirs and vectors of vesicular stomatitis virus. J. Med. Entomol. 40, 957–963 (2003).

    Article  Google Scholar 

  24. Scherer, C. F. et al. Vesicular stomatitis New Jersey virus (VSNJV) infects keratinocytes and is restricted to lesion sites and local lymph nodes in the bovine, a natural host. Vet. Res. 38, 375–390 (2007).

    Article  Google Scholar 

  25. Smith, P. F. et al. Mechanical transmission of vesicular stomatitis New Jersey virus by Simulium vittatum (Diptera: Simuliidae) to domestic swine (Sus scrofa). J. Med. Entomol. 46, 1537–1540 (2009).

    Article  Google Scholar 

  26. Smith, P. F. et al. Host predilection and transmissibility of vesicular stomatitis New Jersey virus strains in domestic cattle (Bos taurus) and swine (Sus scrofa). BMC Vet. Res. 8, 183–188 (2012).

    Article  Google Scholar 

  27. Libersou, S. et al. Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion. J. Cell Biol. 191, 199–210 (2010).

    Article  CAS  Google Scholar 

  28. Nagata, S., Hanayama, R. & Kawane, K. Autoimmunity and the clearance of dead cells. Cell 140, 619–630 (2010).

    Article  CAS  Google Scholar 

  29. Amara, A. & Mercer, J. Viral apoptotic mimicry. Nat. Rev. Microbiol. 13, 461–469 (2015).

    Article  CAS  Google Scholar 

  30. Chen, Y. H. et al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 160, 619–630 (2015).

    Article  CAS  Google Scholar 

  31. Gutiérrez, S., Michalakis, Y. & Blanc, S. Virus population bottlenecks during within-host progression and host-to-host transmission. Curr. Opin. Virol. 2, 546–555 (2012).

    Article  Google Scholar 

  32. Aaskov, J., Buzacott, K., Thu, H. M., Lowry, K. & Holmes, E. C. Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes. Science 311, 236–238 (2006).

    Article  CAS  Google Scholar 

  33. Stack, J. C., Murcia, P. R., Grenfell, B. T., Wood, J. L. & Holmes, E. C. Inferring the inter-host transmission of influenza A virus using patterns of intra-host genetic variation. Proc. Biol. Sci. 280, 20122173 (2013).

    Article  Google Scholar 

  34. Marriott, A. C. & Dimmock, N. J. Defective interfering viruses and their potential as antiviral agents. Rev. Med. Virol. 20, 51–62 (2010).

    Article  CAS  Google Scholar 

  35. Notton, T., Sardanyes, J., Weinberger, A. D. & Weinberger, L. S. The case for transmissible antivirals to control population-wide infectious disease. Trends Biotechnol. 32, 400–405 (2014).

    Article  CAS  Google Scholar 

  36. Vandepol, S. B., Lefrancois, L. & Holland, J. J. Sequences of the major antibody binding epitopes of the Indiana serotype of vesicular stomatitis virus. Virology 148, 312–325 (1986).

    Article  CAS  Google Scholar 

  37. Whelan, S. P., Ball, L. A., Barr, J. N. & Wertz, G. T. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc. Natl. Acad. Sci. USA 92, 8388–8392 (1995).

    Article  CAS  Google Scholar 

  38. Hassan, P. A., Rana, S. & Verma, G. Making sense of Brownian motion: colloid characterization by dynamic light scattering. Langmuir 31, 3–12 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank D. Vie, A. Flores, R. Garijo and S. Torres for technical assistance, and personnel from La Muntanyeta Coop. V. for providing access to cows. This work was financially supported by grants from the European Research Council (ERC-2011-StG- 281191-VIRMUT and ERC-2016-CoG-724519-Vis-à-Vis) and the Spanish MINECO (BFU2013-41329) to R.S. and by a Ramón y Cajal contract to J.M.C.

Author information

Authors and Affiliations

  1. Institute for Integrative Systems Biology (I2SysBio), Universitat de València, C/Catedrático José Beltrán 2, 46980 Paterna, Valencia, Spain

    José M. Cuevas, María Durán-Moreno & Rafael Sanjuán

  2. Department of Genetics, Universitat de València, C/Dr. Moliner 50, 46100 Burjassot, València, Spain

    José M. Cuevas & Rafael Sanjuán

Authors
  1. José M. Cuevas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Durán-Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rafael Sanjuán
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.D.-M. and J.M.C contributed equally to this work and performed all experiments. R.S. conceived the study, supervised the experiments, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Rafael Sanjuán.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–4, Supplementary Table 2. (PDF 503 kb)

Supplementary Table 1

Flow cytometry data used in co-fluorescence tests. (XLSX 21 kb)

Supplementary Video 1

NTA of purified VSV virions untreated. Each dot is the light scattered by an individual VSV particle. NTA uses Brownian motion to infer particle size. Particles flow from left to right because they were pumped by a syringe. This translation movement is discarded for particle size inference. Light intensity increases with particle size, but depends also on the position of the particles in the diluent column (distance to lens) and hence is not used for size inference. (AVI 412 kb)

Supplementary Video 2

NTA of purified VSV virions incubated 1 h at 37 °C. Each dot is the light scattered by an individual VSV particle. NTA uses Brownian motion to infer particle size. Particles flow from left to right because they were pumped by a syringe. This translation movement is discarded for particle size inference. Light intensity increases with particle size, but depends also on the position of the particles in the diluent column (distance to lens) and hence is not used for size inference. (AVI 592 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cuevas, J., Durán-Moreno, M. & Sanjuán, R. Multi-virion infectious units arise from free viral particles in an enveloped virus. Nat Microbiol 2, 17078 (2017). https://doi.org/10.1038/nmicrobiol.2017.78

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.78

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing