scholarly journals Knockdown of piRNA pathway proteins results in enhanced Semliki Forest virus production in mosquito cells

2013 ◽  
Vol 94 (7) ◽  
pp. 1680-1689 ◽  
Author(s):  
Esther Schnettler ◽  
Claire L. Donald ◽  
Stacey Human ◽  
Mick Watson ◽  
Ricky W. C. Siu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Semliki Forest Virus ◽  
Virus Production ◽  
Antiviral Responses ◽  
Mosquito Cells ◽  
Mosquito Cell Lines ◽  
Arbovirus Infection ◽  
Pirna Pathway ◽  
Sirna Pathway

The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production.

scholarly journals Knockdown of piRNA pathway proteins results in enhanced Semliki Forest virus production in mosquito cells

2014 ◽  
Vol 95 (1) ◽  
pp. 244-244
Author(s):  
Esther Schnettler ◽  
Claire L. Donald ◽  
Stacey Human ◽  
Mick Watson ◽  
Ricky W. C. Siu ◽  
...  
Keyword(s):  
Semliki Forest Virus ◽  
Virus Production ◽  
Mosquito Cells ◽  
Pirna Pathway

scholarly journals Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

2009 ◽  
Vol 83 (11) ◽  
pp. 5735-5748 ◽  
Author(s):  
Ghassem Attarzadeh-Yazdi ◽  
Rennos Fragkoudis ◽  
Yi Chi ◽  
Ricky W. C. Siu ◽  
Liane Ülper ◽  
...  
Keyword(s):  
Rna Interference ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Virus Production ◽  
Mosquito Cells ◽  
Infected Cells ◽  
Interference Response ◽  
Interfering Rna ◽  
Rnai Response ◽  
Trigger Cell Death

ABSTRACT In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.

scholarly journals Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Margus Varjak ◽  
Kevin Maringer ◽  
Mick Watson ◽  
Vattipally B. Sreenu ◽  
Anthony C. Fredericks ◽  
...  
Keyword(s):  
Small Rnas ◽  
Virus Genome ◽  
Antiviral Protein ◽  
Independent Manner ◽  
Content Type ◽  
Mosquito Cells ◽  
Antiviral Function ◽  
Pathogenic Viruses ◽  
Pirna Pathway ◽  
Sirna Pathway

ABSTRACT Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions. The small interfering RNA (siRNA) pathway is a major antiviral response in mosquitoes; however, another RNA interference pathway, the PIWI-interacting RNA (piRNA) pathway, has been suggested to be antiviral in mosquitoes. Piwi4 has been reported to be a key mediator of this response in mosquitoes, but it is not involved in the production of virus-specific piRNAs. Here, we show that Piwi4 associates with members of the antiviral exogenous siRNA pathway (Ago2 and Dcr2), as well as with proteins of the piRNA pathway (Ago3, Piwi5, and Piwi6) in an Aedes aegypti-derived cell line, Aag2. Analysis of small RNAs captured by Piwi4 revealed that it is predominantly associated with virus-specific siRNAs in Semliki Forest virus-infected cells and, to a lesser extent, with viral piRNAs. By using a Dcr2 knockout cell line, we showed directly that Ago2 lost its antiviral activity, as it was no longer bound to siRNAs, but Piwi4 retained its antiviral activity in the absence of the siRNA pathway. These results demonstrate a complex interaction between the siRNA and piRNA pathways in A. aegypti and identify Piwi4 as a noncanonical PIWI protein that interacts with members of the siRNA and piRNA pathways, and its antiviral activities may be independent of either pathway. IMPORTANCE Mosquitoes transmit several pathogenic viruses, for example, the chikungunya and Zika viruses. In mosquito cells, virus replication intermediates in the form of double-stranded RNA are cleaved by Dcr2 into 21-nucleotide-long siRNAs, which in turn are used by Ago2 to target the virus genome. A different class of virus-derived small RNAs, PIWI-interacting RNAs (piRNAs), have also been found in infected insect cells. These piRNAs are longer and are produced in a Dcr2-independent manner. The only known antiviral protein in the PIWI family is Piwi4, which is not involved in piRNA production. It is associated with key proteins of the siRNA and piRNA pathways, although its antiviral function is independent of their actions.

scholarly journals A limited role for autophagy during arbovirus infection of mosquito cells

