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Research Groups Group VIP The strange biology of multipartite viruses

The strange biology of multipartite viruses
Leader of the project: Stéphane Blanc

The way of life of multipartite viruses is poorly understood. Using Nanoviruses as biological models, we address the basic biology of multipartite viruses and experimentally investigate the possible benefits and costs in such biological systems.

Staff involved in the project

S. Blanc
Leader of the project
M. Yvon
M.S. Vernerey
J. Di Mattia

J.L. Zeddam

E. Pirolles
Assistant engineer

Master Students:

Anne Sicard (spring 2010, M2 SupAgro., Montpellier, France)
Geoffrey Carpentier (winter-spring 2014, M2 univ. Montpellier, France)
Alexander Dale (Winter 2015, Master 1 Erasmus, Oxford, United Kingdom)
Elise Découvreur (spring 2015, Master 1 Erasmus, Univ. Louvain, Belgium)
Jules Butchacas (winter-spring 2016 Master 1 IMHE Université Montpellier)
Isabelle Russel (spring 2017, Master 1 Erasmus MEME, Université de Montpellier)
Mark Wright (spring 2017, Master 1 Erasmus MEME, Université de Montpellier)
Abdillah Mohamed (winter-spring 2017 Master 2 IMHE Université Montpellier)
Jérémy Dimattia (winter-spring 2017 Master 2 IMHE Université Montpellier)
Yahya Gafaar (fall-winter 2017, visiteur Doctorant JKI institute Brunschweig, Allemagne)

Major collaboration

Yannis Michalakis (Research director, CNRS, Montpellier)


Research project

Background and state of the art

Three main categories of virus genome structure and organization, independently of whether the nucleic acid is single- or double-stranded RNA or DNA, can be distinguished. The “monopartite” viruses have a single chromosome bearing all genetic information and packaged in a single virus particle. The “segmented” viruses have two or more chromosomes, which are all encapsidated together in a single virion. Finally, the “multipartite” viruses also have more than one chromosome, from 2 to 10 depending on the viral species, but each is encapsidated individually in distinct virus particles. Multipartite viruses are extremely frequent in plants, where they induce numerous diseases in major crops around the world. They also infect fungi and have recently been reported in insects.

Because of this puzzling compartmentalization of the genetic information of multipartite viruses into several virus particles, the functioning of their genome is intricately linked to that of their populations. Theoretical studies have proposed putative advantages of such viral systems, like enhanced stability, replication and/or recombination/shuffling of smaller nucleic acid segments; the counterpart costs being the reduced chances to infect new cells and new hosts with at least one copy of each, thus with no information loss. The current view of the way multipartite viral systems can be functional largely suffers from the lack of experimental data that would support any of the proposed benefits or even the actual existence of the above-mentioned cost at new cell and host infection.

The biological model and current results

We have recently demonstrated that the multipartite Faba bean necrotic stunt virus (FBNSV, Family Nanoviridae, Figure 1) reproducibly accumulates its eight genes (or genome segments) within host plants with different and specific relative frequencies, some genes being frequent and others rare (Figure 2 top panel). We here refer to the specific pattern of the frequencies of the eight segments as the “genome formula” (Figure 2 bottom panel). This discovery suggests an unforeseen putative benefit in multipartite viral systems, that is the capability to differentially control the copy number of each gene/segment, which in turn drives the virus population in the situation of maximum costs, due to the increased risk of losing rare segments.

Figure 1: Genome organization of nanoviruses on the example of Faba bean necrotic stunt virus (FBNSV). Eight segments are circular ssDNAs each around 1000 bases : R encodes : Master-Rep assisting replication of all segments, S : coat protein CP separately encapsidating all segments, C : Clink resetting cell cycle, M : movement protein MP mediating in-plant virus migration, N : nuclear shuttle protein NSP regulating aphid transmission (B. Gronenborn, pers. com.), U1-U2-U4 have unknown functions. CR-SL: common region stem loop.
Figure 2 : Gene Copy Number regulation in Nanoviruses
Left Panel
The relative frequency of each FBNSV segment in Vicia faba plants systemically infected after inoculation by aphid vectors is plotted in dark blue and compared to that in agro-inoculated plants (light blue on the left). In Vicia faba, the frequency of the segments was not affected by the mode of inoculation (aphid- versus agro-inoculation), as indicated by ANOVA tests for each of the eight segments. The relative frequency of FBNSV segments in Medicago truncatula plants systemically infected after inoculation by aphid vectors is plotted in red. Vf-At1, Vf-At2, Vf-At3, Mt-At1 and Mt-At2 results from independent aphid-inoculation experiments and the number of infected plants in each set (n) is indicated on the right. A significant effect of the host species could be detected through ANOVA tests for 7 out of 8 segments. (From Sicard et al 2013)

