Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells

The Flavivirus genus includes a large number of medically relevant pathogens that cycle between humans and arthropods. This host alternation imposes a selective pressure on the viral population. Here, we found that dengue virus, the most important viral human pathogen transmitted by insects, evolved a mechanism to differentially regulate the production of viral non-coding RNAs in mosquitos and humans, with a significant impact on viral fitness in each host. Flavivirus infections accumulate non-coding RNAs derived from the viral 3'UTRs (known as sfRNAs), relevant in viral pathogenesis and immune evasion. We found that dengue virus host adaptation leads to the accumulation of different species of sfRNAs in vertebrate and invertebrate cells. This process does not depend on differences in the host machinery; but it was found to be dependent on the selection of specific mutations in the viral 3'UTR. Dissecting the viral population and studying phenotypes of cloned variants, the molecular determinants for the switch in the sfRNA pattern during host change were mapped to a single RNA structure. Point mutations selected in mosquito cells were sufficient to change the pattern of sfRNAs, induce higher type I interferon responses and reduce viral fitness in human cells, explaining the rapid clearance of certain viral variants after host change. In addition, using epidemic and pre-epidemic Zika viruses, similar patterns of sfRNAs were observed in mosquito and human infected cells, but they were different from those observed during dengue virus infections, indicating that distinct selective pressures act on the 3'UTR of these closely related viruses. In summary, we present a novel mechanism by which dengue virus evolved an RNA structure that is under strong selective pressure in the two hosts, as regulator of non-coding RNA accumulation and viral fitness. This work provides new ideas about the impact of host adaptation on the variability and evolution of flavivirus 3'UTRs with possible implications in virulence and viral transmission.Fil: Filomatori, Claudia Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Carballeda, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Villordo, Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Aguirre, Sebastian. Cedars Sinai Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pallarés, Horacio Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Maestre, Ana M.. Cedars Sinai Medical Center; Estados UnidosFil: Sánchez Vargas, Irma. State University of Colorado - Fort Collins; Estados UnidosFil: Blair, Carol D.. State University of Colorado - Fort Collins; Estados UnidosFil: Fabri, Cintia. Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I. Maiztegui"; ArgentinaFil: Morales, Maria A.. Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I. Maiztegui"; ArgentinaFil: Fernandez Sesma, Ana. Cedars Sinai Medical Center; Estados UnidosFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

RNA Structure Duplication in the Dengue Virus 3 ' UTR: Redundancy or Host Specificity?

Flaviviruses include a diverse group of medically important viruses that cycle between mosquitoes and humans. During this natural process of switching hosts, each species imposes different selective forces on the viral population. Using dengue virus (DENV) as model, we found that paralogous RNA structures originating from duplications in the viral 3' untranslated region (UTR) are under different selective pressures in the two hosts. These RNA structures, known as dumbbells (DB1 and DB2), were originally proposed to be enhancers of viral replication. Analysis of viruses obtained from infected mosquitoes showed selection of mutations that mapped in DB2. Recombinant viruses carrying the identified variations confirmed that these mutations greatly increase viral replication in mosquito cells, with low or no impact in human cells. Use of viruses lacking each of the DB structures revealed opposite viral phenotypes. While deletion of DB1 reduced viral replication about 10-fold, viruses lacking DB2 displayed a great increase of fitness in mosquitoes, confirming a functional diversification of these similar RNA elements. Mechanistic analysis indicated that DB1 and DB2 differentially modulate viral genome cyclization and RNA replication. We found that a pseudoknot formed within DB2 competes with long-range RNA-RNA interactions that are necessary for minus-strand RNA synthesis. Our results support a model in which a functional diversification of duplicated RNA elements in the viral 3' UTR is driven by host-specific requirements. This study provides new ideas for understanding molecular aspects of the evolution of RNA viruses that naturally jump between different species.IMPORTANCE Flaviviruses constitute the most relevant group of arthropod-transmitted viruses, including important human pathogens such as the dengue, Zika, yellow fever, and West Nile viruses. The natural alternation of these viruses between vertebrate and invertebrate hosts shapes the viral genome population, which leads to selection of different viral variants with potential implications for epidemiological fitness and pathogenesis. However, the selective forces and mechanisms acting on the viral RNA during host adaptation are still largely unknown. Here, we found that two almost identical tandem RNA structures present at the viral 3' untranslated region are under different selective pressures in the two hosts. Mechanistic studies indicated that the two RNA elements, known as dumbbells, contain sequences that overlap essential RNA cyclization elements involved in viral RNA synthesis. The data support a model in which the duplicated RNA structures differentially evolved to accommodate distinct functions for viral replication in the two hosts.status: publishe

Production of sfRNAs in Zika virus infections.

