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Sanjuán lab

Viral mutation and evolution

Experimental Evolution, Virology, Population Genetics

 

Rafael Sanjuán, Ph.D. in Evolutionary Genetics (2005)

E-mail: rafael.sanjuan@uv.es

Tel: +34 963 543 270

Fax: +34 963 543 670

Address:

Institut Cavanilles de Biodiversitat i Biologia Evolutiva

Parc Cientific de la Universitat de València

C/ Catedrático José Beltrán n°2

46980 Paterna, Valencia

Spain

 

 

Presentation

   In our lab at the Scientific Campus of the University of Valencia (find us), we study virus evolution from an experimental approach. Currently, we are investigating how the mutation rate and the fitness effects of mutations shape the variability and evolution of viruses.

We are currently looking for postdocs interested in viral mutation and evolution and with expertise in bioinformatics, next-generation sequencing, and HIV.

 

 Model systems

   Viruses are excellent model systems for studying evolution because they evolve fast under lab conditions, which allows us to do 'real time' analyses. Moreover, their small genomes facilitate genetic manipulation and the study of the genetic bases of adaptation. We have worked both at the computational and experimental levels with several viruses:

  1. Bacteriophages. RNA (Qb, MS2, SP) and single-stranded DNA (FX174, G4, F1) coliphages, all of which infect the same host strain, thus allowing us to perform direct comparisons between viral species.

  2. Vesicular stomatitis virus (VSV), a negative-stranded RNA virus belonging to the family Rhabdoviridae. As most RNA viruses, it has a small genome size (11 kb), a high per-base mutation rate (~10-4-10-5), and low tolerance to mutations (~40% random nucleotide substitutions are lethal to the virus). In the wild, it is of particular importance to farmers in regions where it can infect livestock. In the lab, it has been extensively used for experimental evolution.

  3. Transmissible gastroenteritis virus (TGEV), a model coronavirus. Using samples kindly provided by Prof. Luis Enjuanes (CSIC, Madrid), we are measuring the mutation rate of this virus. Coronaviruses are probably the only RNA viruses whose polymerases are able of proofreading, and this should result in relatively low mutation rates.

  4. Hepatitis C virus (HCV) constitutes a major global health concern. We have analyzed sequence datasets in collaboration with Prof. Fernando González-Candelas to investigate the effect of the interferon-ribavirin therapy on the viral mutation rate.

  5. HIV-1.  Given the huge amount of  information available for this virus, it constitutes an excellent model for studying  evolution at the molecular level. For instance, in collaboration with Dr. Antonio V. Borderia (Institut Pasteur), we have shown that HIV-1 evolution is constrained by  selection operating simultaneously at the RNA structure and protein levels. We are also investigating how immune pressure shapes HIV-1 sequence variability.

  6. Viroids are plant infectious agents constituted by a minimal (200-400 nt) RNA genome that does not code for any protein. In collaboration with the groups of the Profs. Santiago F. Elena and Ricardo Flores, we have performed several in silico analyses of their RNA secondary structure and  have measured the per-base mutation rate of a hammerhead viroid, which turned out to be the highest described for any biological entity.

 Research tools

  • Experimental evolution
       Experimental evolution is aimed at testing evolutionary hypotheses under  controlled conditions. As such, it can help us to disentangle the contribution of different  evolutionary processes. Cultured viruses, bacteria, yeast, or even higher eukaryotes with sufficiently short generation times can be used as model systems. Serial transfers of these organisms are performed in the lab under a variety of conditions, including different population sizes, physical environments, transmission modes, or mutation rates. Typically, we estimate biological fitness using growth rates or competition assays, but other parameters (e.g. virulence) can be studied as well.

  • Molecular biology
       We use several molecular biology techniques, including RT-PCR, in vitro replication, sequencing, molecular cloning, site-directed mutagenesis, or chemical mutagenesis, as well as microbiological and cellular biology basic techniques (cell culturing, monoclonal antibody production, viral transfers).

  • Comparative biology
       Comparing species is the classical approach in biology. However, this has not very often been combined with experimentation. One of our main research goals is to compare the evolutionary properties of different species, including their ability to generate genetic variation, adapt to novel environments, tolerate deleterious mutations, or evolve new functional capabilities, in the laboratory.

  • Computational biology and modeling
       We use computational tools for phylogenetic analysis, statistical analysis, modeling,  prediction of RNA secondary structures, or digital evolution.

 Main findings

  • Mutation rates
             Mutation is the ultimate source of genetic variation and, as such, a key factor explaining the high variability and fast evolution of RNA viruses.

  1. We have recently published an extensive review of viral mutation rate estimates (2010) (see associated on-line resource ).
  2. We are exploiting the well-known E.coli methyl-directed mistmatch repair system to engineer ssDNA phages with altered mutation rates.
  3. Using a large dataset of molecular clone sequences from a local hepatitis C virus outbreak, we showed that the current ribavirin plus interferon treatment increases the mutation rate of the virus by aprox. threefold, and thus that viral mutagenesis might contribute to the therapeutic effect of this treatment (2009).
  4. We measured the mutation rate of Chrysanthemum chlorotic mottle viroid (CChMVd) and shown that it is the highest described for any biological entity (2009).
  5. We measured the mutation rate of the  bacteriophage FX174 using the Luria-Delbrück fluctuation test (2009). Our estimate confirmed Drake's rule, which states that DNA-based mircoorganisms (DNA viruses, bacteria, unicellular eukaryotes) show a constant genomic mutation rate of ~0.003 substitutions per genome per round of copying, despite large variations in lifestyle and genome complexity.
  6. Estimation of the mutation rate of VSV using the Luria-Delbrück fluctuation test (2005).
  7. Upper-limit estimation of the mutation rate of Tobacco etch virus (2009).
  8. Fitness costs of replication fidelity in RNA viruses (2005, 2007).
  9. Selection fails to optimize mutation rates in digital organisms (2008).

  • Mutational robustness
         The ability of organisms to tolerate mutations (mutational or genetic robustness) determines the strength of natural selection and as such, plays an important role in evolution.

  1. Using a series of VSV single-nucleotide mutants obtained by site-directed mutagenesis, we were the first to directly characterize the distribution of mutational fitness effects in an RNA virus (2004). This work established that RNA viruses show remarkably low robustness, roughly 40% of random point mutations being lethal in the case of VSV.

  2. The above results have been extended to other RNA viruses and single-stranded DNA viruses (2007-2010). Recently, I  published a review on the use of site-directed mutagenesis as a valuable tool for characterizing the fitness effects of random mutations in viruses (2010).

  3. We have also characterized the fitness effects of synonymous substitutions in RNA viruses and found that they can contribute significantly to shaping genetic variability and evolution. The effects of synonymous substitutions are much weaker in DNA viruses (2011).

  4. Due to their high mutation rates and low robustness, RNA viruses are a good target for lethal mutagenesis. We developed a population genetics theory of lethal mutagenesis in viruses, in collaboration with Prof. Jim Bull and Dr. Claus Wilke (University of Texas) (2007).

  5. We demonstrated that increased robustness offers a selective advantage in viral populations subjected to chemical mutagenesis (2007). This work also provided support for a prediction of the quasispecies theory known as " the survival of the flattest ".

  6. The relationship between robustness and evolvability remains controversial.  We showed that, in VSV, increased robustness appears to hamper adaptation (2009).

  7. We showed that selection for thermostability can lead to the emergence of mutational robustness in the bacteriophage Qb (2010).

  8. We have also studied robustness in viroids. Using RNA folding algorithms, we predicted the  effect of all possible single substitutions on the secondary structure of 29 viroid species (2006). We also developed a one-step site-directed mutagenesis lab protocol for viroids which facilitates the creation of mutant collections (2007).

  • Epistasis
             Epistasis (the interaction between genes or loci) is central to several population-genetic theories, including those seeking to explain sexual reproduction, ploidy, or speciation. More recently, systems biology has opened new avenues for the study of epistasis.

  1. In collaboration with Prof. Santiago Elena (CSIC), we proposed the existence of a general correlation between  epistasis and genome complexity, and supported it using experimental data from viruses, prokaryotes, unicellular eukaryotes and higher eukaryotes (2006).

  2. In collaboration with Dr. Miguel R. Nebot (University of Valencia), we developed a simple gene network model for explaining the above correlation between epistasis and genome complexity (2008).

  3. We characterized the distribution of epistasis coefficients between random pairs of mutations obtained by site-directed mutagenesis in VSV (2004).

  4. We studied epistasis in viroids using RNA folding algorithms. In this work, we provided evidence for the hypothesis that epistasis and robustness are correlated traits (2006).