Right Panel
The genome formulae of FBNSV in Vicia faba (GF Vf) and in Medicago truncatula (GFMt) were respectively calculated by pooling all Vf-At and Mt-At data sets, and are indicated below the graph.
The relative frequency of the segments (segment name below panels) was estimated by qPCR. The rarest is arbitrarily considered as 1 copy, and the copy number of the others is represented relative to it. Genome formula in Vicia faba (V.f. green left), Medicago truncatula (M.t. blue center) and Acyrtosiphon pisum (A.p. grey right). Adapted from Sicard et al 2013 and Sicard et al 2015.

Main questions of the project

We here develop a research program that experimentally addresses in parallel the relationship between the various genome segments and the population dynamics/genetics of multipartite viruses, using FBNSV as a model system. We investigate:

i) the link between the gene/segment copy number (genome formula), the regulation of gene expression, and the expression of viral phenotypes (accumulation, aggressiveness, transmission by aphid vectors);

ii) the actual distribution of distinct genomic segments in cells of the host and of the vectors, in order to reveal whether they are all always together in individual cells at all steps of the life cycle, or whether the genetic information can be temporarily separated in distinct locations of the host or vector, or even in distinct hosts in the landscape (Figure 3);

Figure 3 : Localization of distinct genome segments in host plant and aphid vector cells
The fluorescent labeling of pairs of segments (here U2-red and U4-green) demonstrates that the segments do not necessarily localize together in individual cells of the host plant where the virus replicates (left). This suggests that the multipartite viral systems can be functional when distinct genes accumulate in distinct cells and thus that the system coordinates across distinct cells in a tissue, defining a pluricellular way of life. In sharp contrast, in the gut cells of insect vectors (right), the virus does not replicate and undergoes massive transcytosis to traverse the cellular gut barrier. We observe countless cytoplasmic granules, each containing all segments of the tested pairs. This illustrates a very intriguing counterintuitive situation where all segments accumulate together in cells where no viral replication occurs (aphid gut cells), whereas they accumulate separately in distinct cells where the virus replicates (plant cells). Both images show merged green and red channels, and colocalization appears yellow. Nucleus are DAPI-stained.
(Unpublished results)

iii) the way ssDNA segments, mRNA and proteins actually traffic within hosts and vectors to investigate whether the various segments can complement each other only within cells or also across cells; iv) the plastictity of the genome formula in response to environmental changes and its evolution during adaptation to new host species.

This project provides key information on the basic “way of life” of multipartite viruses. In particular, the proposed experiments inform (or will inform) on both the putative benefits and costs in these enigmatic biological systems, for which decades of theoretical studies led to the recent conclusion that the current conceptual framework of virology cannot explain their existence.

Our ultimate goal is to generate seminal results that will inspire increased research efforts on multi-component viral systems beyond multipartite viruses plant, also on satellites, defective interfering particles and segmented viruses of animals.


The project is currently funded by INRA SPE division and by French ANR (ANR-nano project)

We are trying to attract additional external funds to further develop our project by investing more into:

-Our main model to acquire more details and deep understanding of the biology of nanoviruses
-other plant multipartite viral systems
-Satellites and defective interfering particles of monopartite viruses
-Segmented viruses encapsidating their segments in a non-selective manner (bunyaviruses)

Publication list of the PI: Stéphane Blanc (* corresponding author)

Up coming stories:

- Blanc S and Y Michalakis (editors). « Multicomponent viral systems ». We are preparing and will edit a full section comprising 16 complementary review articles each related to the topic. Current Opinion in Virology 2019.