<p>(<b>A</b>) Northern blot hybridization showing the accumulation sfRNA in C6/36 and A549 cells infected with a Zika isolate from Argentina (INEVH). (<b>B</b>) RNA structure model predicted for the ZIKV 3’UTR. Conserved SLI, SLII, pseudo-dumbbell (ψ-DB) and DB structures are indicated. Pseudoknots are indicated with red lines. Location and size of the two sfRNAs identified in infected cells are shown (sfRNA1 and sfRNA2). (<b>C and D</b>) Northern blots showing the pattern of sfRNAs in Raji, A549 and C6/36 cells infected with ZIKV from different origins (Cambodia, Senegal and Puerto Rico) as indicated on the top.</p

Mosquito adapted DENV variant exhibit an exacerbated antiviral response in human cells.

<p>Time course analysis of antiviral response in dendritic cells (DCs). DCs of three independent donors were infected with PT or MS3 virus, and at 3, 12, and 24 hpi IFNβ (<b>A</b>) or ISG15 (<b>B</b>) mRNA were determined by real time PCR. (<b>C</b>) The levels of IP-10 in the supernatant of infected cells was assessed by ELISA at 3, 12, 24, 48 and 72 hpi. Data are the means and standard deviations of three replicates from a representative donor.</p

Mapping the sfRNAs generated in DENV infected human and mosquito cells.

<p>(<b>A</b>) Secondary structure of DENV 3’UTR indicating the location and size of sfRNAs identified by sequencing analysis. Below, plots representing relative amounts of sfRNA species produced using human or mosquito infected cells. The amount of each sfRNA was estimated by ImageJ-quantitation and expressed as the mean +/- SD of the relative percentage from total sfRNAs (n = 3). (<b>B</b>) Northern blot hybridization using specific probes complementary to the viral 3’UTR employing RNA extracted at 30 and 50 hpi from C6/36 or A549 cells infected with either DENV-M or DENV-H stocks, as indicated. (<b>C</b>) DENV sfRNAs produced in infected <i>Ae</i>. <i>albopictus</i> and <i>Ae</i>. <i>aegypi</i> mosquitos. Northern blot hybridization using RNA extracted from mosquitos infected with DENV-M or DENV-H. A probe complementary to the viral 3’SL was used for detection.</p

Comparative analysis of predicted RNA structures of the 3’UTR of mosquito-borne flaviviruses (MBFV).

<p>(<b>A</b>) Conserved stem loops of selected viruses from each subgroup of MBFV are shown in red. Predicted H-type pseudoknots are indicated with red dashed lines and pseudoknots including nucleotides present in the three-way junction are shown in black dashed lines. Information obtained from crystallographic studies using MVEV and ZIKV RNAs was included [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006265#ppat.1006265.ref025" target="_blank">25</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006265#ppat.1006265.ref039" target="_blank">39</a>]. Group-specific RNA structures are indicated in grey. The distance tree was drawn using the neighbor joining method of all complete genome sequences for each virus available in GenBank. (<b>B</b>) Representation of the complete 3’UTR structure of different MBFVs showing the conserved elements involved in Xrn1 stalling. Conserved stem loop (SL) and dumbbell (DB) structures are shown in red and blue, respectively. Arc plots of RNA structures corresponding to the conserved xrRNA1, xrRNA2, xrRNA3 and xrRNA4 are shown for DENV1 to 4, ZIKV, WNV, MVEV, SLEV and BAGV. The conserved 3’SL structure is shown in dark blue.</p

Dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding RNAs and fitness in human cells - Fig 1

<p>Different patterns of sfRNA produced during dengue virus host adaptation (<b>A</b>) Schematic representation of DENV 3’UTR diversification during host switch. Variants in viral populations obtained in mosquito or human cells are represented by circles. The size and color of circles represent the frequency and number of mutations of variants, respectively. The distance between variants is also shown by a fan dendrogram. (<b>B</b>) Genomic and subgenomic DENV RNAs produced in infections using viruses generated in human (DENV-H) or mosquito (DENV-M) cells. Cells were infected at MOI of 1 with DENV-H or DENV-M. Northern blots using a mix of radiolabeled probes that recognize the viral 3’UTR, capsid and NS5 (left), only the 3’UTR (middle) or only the 3’SL (right) are shown. The species of sfRNAs detected are indicated on the right. (<b>C</b>) Northern blot showing sfRNAs produced during the process of DENV adaptation to human cells. Specific probes complementary to the viral 3’UTR were used to hybridize RNA isolated from A549 cells infected with the indicated viruses. P5H, P10H and P15H indicate viral stocks passaged 5, 10 and 15 times in human cells, respectively. (<b>D</b>) Northern blot showing sfRNAs produced during the process of DENV adaptation to mosquito cells. Specific probes complementary to the viral 3’UTR were used to hybridize RNA isolated from C6/36 cells infected with the indicated viruses.</p

Dissecting the molecular determinants for sfRNA accumulation during DENV host change.