  5. Selection and epistasis coefficients for an essential regulatory RNA secondary structure of the Rous sarcoma virus (2006).

  • Other studies 
  1. Assessment of the evolvability of RNA and DNA viruses in the laboratory: RNA viruses evolve faster but the difference is less  than expected from differences in mutation rates (2011).
  2. Selection acting at the level of RNA struture can constrain the evolution of HIV-1 at other levels, such as the emergence of drug resistance or immune escape (2011).
  3. Co-infection with Vaccinia virus enhances VSV adaptability (2008)
  4. Development of a least-squares statistical test for assessing the confidence of distance-based phylogenetic trees (2005)
  5. Effect of ribavirin/interferon treatment on VSV fitness and evolvability (2005)
  6. Assessment of the relative importance of compensatory evolution and reversion in VSV experimental populations undergoing fitness recovery (2005)
  7. Identification of trade-offs between fitness-related traits in VSV in cell cultures (2005)
  8. The role of natural selection in the organ compartmentalization of HIV-1 within patients (2004)
  9. Study of the role played by gene duplications in the evolution of novel functions (2001)
  10. Effects of transmission mode and genetic bottlenecking on the fitness of VSV in the laboratory (2001)

 

 List of publications

  • Cuevas J.M., Domingo-Calap P., Sanjuán R. (2012). The fitness effects of synonymous mutations in DNA and RNA viruses. Mol. Biol. Evol. 29: 17-20.
  • Belshaw R., Sanjuán R., Pybus O.G. (2011). Viral mutation and substitution: units and levels. Curr. Opin. Virol. 1: 430-435.
  • Cuevas J.M., Pereira-Gómez M., Sanjuán R. (2011). Mutation rate of bacteriophage fX174 modified through changes in GATC sequence context. Infect. Genet. Evol. 11:1820-1822.
  • Domingo-Calap P., Sanjuán R. (2011). Experimental evolution of RNA versus DNA viruses. Evolution 65: 2987-2994.
  • Sanjuán R., Domingo-Calap P. Experimental evolution in viruses (2011) in: Encyclopedia of Life Sciences. (doi: 10.1002/9780470015902.a0022857).
  • Sanjuán R., Bordería A.V. (2011). Interplay between RNA structure and protein evolution in HIV-1. Mol. Biol. Evol. 28: 1333-1338.
  • Sanjuán R., Nebot M.R., Chirico N., Mansky L.M., Belshaw R. (2010). Viral mutation rates. J. Virol. 84: 9733-9748 .
  • Domingo-Calap P., Pereira-Gómez M., Sanjuán R. (2010). Selection for thermostability can lead to the emergence of mutational robustness in an RNA virus. J. Evol. Biol. 23: 2453-2460 .
  • Sanjuán R., Bradwell K. (2010). Book review: The Evolution and Emergence of RNA viruses (Author: E.C. Holmes). Syst. Biol. 59: 610-612.
  • Cuevas J.M., Sanjuán R., Moya A. Evolución experimental en virus (2010) in: Adaptación y Evolución (Eds: H. Dopazo, A. Navarro), p. 439-446. Obrapropia.
  • González-Candelas F., Sanjuán R. The population genetics of human viral pathogens (2010) in: Microbial Population Genetics (Ed: Jianping Xu) p. 167-188.
  • Sanjuán R. (2010). Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies. Philos. Trans. Roy. Soc. Lond. B 365: 1975-1982 .
  • Peris J.B., Davis P., Cuevas J.M., Nebot M.R., Sanjuán R. (2010). Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1. Genetics 185: 603-609.
  • Domingo-Calap P., Cuevas J.M., Sanjuán R. (2009). The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet. 5: e1000742 .
  • Cuevas J.M., Duffy S., Sanjuán R. (2009). Point muation rate of bacteriophage FX174. Genetics 183: 747-749 .
  • Cuevas J.M., Moya A., Sanjuán R. (2009). A genetic background with low mutational robustness is associated with increased adaptability to a novel host in an RNA virus. J. Evol. Biol. 22: 2041-2048 .