-Gallet R, Fabre F, Thébaud G, Blanc S and Y Michalakis. Small bottleneck size in Faba bean necrotic stunt virus populations during aphid transmission and plant colonization. Manuscript in preparation.

-Sicard A, Pirolles E., Gallet R, Vernerey M-S, Yvon M, Urbino C, Peterschmitt M, Michalakis Y and S Blanc*. A pluricellular way of life for a multipartite virus. Manuscript in preparation

-Yvon M, Wright M, Michalakis Y and S Blanc*. A plant virus induces a switch in the investment of its insect vector from fecundity to survival in the absence of ressources. Manuscript in preparation

-Webster C, van Munster M, Monsion B, Gargani D, Hoh F, Barthe P, Padilla A, Bron P, Blanc S and M Uzest. Identification of plant virus receptor candidates in the stylets of aphid vectors. Manuscript in preparation

-Gallet* R, Fabre F, Michalakis Y, and S Blanc* (2017). The number of target molecules of amplification step limits accuracy and sensitivity in ultradeep-sequencing viral population studies. J. Virol 2016, doi:10.1128/JVI.00561-17

-van Munster M, Yvon M, Vile D, Dader B, Fereres A and S Blanc*. Water deficit enhances the transmission of plant viruses bu insect vectors. PLoS One 2017, 3;12(5). Doi:10.1371/journal .pone0174398. eCollection2017

-Yvon M, Vile D, Brault V, Blanc S and M van Munster. Drought reduces transmission of turnip yellows virus, an insect-vectored circulative virus. Virus research 2017, doi: 10.1016/j.virusres.2017.07.009

-Webster C, Thillier M, Priolles E, Cayrol B, Blanc S and M Uzest. Proteomic composition of the acrostyle: Novel approaches to identify cuticular proteins involved in virus-insect interactions. Insect Science 2017,

- Sicard A, Michalakis Y, Gutierrez S and S Blanc*. The strange lifestyle of multipartite viruses. PLoS Pathog 2016, 12(11): e1005819.

- Stéphane Blanc* and Yannis Michalakis : Manipulation of hosts and vectors by plant viruses and impact of the environment. Current Opinion in Insect Science 2016, 16:36–43. doi.org/10.1016/j.cois.2016.05.007

- Blanc* S. & M. Uzest. Non-circulative virus-vector interactions, In «Microbe-Arthropod vector interactions» 2016, ed J. Brown, American Phytopathological Society Press.

-Roumagnac P, Granier M, Bernardo P, Deshoux M, Ferdinand R, Galzi S, Fernandez E, Julian C, Abt I, Filloux D, Varsani A, Blanc S, Martin DP and M. Peterschmitt: Alfalfa leaf curl virus: An pahid transmitted geminivirus. J Virol 2015, 89:9683–9688. doi:10.1128/JVI.00453-15.

-Sicard A, Zeddam JL, Yvon M, Michalakis Y, Gutierrez S, Blanc* S: Circulative non propagative aphid-transmission of nanoviruses: an oversimplified view. J Virol 2015, 89:9719 –9726. doi:10.1128/JVI.00780-15..

- Gutiérrez S, Pirolles E, Yvon M, Baecker V, Michalakis Y and S Blanc*: The multiplicity of cellular infection changes depending on the route of cell infection in a plant virus. J Virol 2015, 89:9665–9675. doi:10.1128/JVI.00537-15

- Blanc* S and S Gutiérrez: The specifics of vector transmission of arboviruses of vertebrates and plants. Current Opinion in Virology 2015, 15:27–33, doi.org/10.1016/j.coviro.2015.07.003

-Blanc* S, Drucker M, Uzest M: Localizing viruses in their insect vectors. Annu Rev Phytopathol 2014, 52:403-425.

-Bak A, Gargani D, Macia JL, Malouvet E, Vernerey MS, Blanc S, Drucker M: Virus factories of cauliflower mosaic virus are virion reservoirs that engage actively in vector transmission. J Virol 2013, 87:12207-12215.