<p>(<b>A</b>) Schematic representation of mosquito-selected viral variants with mutations and deletions within the viral 3’UTR. DENV variants were found with deletions, variant S1 and S2, or point mutations variants S3 to S6. The location of the mutations is indicated. (<b>B</b>) Replication of parental (PT) and recombinant viruses carrying the mutations identified. Immunofluorescence of cells infected PT and the mutants S1 to S6 as indicated at the top. Viral RNA detection using specific radiolabeled probes complementary to capsid and NS5 coding sequences is shown at 72hpi of A549 cells (bottom). (<b>C</b>) Northern blot hybridization for sfRNA detection using transfected cells with each DENV mutant as indicated on the top. <b>(D)</b> The structure of xrRNA2 is necessary for accumulation of short sfRNA species. Schematic representation of changes in the xrRNA2 structure of mutant S6 and restored R6, and Northern blot shown the accumulated sfRNAs in each case. <b>(E)</b> Point mutations within xrRNA2 are sufficient for the switch of sfRNA pattern produced during DENV infections. Schematic representation of mutation abrogating PK formation (M-PKII) is shown on the left and Northern blot comparing the accumulation of sfRNAs detected in infected cells with M-PKII or a virus with a complete deletion of xrRNA2 (ΔSLII).</p

La Educación en América Latina: Problemas, propuestas y desafíos Tomo I: Educación y Sociedad

El libro “La Educación en América Latina: Problemas, propuestas y desafíos”, Tomo I: Educación y Sociedad, es resultado de investigaciones de estudiantes y docentes de Panamá, Colombia y Ecuador. Los capítulos del libro son resultados de investigaciones desarrolladas por sus autores y fueron arbitrados por especialistas en las temáticas respectivas, bajo el sistema revisión ciega simple.Capítulo 1. El aula invertida como herramienta de aprendizaje, mediado por el uso de las TIC / Jonathan M. Guallpa C., Rainer O. Rubín R., Willian E. Guallpa CCapítulo 2. Las paradojas del profesorado de educación física en Bogotá frente al género y el cuerpo – avance de investigación / Carlos Orlando Guavita OcampoCapítulo 3. Estado del arte: Currículo SER+STEM en el ciclo de formación K-12 de Medellín / Elena HernándezCapítulo 4. Beneficios del ajedrez en la educación preescolar en Colombia: Una revisión sistemática / Juan Manuel García SuárezCapítulo 5.Impacto de la actividad física en el contexto escolar, una visión holística desde la neuroeducación: Revisión bibliográfica / Rita Vianney Mateus RodríguezCapítulo 6. Modelo didáctico para la conservación de fuentes hídricas, un enfoque basado en roles funcionales de macroinvertebrados acuáticos / Nicolás Martínez GarcíaCapítulo 7. Avances en indagacion sobre desarrollo de capacidades cognitivas en enseñanza de cursos en producción animal / María Ligia Roa VegaCapítulo 8. La comunicación en el proceso enseñanza- aprendizaje de la matemática: un elemento diferenciador / César Augusto García EscobarCapítulo 9. Propuesta pedagógica ambiental para la apropiación de la biodiversidad de la comunidad del Colegio Eduardo Umaña: Tyguas / Nury Stella Penagos CabuyaCapítulo 10. Narrativas digitales y educación: Un camino hacia la transformación educativa / César Augusto García EscobarCapítulo 11. Percepción visual y Gestalt, una aproximación al aprendizaje de sujetos con esquizofrenia / María A. CarballedaCapítulo 12. Escuela Batuta de el Tambo: Incidencia en el proyecto de vida de exalumnos / Milton Gerardo Salas MoncayoCapítulo 13. Modelos de formación ambiental para jóvenes en Latinoamérica: Un enfoque reflexivo, crítico, social y transformacional / Yeimy Paola Lizcano PabónCapítulo 14. Estrategias metodológicas activas participativas para mejorar el proceso de enseñanza - aprendizaje de los estudiantes de bachillerato / Olga María Ojeda OjedaCapítulo 15. Aprendizaje eficaz en estudiantes de secundaria con fracaso escolar en post pandemia / Luis Alberto Barreno CubillaCapítulo 16. El pensamiento crítico y las estrategias didácticas de docentes de Panamá y Colombia / Marisela Valencia GrisalesCapítulo 17. Revisión de la literatura sobre el pensamiento divergente en la educación matemática / Luz Enid Ramírez CastañoCapítulo 18. Estilos de crianza y su influencia en conductas desadaptativas y rendimiento académico de estudiantes adolescentes / Celina PabónCapítulo 19. La enseñanza del inglés mediada por las TIC las TAC y las TEP como una propuesta metodológica en la mejora de la gestión académica / Joconda Paulina Hallo VargasCapítulo 20. Pedagogía y narrativas transmedia: Del consumo a la producción de contenidos en el aula / Nancy Rojas Martíne