  • Domingo-Calap P., Sentandreu V., Bracho A., González-Candelas F., Moya A., Sanjuán R. (2009). Unequal distribution of RT-PCR artifacts along the E1-E2 region of hepatitis C virus. J. Virol. Methods 161: 136-140.
  • Cuevas J.M., Domingo-Calap P., Pereira-Gómez M., Sanjuán R. (2009). Experimental evolution and population genetics of RNA viruses.  The Open Evolution Journal 3: 9-16.
  • Cuevas J.M., González-Candelas F., Moya A., Sanjuán R. (2009), Effect of ribavirin on the mutation rate and spectrum of hepatitis C virus in vivo. J. Virol. 83: 5760-5764.
  • Cuevas J.M., Sanjuán R. (2009). Evolución experimental: evolución en tiempo real. Apuntes de Ciencia y Tecnología 30: 30-34.
  • Sanjuán R., Agudelo-Romero P., Elena S.F. (2009). Upper-limit mutation rate estimation for a plant virus. Biol. Lett. 5: 394-396.
  • Gago S., Elena S.F., Flores R., Sanjuán R. (2009). Extremely high mutation rate of a hammerhead viroid. Science 323: 1308.
  • Elena S.F., Sanjuán R. (2008) The effect of genetic robustness on evolvability in digital organisms. BMC Evol. Biol. 8: 284.
  • Clune J., Misevic D., Ofria C., Lenski R., Elena S.F., Sanjuán R. (2008). Selection fails to optimize mutation rates in rugged fitness landscapes. PLoS Comput. Biol. 4: e1000187 .
  • Carrillo F.Y., Sanjuán R., Moya A., Cuevas J.M. (2008). Enhanced adaptation of vesicular stomatitis virus in cells infected with vaccinia virus. Infect. Genet. Evol. 8: 614-620.
  • Sanjuán R., Nebot M.R. (2008) A network model for the correlation between epistasis and genomic complexity. PLoS ONE 3: e2663.
  • Bull J.J., Sanjuán R., and Wilke C.O. Lethal mutagenesis (2008) in: Origin and Evolution of Viruses (Eds: E. Domingo, C. Parrish, J. Holland), p. 207-218.
  • Sanjuán R. Quasispecies and experimental evolution of RNA viruses (2008) in: Enciclopedia of Virology (Eds: B.W.J. Mahy, M.H.V. Van Regenmortel), p. 359-365.
  • Elena S.F., Agudelo-Romero P., Carrasco P., Codoñer F.M., Martín S., Torres C., and Sanjuán R. (2008). Experimental evolution of plant RNA viruses. Heredity 100: 478-483.
  • Serrani J.C., Sanjuán R., Fos M., García-Martínez J.L. (2007). Gibberellin regulation of fruit-set and growth in tomato. Plant Physiol. 145: 246-257 .
  • Elena S.F. and Sanjuán R. (2007). Virus evolution: insights from an experimental approach. Annu. Rev. Ecol. Evol. Syst. 38: 27-52 .
  • Sanjuán R. and Daròs J.A. (2007). Site-directed mutagenesis of viroid dimeric cDNA. J. Virol. Methods 145: 71-75.
  • Sanjuán R., Cuevas J.M., Furió V., Holmes E.C., and Moya A. (2007). Selection for robustness in mutagenized RNA viruses. PLOS Genet. 15: e93.
  • Bordería A.V., Codoñer F.M., and Sanjuán R. (2007) Selection promotes organ compartmentalization in HIV-1: evidence form gag and pol genes. Evolution 61: 272-279.
  • Bull J.J., Sanjuán R., and Wilke C.O. (2007) Theory of lethal mutagenesis for viruses. J. Virol. 81: 2930-2939 .
  • Furió V., Moya A., and Sanjuán R. (2007). The cost of replication fidelity in human immunodeficiency virus type 1. Proc. Biol. Sci. 274: 225-230.
  • Carrillo F.Y., Sanjuán R., Moya A., and Cuevas F.M. (2007). The effect of co- and superinfection on the adaptive dynamics of vesicular stomatitis virus. Infect. Genet. Evol. 7: 69-73.
  • Czarna A., Sanjuán R., González-Candelas F., and Wróbel B. (2006). Topology testing of phylogenies using least squares methods. BMC Evol. Biol., 6: 105.
  • Sanjuán R. and Elena S.F. (2006). Epistasis correlates to genomic complexity. Proc. Natl. Acad. Sci. USA 103: 14402-14405.
  • Sanjuán R., Forment J., and Elena S.F. (2006). In silico predicted robustness of viroids RNA secondary structures. II. Interaction between mutation pairs. Mol. Biol. Evol. 23: 2123-2130.