-Gutiérrez S, Michalakis Y, Van Munster M, Blanc* S: Plant feeding by insect vectors can affect life cycle, population genetics and evolution of plant viruses. Functional Ecology 2013, 27:610-622.

-Martiniere A, Bak A, Macia JL, Lautredou N, Gargani D, Doumayrou J, Garzo E, Moreno A, Fereres A, Blanc* S and M. Drucker*: A virus responds instantly to the presence of the vector on the host and forms transmission morphs. Elife 2013, 2:e00183.

-Sicard A, Yvon M, Timchenko T, Gronenborn B, Michalakis Y, Gutierrez S, Blanc* S: Gene copy number is differentially regulated in a multipartite virus. Nature Commununications 2013, 4:2248. doi: 10.1038/ncomms3248

-Gutierrez S, Michalakis Y, Blanc* S: Virus population bottlenecks during within-host progression and host-to-host transmission. Curr Opin Virol 2012, 2:546-555.

-Gutierrez S, Yvon M, Pirolles E, Garzo E, Fereres A, Michalakis Y, Blanc* S: Circulating virus load determines the size of bottlenecks in viral populations progressing within a host. PLoS Pathog 2012, 8:e1003009.

-Bak A, Irons SL, Martiniere A, Blanc S, Drucker M: Host cell processes to accomplish mechanical and non-circulative virus transmission. Protoplasma 2011, 249 :529-539

-Blanc* S, Uzest M and M Drucker. New research horizon in vector-transmission of plant viruses. Current Opinion in Microbiology 2011, 14 :483-491

-Blanc* S, Drucker M: Functions of virus and host factors during vector-mediated transmission. In Recent Advances in Plant Virology. Edited by Caranta C, Aranda MA, Tepfer M, Lopez-Moya JJ: Caister Academic Press; 2011:103-120.

-Martiniere A, Macia JL, Bagnolini G, Jridi C, Bak A, Blanc S, Drucker M: VAPA, an Innovative "Virus-Acquisition Phenotyping Assay" Opens New Horizons in Research into the Vector-Transmission of Plant Viruses. PLoS One 2011, 6:e23241.

-Vuillaume F, Thebaud G, Urbino C, Forfert N, Granier M, Froissart R, Blanc S, Peterschmitt M: Distribution of the phenotypic effects of random homologous recombination between two virus species. PLoS Pathog 2011, 7:e1002028.

-Brault V, Uzest M, Monsion B, Jacquot E, Blanc S: Aphids as transport devices for plant viruses. C R Biol 2010, 333:524-538.

-Gutierrez S, Yvon M, Thebaud G, Monsion B, Michalakis Y, Blanc* S: Dynamics of the multiplicity of cellular infection in a plant virus. PLoS Pathog 2010, 6(9): e1001113

-Hoh F, Uzest M, Drucker M, Plisson-Chastang C, Bron P, Blanc S, Dumas C: Structural insights into the molecular mechanisms of cauliflower mosaic virus transmission by its insect vector. J Virol 2010, 84:4706-4713.

-Khelifa M, Masse D, Blanc S, Drucker M: Evaluation of the minimal replication time of Cauliflower mosaic virus in different hosts. Virology 2010, 396:238-245.

-Uzest M, Gargani D, Dombrovsky A, Cazevieille C, Cot D, Blanc* S: The "acrostyle": a newly described anatomical structure in aphid stylets. Arthropod Struct Dev 2010, 39:221-229.

-Martinière A, Gargani D, Uzest M, Lautredou N, Blanc S, Drucker M: A Role for Plant Microtubules in the Formation of Transmission-specific inclusion bodies of Cauliflower mosaic virus. Plant J 2009, 58:135-146.

-Yvon M, Monsion B, Martin JP, Gutiérrez S, Blanc* S: PCR-based amplification and analysis of specific viral sequences from individual plant cells. Journal of Virological Methods 2009, doi:10.1016/j.jviromet.2009.04.016

-Blanc* S: Vector transmission of plant viruses. In Encyclopedia of Virology, edn 3rd. Edited by Mahy BWJ, van regenmortel MHV: Elsevier Ltd.; 2008:274-282.