2019 ◽  
Author(s):  
Doug E. Brackney ◽  
Maria A. Correa
Keyword(s):  
Aedes Aegypti ◽  
Viral Infections ◽  
Mosquito Control ◽  
Mosquito Cell ◽  
Mammalian Host ◽  
Transmission Cycle ◽  
Limited Role ◽  
Mosquito Cells ◽  
Arbovirus Infection

AbstractMacroautophagy is an evolutionarily conserved cellular process critical for maintaining cellular homeostasis. It can additionally function as an innate immune response to viral infection as has been demonstrated for a number of arthropod-borne (arbo-) viruses. Arboviruses are maintained in a transmission cycle between vertebrate hosts and invertebrate vectors yet the majority of studies assessing autophagy-arbovirus interactions have been limited to the mammalian host. Therefore we evaluated the role of autophagy during arbovirus infection of the invertebrate vector using the tractable Aag2 Aedes aegypti mosquito cell culture system. Our data demonstrates that autophagy is significantly induced in mosquito cells upon infection with two flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), as well as an unrelated mosquito-borne virus, chikungunya virus (CHIKV; Togaviridae). While assessing the role of autophagy during arbovirus infection, we observed a somewhat paradoxical outcome. Both induction and suppression of autophagy via torin 1 and spautin-1, respectively, resulted in increased viral titers for all three viruses, yet suppression of autophagy-related genes had no effect. Interestingly, chemical modulators of autophagy had either no effect or opposite effects in another widely used mosquito cell line, C6/36 Aedes albopictus cells. Together, our data reveals a limited role for autophagy during arbovirus infection of mosquito cells. Further, our findings suggest that commonly used chemical modulators of autophagy alter mosquito cells in such a way as to promote viral replication; however, it is unclear if this occurs directly through autophagic manipulation or other means.Author SummaryArthropod-borne (arbo) viruses, specifically those transmitted by Aedes aegypti mosquitoes, cause significant morbidity and mortality and pose a continued public health threat worldwide. Many of these viruses lack vaccines or therapeutics and current mosquito control strategies are underperforming. For these reasons, identifying vulnerabilities within the transmission cycle that can be targeted will be critical to the development of novel control interventions. Autophagy is a highly conserved cellular pathway and previous studies manipulating this pathway have shown promise in minimizing viral infections in mammalian hosts. In this study we examined arbovirus-autophagy interactions within vector mosquitoes. The goal was to elucidate the role of autophagy during infection of mosquitoes in hopes of identifying critical interactions that can be targeted by novel approaches to block infection of and transmission by vector mosquitoes.

scholarly journals Cholesterol is required in the exit pathway of Semliki Forest virus.

1993 ◽  
Vol 123 (1) ◽  
pp. 57-65 ◽  
Author(s):  
M T Marquardt ◽  
T Phalen ◽  
M Kielian
Keyword(s):  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Virus Production ◽  
Virus Particles ◽  
Target Membrane ◽  
Vesicular Stomatitis ◽  
Lipid Requirement

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.

scholarly journals Characterization of a novel insect-specific flavivirus from Brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses

2014 ◽  
Vol 95 (12) ◽  
pp. 2796-2808 ◽  
Author(s):  
Joan L. Kenney ◽  
Owen D. Solberg ◽  
Stanley A. Langevin ◽  
Aaron C. Brault
Keyword(s):  
Japanese Encephalitis ◽  
Virus Production ◽  
Inhibitory Effect ◽  
Encephalitis Virus ◽  
Growth Potential ◽  
Conserved Sequence ◽  
Mosquito Cells ◽  
Sequence Elements ◽  
Mosquito Cell Lines

In the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated ‘Nhumirim virus’; NHUV) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential and 3′ untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus’s capacity to suppress replication of representative Culex spp.-vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3′ UTR indicated NHUV to be most similar to viruses within the yellow fever serogroup and Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared with traditional insect-specific flaviviruses. This suggests that, despite apparently being insect specific, this virus probably diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in a significant reduction in virus production of West Nile virus (WNV), St Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a 106-fold and 104-fold reduction in peak titres, respectively.

scholarly journals Antiviral RNAi response against the insect-specific Agua Salud alphavirus