  •  Sanjuán R., Forment J., and Elena S.F. (2006). In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations. Mol. Biol. Evol. 23: 1427-1436.
  • Sanjuán R. (2006) Quantifying antagonistic epistasis in a multifunctional RNA secondary structure of the Rous sarcoma virus. J. Gen. Virol., 87: 1595-1602 .
  • Elena SF., Carrasco P., Daròs J.A., and Sanjuán R. (2006). Mechanisms of genetic robustness in RNA viruses. EMBO R. 7: 168-173.
  • Furió V., Moya A., and Sanjuán R. (2005). The cost of replication fidelity in an RNA virus. Proc. Natl. Acad. Sci. USA 102: 10233-10237.
  • Elena S.F. and Sanjuán R. (2005). On the adaptive value of high mutation rates in RNA viruses: separating causes from consequences. J. Virol., 79, 11555-11558.
  • Sanjuán R., Cuevas J.M., Moya A., and Elena S.F. (2005). Epistasis and the adaptability of an RNA virus. Genetics 170: 1001-1008.
  • Elena S.F. and Sanjuán R. (2005). RNA viruses as complex adaptive systems. Biosystems 81: 31-41.
  • Sanjuán R. and Wróbel B. (2005). Weighted least-squares likelihood ratio test for branch testing in phylogenies reconstructed from distance methods. Syst. Biol., 54: 218-229.
  • Cuevas J.M., Sanjuán R., Moya A., and Elena S.F. (2005). Mode of selection and experimental evolution of antiviral drugs resistance in vesicular stomatitis virus. Infect. Genet. Evol. 5: 55-65.
  • Cuevas J.M., Moya A., and Sanjuán R. (2005). Following the very initial growth of biological RNA viral clones. J Gen. Virol. 86: 435-443.
  • Sanjuán R., Moya A., and Elena S.F. (2004). The contribution of epistasis to the architecture of fitness in an RNA virus. Proc. Natl. Acad. Sci. USA 101: 15376-15379 .
  • Sanjuán R., Moya A., and Elena S.F. (2004). The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc. Natl. Acad. Sci. USA 101: 8396-8401.
  • Sanjuán R., Codoner F.M., Moya A., and Elena S.F. (2004). Natural selection and the organ-specific differentiation of HIV-1 V3 hypervariable region. Evolution 58: 1185-1194.
  • Elena S.F. and Sanjuán R. (2003). Evolution: Climb every mountain? Science 302: 2074-2075.
  • Elena S.F., Codoner F.M., Cuevas J.M., and Sanjuán R. (2003). Adaptive dynamics during experimental evolution of RNA viruses. Biology International 44: 75-78.
  • Elena S.F., Codoner F.M., and Sanjuán R. (2003). Intraclonal variation in RNA viruses: generation, maintenance and consequences. Biol. J. Linn. Soc. 79: 17-26.
  • Elena S.F., Sanjuán R., Borderia A.V., and Turner P.E. (2002). Differential effects of horizontal and vertical transmission in the fitness of an RNA virus: A reanalysis. Infect. Genet. Evol. 1: 307-309.
  • Sanjuán R. and Marín I. (2001). Tracing the origin of the compensasome: evolutionary history of DEAH helicase and MYST acetyltransferase gene families. Mol. Biol Evol. 18: 330-343.
  • Elena S.F., Sanjuán R., Borderia A.V., and Turner P.E. (2001). Transmission bottlenecks and the evolution of fitness in rapidly evolving RNA viruses. Infect. Genet. Evol.: 1: 41-48.

 

 

 Lab people

 

Position

Name

E-mail

Ph.D. Student

Pilar Domingo-Calap

pilar.domingo@uv.es

Ph.D. Student

Joan Peris

hoeman5beta@hotmail.com

Ph.D. Student

Marianoel Pereira-Gómez

marianoel.pereira@uv.es

Research Assistant

Raquel Garijo

raquel.garijo@uv.es

Master Student

Pablo Hernández

 

 

VISITORS AND PAST MEMBERS

Temesgen Woldezion (Student of the MHIRT program), July-August 2008.

Loles Catalán (Research Assistant), December 2008 - July 2009.

José M. Cuevas (Postdoc), January 2008 - December 2009.

Paulina Davis (Student of the MHIRT program), July-August 2009.

Katie Bradwell (Master Student), September 2009 – September 2010.

Antonio V. Borderia (Postdoc), December 2009 - February 2010.


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Last modified February 2, 2012