-Monsion B, Duborjal H, Blanc* S: Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples. BMC Genomics 2008, 9:85.

-Monsion B, Froissart R, Michalakis Y, Blanc* S: Large bottleneck size in Cauliflower Mosaic Virus populations during host plant colonization. PLoS Pathog 2008, 4:e1000174.

-Urbino C, Thebaud G, Granier M, Blanc S, Peterschmitt M: A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses. Virol J 2008, 5:135.

-Blanc* S: Virus transmission-getting out and in., vol 7. Edited by Heinlein EWaM. Berlin-Heidelberg: Springer-Verlag; 2007.

-Brault V, Blanc S, Jacquot E: Comment les pucerons transmettent des maladies virales aux plantes. Biofutur 2007, 279:40-44.

-Khelifa M, Journou S, Krishnan K, Gargani D, Esperandieu P, Blanc S, Drucker M: Electron-lucent inclusion bodies are structures specialized for aphid transmission of cauliflower mosaic virus. J Gen Virol 2007, 88:2872-2880.

-Uzest M, Gargani D, Drucker M, Hebrard E, Garzo E, Candresse T, Fereres A, Blanc* S: A protein key to plant virus transmission at the tip of the insect vector stylet. Proc Natl Acad Sci U S A 2007, 104:17959-17964.

-Jridi C, Martin JF, Marie-Jeanne V, Labonne G, Blanc* S: Distinct viral populations differentiate and evolve independently in a single perennial host plant. J Virol 2006, 80:2349-2357.

-Ballut L, Drucker M, Pugniere M, Cambon F, Blanc S, Roquet F, Candresse T, Schmid HP, Nicolas P, Gall OL, et al.: HcPro, a multifunctional protein encoded by a plant RNA virus, targets the 20S proteasome and affects its enzymic activities. J Gen Virol 2005, 86:2595-2603.

-Froissart R, Roze D, Uzest M, Galibert L, Blanc S, Michalakis Y: Recombination every day: abundant recombination in a virus during a single multi-cellular host infection. PLoS Biol 2005, 3:e89.

-Moreno A, Hebrard E, Uzest M, Blanc* S, Fereres A: A single amino acid position in the helper component of cauliflower mosaic virus can change the spectrum of transmitting vector species. J Virol 2005, 79:13587-13593.

-Plisson C, Uzest M, Drucker M, Froissart R, Dumas C, Conway J, Thomas D, Blanc S, Bron P: Structure of the mature P3-virus particle complex of cauliflower mosaic virus revealed by cryo-electron microscopy. J Mol Biol 2005, 346:267-277.

-Blanc* S: Insect transmission of viruses. In Microbe-vector interactions in vector-borne diseases. Edited by Gillespie SH, Smith GL, Osbourn A: Cambridge University Press; 2004:42-61. [Symposium S (Series Editor), vol 63.]

-Froissart R, Uzest M, Ruiz-Ferrer V, Drucker M, Hebrard E, Hohn T, Blanc* S: Splicing of Cauliflower mosaic virus 35S RNA serves to downregulate a toxic gene product. J Gen Virol 2004, 85:2719-2726.

-Plisson C, Drucker M, Blanc S, German-Retana S, Le Gall O, Thomas D, Bron P: Structural characterization of HC-Pro, a plant virus multifunctional protein. J Biol Chem 2003, 278:23753-23761.

-Drucker M, Froissart R, Hebrard E, Uzest M, Ravallec M, Esperandieu P, Mani JC, Pugniere M, Roquet F, Fereres A, and S Blanc* : Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector. Proc Natl Acad Sci U S A 2002, 99:2422-2427.

-Drucker M, German-Retana S, Espérandieu P, Le Gall O, Blanc* S: Purification of a viral protein from infected plant tissues using the strep tag II affinity tag. BioTech Intl. 2002, 14:16-18.

-Froissart R, Michalakis Y, Blanc* S: Helper component-transcomplementation in the vector transmission of plant viruse. Phytopathology 2002, 92:576-579.

