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Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes.
J Virol. 2016 Nov 15; 90(22):10145-10159.JV

Abstract

Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production.

IMPORTANCE

Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.

Authors+Show Affiliations

Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.Laboratory of Virology, Wageningen University, Wageningen, The Netherlands. Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom.Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.Laboratory of Entomology, Wageningen University, Wageningen, The Netherlands.Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.Laboratory of Entomology, Wageningen University, Wageningen, The Netherlands.Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.Laboratory of Virology, Wageningen University, Wageningen, The Netherlands gorben.pijlman@wur.nl.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27581979

Citation

Göertz, G P., et al. "Noncoding Subgenomic Flavivirus RNA Is Processed By the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission By Culex Pipiens Mosquitoes." Journal of Virology, vol. 90, no. 22, 2016, pp. 10145-10159.
Göertz GP, Fros JJ, Miesen P, et al. Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes. J Virol. 2016;90(22):10145-10159.
Göertz, G. P., Fros, J. J., Miesen, P., Vogels, C. B. F., van der Bent, M. L., Geertsema, C., Koenraadt, C. J. M., van Rij, R. P., van Oers, M. M., & Pijlman, G. P. (2016). Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes. Journal of Virology, 90(22), 10145-10159. https://doi.org/10.1128/JVI.00930-16
Göertz GP, et al. Noncoding Subgenomic Flavivirus RNA Is Processed By the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission By Culex Pipiens Mosquitoes. J Virol. 2016 Nov 15;90(22):10145-10159. PubMed PMID: 27581979.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes. AU - Göertz,G P, AU - Fros,J J, AU - Miesen,P, AU - Vogels,C B F, AU - van der Bent,M L, AU - Geertsema,C, AU - Koenraadt,C J M, AU - van Rij,R P, AU - van Oers,M M, AU - Pijlman,G P, Y1 - 2016/10/28/ PY - 2016/05/15/received PY - 2016/08/23/accepted PY - 2016/10/30/pubmed PY - 2017/5/10/medline PY - 2016/9/2/entrez SP - 10145 EP - 10159 JF - Journal of virology JO - J. Virol. VL - 90 IS - 22 N2 - : Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production. IMPORTANCE: Understanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved. SN - 1098-5514 UR - https://www.unboundmedicine.com/medline/citation/27581979/Noncoding_Subgenomic_Flavivirus_RNA_Is_Processed_by_the_Mosquito_RNA_Interference_Machinery_and_Determines_West_Nile_Virus_Transmission_by_Culex_pipiens_Mosquitoes_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/27581979/ DB - PRIME DP - Unbound Medicine ER -