2021 ◽  
Author(s):  
Mine Altinli ◽  
Mayke Leggewie ◽  
Marlis Badusche ◽  
Rashwita Gyanwali ◽  
Christina Scherer ◽  
...  
Keyword(s):  
Rnai Pathway ◽  
Nucleotide Bias ◽  
Knock Out ◽  
Antiviral Responses ◽  
Ping Pong ◽  
Infected Cells ◽  
Sirna Pathway ◽  
Antiviral Role ◽  
Rnai Response

Arboviruses transmitted by mosquitoes are responsible for the death of millions of people each year. In addition to arboviruses, many insect-specific viruses (ISVs) have been discovered in mosquitoes in the last decade. ISVs, in contrast to arboviruses transmitted by mosquitoes to vertebrates, cannot replicate in vertebrate cells even when they are evolutionarily closely related to arboviruses. The alphavirus genus includes many arboviruses, although only a few ISVs have been discovered from this genus so far. Here, we investigate the interactions of a recently isolated insect-specific alphavirus, Agua-Salud alphavirus (ASALV), with its mosquito host. RNAi is one of the essential antiviral responses against arboviruses, although there is little knowledge on the interactions of RNAi with ISVs. Through knock-down of transcripts of the different key RNAi pathway (siRNA, miRNA and piRNA) proteins, we show the antiviral role of Ago2 (siRNA), Ago1 (miRNA), and Piwi4 proteins against ASALV in Aedes aegypti derived cells. ASALV replication increased in Dicer2 and Ago2 knock-out cells, confirming the antiviral role of the siRNA pathway. In infected cells, mainly ASALV-specific siRNAs are produced while piRNAs, with the characteristic nucleotide bias resulting from ping-pong amplification, are only produced in Dicer2 knock-out cells. Taken together, ASALV interactions with the mosquito RNAi response differs from arthropod-borne alphaviruses in some aspects, although they also share some commonalities. Further research is needed to understand whether the identified differences can be generalised to other insect-specific alphaviruses.

scholarly journals Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghuvir Viswanatha ◽  
Enzo Mameli ◽  
Jonathan Rodiger ◽  
Pierre Merckaert ◽  
Fabiana Feitosa-Suntheimer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Mosquito Species ◽  
Mosquito Cell ◽  
Exchange System ◽  
Public Health Burden ◽  
Cassette Exchange ◽  
Mosquito Cells ◽  
Mosquito Cell Lines ◽  
Genome Scale

AbstractMosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.

Glioblastoma invasion and NMDA receptors: A novel prospect

2019 ◽  
Vol 106 (3) ◽  
pp. 250-260 ◽  
Author(s):  
DN Nandakumar ◽  
P Ramaswamy ◽  
C Prasad ◽  
D Srinivas ◽  
K Goswami
Keyword(s):  
Glutamate Receptors ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Transcript Level ◽  
Glioblastoma Cells ◽  
Invasion And Migration ◽  
Matrigel Invasion ◽  
Nmdar Activation ◽  
And Migration

Purpose Glioblastoma cells create glutamate-rich tumor microenvironment, which initiates activation of ion channels and modulates downstream intracellular signaling. N-methyl-D-aspartate receptors (NMDARs; a type of glutamate receptors) have a high affinity for glutamate. The role of NMDAR activation on invasion of glioblastoma cells and the crosstalk with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is yet to be explored. Main methods LN18, U251MG, and patient-derived glioblastoma cells were stimulated with NMDA to activate NMDAR glutamate receptors. The role of NMDAR activation on invasion and migration and its crosstalk with AMPAR were evaluated. Invasion and migration of glioblastoma cells were investigated by in vitro trans-well Matrigel invasion and trans-well migration assays, respectively. Expression of NMDARs and AMPARs at transcript level was evaluated by quantitative real-time polymerase chain reaction. Results We determined that NMDA stimulation leads to enhanced invasion in LN18, U251MG, and patient-derived glioblastoma cells, whereas inhibition of NMDAR using MK-801, a non-competitive antagonist of the NMDAR, significantly decreased the invasive capacity. Concordant with these findings, migration was significantly augmented by NMDAR in both cell lines. Furthermore, NMDA stimulation upregulated the expression of GluN2 and GluA1 subunits at the transcript level. Conclusions This study demonstrated the previously unexplored role of NMDAR in invasion of glioblastoma cells. Furthermore, the expression of the GluN2 subunit of NMDAR and the differential overexpression of the GluA1 subunit of AMPAR in both cell lines provide a plausible rationale of crosstalk between these calcium-permeable subunits in the glutamate-rich microenvironment of glioblastoma.

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