-Lett JM, Granier M, Hippolyte I, Grondin M, Royer M, Blanc S, Reynaud B, Peterschmitt M: Spatial and temporal distribution of geminiviruses in leafhoppers of the genus Cicadulina monitored by conventional and quantitative polymerase chain reaction. Phytopathology 2002, 92:65- 74.

-Palacios I, Drucker M, Blanc S, Leite S, Moreno A, Fereres A: Cauliflower mosaic virus is preferentially acquired from the phloem by its aphid vectors. J Gen Virol 2002, 83:3163-3171.

-Blanc* S, Hébrard E, Drucker M, Froissart R: Molecular basis of vector transmission : Caulimoviruses. In Virus-Insect-Plant interactions. Edited by Harris K, Smith OP, Duffus JE: Academic Press; 2001:143-166.

-Hebrard E, Drucker M, Leclerc D, Hohn T, Uzest M, Froissart R, Strub JM, Sanglier S, van Dorsselaer A, Padilla A, C. Dumas and S Blanc* : Biochemical characterization of the helper component of Cauliflower mosaic virus. J Virol 2001, 75:8538-8546.

-Leh V, Jacquot E, Geldreich A, Haas M, Blanc S, Keller M, Yot P: Interaction between the open reading frame III product and the coat protein is required for transmission of cauliflower mosaic virus by aphids. J Virol 2001, 75:100-106.

-Raccah B, Huet H, Blanc S: Potyviruses. In Virus-Insect-Plant interactions. Edited by Harris K, Duffus JE, Smith OP: Academic Press; 2001:181-206.

-Héricourt F, Blanc S, Redeker V, Jupin I: Evidence for phosphorylation and ubiquitinylation of turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. Biochem. J. 2000, 349:417-425.

-Blanc S, Dolja VV, Llave C, T.P. P: Histidine-tagging and purification of tobacco etch potyvirus helper component protein. Journal of Virological Methods 1999, 77:11-15.

-Hébrard E, Froissart R, Louis C, Blanc* S: Les modes de transmission des virus phytopathogènes par vecteurs. Virologie 1999, 3:35-48.

-Leh V, Jacquot E, Geldreich A, Hermann T, Leclerc D, Cerutti M, Yot P, Keller M, Blanc* S: Aphid transmission of cauliflower mosaic virus requires the viral PIII protein. Embo J 1999, 18:7077-7085.

-Blanc S, Ammar ED, Garcia-Lampasona S, Dolja VV, Llave C, Baker J, Pirone TP: Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. J Gen Virol 1998, 79 ( Pt 12):3119-3122.

-Blanc S, Lopez-Moya JJ, Wang R, Garcia-Lampasona S, Thornbury DW, Pirone TP: A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 1997, 231:141-147.

-Blanc S, Schmidt I, Vantard M, Scholthof HB, Kuhl G, Esperandieu P, Cerutti M, Louis C: The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells. Proc Natl Acad Sci U S A 1996, 93:15158-15163.

-Pirone TP, Blanc S: Helper-dependent vector transmission of plant viruses. Annu Rev Phytopathol 1996, 34:227-247.

-Kiss-Laszlo Z, Blanc S, Hohn T: Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity. Embo J 1995, 14:3552-3562.

-Schmidt I, Blanc S, Esperandieu P, Kuhl G, Devauchelle G, Louis C, Cerutti M: Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission. Proc Natl Acad Sci U S A 1994, 91:8885-8889.

-Blanc S, Cerutti M, Chaabihi H, Louis C, Devauchelle G, Hull R: Gene II product of an aphid-nontransmissible isolate of cauliflower mosaic virus expressed in a baculovirus system possesses aphid transmission factor activity. Virology 1993, 192:651-654.

-Blanc S, Cerutti M, Usmany M, Vlak JM, Hull R: Biological activity of cauliflower mosaic virus aphid transmission factor expressed in a heterologous system. Virology 1993, 192:643-650.

-Blanc S, Schmidt I, Kuhl G, Esperandieu P, Lebeurier G, Hull R, Cerutti M, Louis C: Paracrystalline structure of cauliflower mosaic virus aphid transmission factor produced both in plants and in a heterologous system and relationship with a solubilized active form. Virology 1993, 197:283-292.