Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness

Manokaran, Gayathri; Finol, Esteban; Wang, Chunling; Gunaratne, Jayantha; Bahl, Justin; Ong, Eugenia Z.; Tan, Hwee Cheng; Sessions, October M.; Ward, Alex M.; Gubler, Duane J.; Harris, Eva; García-Blanco, Mariano A.; Ooi, Eng Eong

doi:10.1126/science.aab3369

Cited by 343 publications

(457 citation statements)

References 39 publications

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“…Three days after dsRNA injection, 24-h-starved mosquitoes were offered a blood meal comprised of 40% washed erythrocytes from specific-pathogen-free pig's blood (PWG Genetics), 5% 5 mM ATP (Thermo Fisher Scientific), 5% human serum (Sigma), and a 50% volume of RPMI containing virus diluted to 10 7 PFU/ml. Blood titers were validated in a plaque assay as described previously (69). The infectious blood meal was maintained at 37°C using a hemotek feeder system (Discovery Workshops) covered by a stretched pig's intestine.…”

Section: Methodsmentioning

confidence: 99%

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RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

Campos

Wong

Xie

et al. 2017

J Virol

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The Flavivirus genus contains several arthropod-borne viruses that pose global health threats, including dengue viruses (DENV), yellow fever virus (YFV), and Zika virus (ZIKV). In order to understand how these viruses replicate in human cells, we previously conducted genome-scale RNA interference screens to identify candidate host factors. In these screens, we identified ribosomal proteins RPLP1 and RPLP2 (RPLP1/2) to be among the most crucial putative host factors required for DENV and YFV infection. RPLP1/2 are phosphoproteins that bind the ribosome through interaction with another ribosomal protein, RPLP0, to form a structure termed the ribosomal stalk. RPLP1/2 were validated as essential host factors for DENV, YFV, and ZIKV infection in two human cell lines: A549 lung adenocarcinoma and HuH-7 hepatoma cells, and for productive DENV infection of Aedes aegypti mosquitoes. Depletion of RPLP1/2 caused moderate cell-line-specific effects on global protein synthesis, as determined by metabolic labeling. In A549 cells, global translation was increased, while in HuH-7 cells it was reduced, albeit both of these effects were modest. In contrast, RPLP1/2 knockdown strongly reduced early DENV protein accumulation, suggesting a requirement for RPLP1/2 in viral translation. Furthermore, knockdown of RPLP1/2 reduced levels of DENV structural proteins expressed from an exogenous transgene. We postulate that these ribosomal proteins are required for efficient translation elongation through the viral open reading frame. In summary, this work identifies RPLP1/2 as critical flaviviral host factors required for translation.IMPORTANCE Flaviviruses cause important diseases in humans. Examples of mosquito-transmitted flaviviruses include dengue, yellow fever and Zika viruses. Viruses require a plethora of cellular factors to infect cells, and the ribosome plays an essential role in all viral infections. The ribosome is a complex macromolecular machine composed of RNA and proteins and it is responsible for protein synthesis. We identified two specific ribosomal proteins that are strictly required for flavivirus infection of human cells and mosquitoes: RPLP1 and RPLP2 (RPLP1/2). These proteins are part of a structure known as the ribosomal stalk and help orchestrate the elongation phase of translation. We show that flaviviruses are particularly dependent on the function of RPLP1/2. Our findings suggest that ribosome composition is an important factor for virus translation and may represent a regulatory layer for translation of specific cellular mRNAs. (1)(2)(3)(4). Dengue viruses (DENV-1 to -4), the most prevalent of all arthropod-borne viruses, infect approximately 390 million people per year (1); yellow fever virus (YFV), which causes life-threatening disease (5) has reemerged this year in Africa (6); Zika virus (ZIKV) is currently responsible for a pandemic in the Americas that has caused grave concern because of associations with birth defects and Guillain-Barré syndrome (3). KEYWORDS flavivirus, ribosomal proteins, tran...

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Section: Methodsmentioning

confidence: 99%

“…Mosquitoes were allowed to feed for 2 h. Engorged females were selected and maintained with access to sugar in the insectary. Six days post-oral infection, individual mosquitoes were homogenized in 500 l of RPMI, filtered through a 0.22-m filter (Sartorius) and determined the titer of by plaque assay (69).…”

Section: Methodsmentioning

confidence: 99%

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

Campos

Wong

Xie

et al. 2017

J Virol

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“…In mammalian cells, sfRNA is essential for flavivirus-induced cytopathicity and pathogenicity (25). Importantly, sfRNA acts as an antagonist of both interferon-and retinoic acid-inducible gene-I-like receptor-dependent innate immune responses (31,32). Accordingly, WNV mutants deficient in sfRNA production are attenuated in mice (25,29).…”

mentioning

confidence: 99%

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

Göertz

Fros

Miesen

et al. 2016

J Virol

107

158

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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. IMPORTANCEUnderstanding 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. Viruses from the genus Flavivirus (family Flaviviridae), such as the highly pathogenic dengue virus (DENV...

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“…This sub genomic RNA seems to be responsible for regulating the cellular cytosolic receptor RIG-I that recognizes viral RNA. This signaling pathway would be similar to the one adopted by Dengue virus [17].…”

Section: Mechanism Of Action Of Zika Virusmentioning

confidence: 83%

Brief Insights into Zika-Microcephaly Mechanism

Gm¹,

Itg²,

Aa³

2017

Clin Exp Pharmacol

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After recent reports attesting a straight correlation between Zika infection and brain disorders in new-borns, research questions now focus on establishing causality and the mechanisms underlying it. Studies involving monolayer cultures, murine and human brain tissue slices and the cerebral organoid system have provided important information regarding neuronal damage, but the precise mechanisms underlying neuronal cell tropism and cell damage have not been elucidated. Herein, we discuss the possibility that Zika virus proteins enter the nucleus by a brief search for nuclear signal localization and for potential nucleosome binding motifs using a bioinformatics approach and point to other questions that should be the focus of research aiming to understand Zika-virus associated cell-damage.

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Dengue subgenomic RNA binds TRIM25 to inhibit interferon expression for epidemiological fitness

Cited by 343 publications

References 39 publications

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

RPLP1 and RPLP2 Are Essential Flavivirus Host Factors That Promote Early Viral Protein Accumulation

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

Brief Insights into Zika-Microcephaly Mechanism

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