Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus
- Jelke J. Fros†1,
- Corinne Geertsema†1,
- Karima Zouache2,
- Jim Baggen1,
- Natalia Domeradzka1,
- Daniël M. van Leeuwen1,
- Jacky Flipse1,
- Just M. Vlak1,
- Anna-Bella Failloux2 and
- Gorben P. Pijlman1Email author
© Fros et al. 2015
Received: 30 June 2015
Accepted: 3 September 2015
Published: 17 September 2015
Chikungunya virus (CHIKV) is an arthritogenic alphavirus (family Togaviridae), transmitted by Aedes species mosquitoes. CHIKV re-emerged in 2004 with multiple outbreaks worldwide and recently reached the Americas where it has infected over a million individuals in a rapidly expanding epidemic. While alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsP3 remain elusive. CHIKV nsP3 modulates the mammalian stress response by preventing stress granule formation through sequestration of G3BP. In mosquitoes, nsP3 is a determinant of vector specificity, but its functional interaction with mosquito proteins is unclear.
In this research we studied the domains required for localization of CHIKV nsP3 in insect cells and demonstrated its molecular interaction with Rasputin (Rin), the mosquito homologue of G3BP. The biological involvement of Rin in CHIKV infection was investigated in live Ae. albopictus mosquitoes.
In insect cells, nsP3 localized as cytoplasmic granules, which was dependent on the central domain and the C-terminal variable region but independent of the N-terminal macrodomain. Ae. albopictus Rin displayed a diffuse, cytoplasmic localization, but was effectively sequestered into nsP3-granules upon nsP3 co-expression. Site-directed mutagenesis showed that the Rin-nsP3 interaction involved the NTF2-like domain of Rin and two conserved TFGD repeats in the C-terminal variable domain of nsP3. Although in vitro silencing of Rin did not impact nsP3 localization or CHIKV replication in cell culture, Rin depletion in vivo significantly decreased the CHIKV infection rate and transmissibility in Ae.albopictus.
We identified the nsP3 hypervariable C-terminal domain as a critical factor for granular localization and sequestration of mosquito Rin. Our study offers novel insight into a conserved virus-mosquito interaction at the molecular level, and reveals a strong proviral role for G3BP homologue Rin in live mosquitoes, making the nsP3-Rin interaction a putative target to interfere with the CHIKV transmission cycle.
Chikungunya virus (CHIKV) is a member of the genus Alphavirus (family Togaviridae), a group of widely distributed human and animal pathogens. The New world alphaviruses can cause encephalitic disease in humans, while the Old world alphaviruses, including CHIKV, Sindbis virus (SINV), O’nyong nyong virus (ONNV) and Semliki Forest virus (SFV), are associated with rash, fever and (sometimes chronic) arthritis . CHIKV is transmitted by vector mosquitoes and actively replicates in mosquitoes of the genus Aedes, in particular Ae. aegypti and Ae. albopictus. CHIKV is endemic in most of Central Africa and South-East Asia. In 2005-2006, major outbreaks of CHIKV occurred on the Indian Ocean islands of Mayotte, Seychelles, Mauritius and La Réunion, where more than one-third of the population was infected and resultant deaths were reported  (Schwartz & Albert, 2010). In 2006-2007 CHIKV caused a major outbreak in India (~1.3 million cases), followed by outbreaks in the rest of South-East Asia . The first autochthonous CHIKV outbreak in Europe occurred in Italy in 2007, where more than 200 people were infected . Likewise, a local CHIKV transmission by Ae. albopictus occurred in France in 2010 (2 cases) and 2014 (4 cases) [4, 5]. The outbreak that started in the Caribbean in 2013 has spread to the American main land and by December 2014 over a million cases have been reported throughout the Americas [6, 7]. No licensed vaccine or antiviral treatment against CHIKV is available at present, but many prototype vaccines are in development [8–14].
CHIKV proteins are translated from a viral single-stranded positive-sense RNA of approximately 11.8 kb [15, 16]. The four alphavirus non-structural proteins (nsP1-4) are directly translated from genomic RNA and form a replication complex (RC), which is associated with the plasma membrane and endosomal membranes . A number of functions has been assigned to alphavirus nsPs: nsP1 is involved in capping of RNA  and is the membrane anchor of the RC , nsP2 has protease and helicase activity, causes host shut-off and inhibits interferon-induced JAK-STAT signaling and the unfolded protein response [18–21]. NsP4 serves as RNA-dependent RNA polymerase . The functions of nsP3 are more enigmatic, but the protein is highly phosphorylated on serine and threonine residues [22, 23] and is essential for RNA synthesis  as part of the viral RC . CHIKV nsP3 can be divided into three regions; the macrodomain (amino acids 1-160) is conserved among alphaviruses, Coronaviridae, rubella and hepatitis E viruses and can bind ADP-ribose, RNA and DNA in vitro . The central, zinc-binding domain (amino acids 161-324) is conserved among alphaviruses, while the C-terminal region is highly variable and even shows substantial dissimilarity between CHIKV strains [26, 27].
SINV nsP3 is found in cytoplasmic granules or foci which are also comprised of various host proteins [28, 29]. In both mammalian and mosquito cells the cellular protein Ras-GAP SH3 domain binding proteins (G3BPs) were found in nsP3-granules [28, 29]. G3BPs are ubiquitously expressed proteins conserved among eukaryotes. Mammals have three G3BPs: G3BP1, 2a and 2b, which are expressed from 2 distinct genes, while insects have one, named Rasputin (Rin) . Mammalian G3BP is a widely used marker for stress granules (SGs) , which are cytoplasmic messenger ribonucleoproteins (mRNPs) that form when translation is impaired in response to several types of cellular stress . NsP3-G3BP-granules are the explicit phenotype of the first reported function of alphavirus nsP3, as we have recently shown that CHIKV nsP3-G3BP granule formation prevents the establishment of bona fide SGs . These nsP3-G3BP-granules did not contain other crucial SG marker eIF3 and cells expressing nsP3 were unable to respond normally to oxidative stress . The inhibition of SGs via an interaction between nsP3 and G3BP has now also been confirmed for SFV . Details on the interaction between nsP3 and mosquito Rin are currently lacking.
Mosquito vectors display different degrees of vector competence for different CHIKV isolates . Vector competence is a complex trait involving an interplay between vectors, pathogens and environmental factors  but the molecular details are not well understood. While it has been firmly established that antiviral RNAi pathways play a major role in controlling CHIKV and other arboviral infections in the mosquito [37, 38], other mechanisms of virus-host interactions that influence vector competence and the roles therein of viral (non) structural proteins need to be examined. Recently, however, nsP3 of ONNV (transmitted by Anopheles mosquitoes), has been uncovered as an important determinant for vector specificity. CHIKV does not normally infect An. gambiae, however, a chimeric virus containing ONNV nsP3 in a CHIKV infectious clone backbone became infectious for An. gambiae mosquitoes . Thus, it is hypothesized that specific molecular interactions between mosquito host factors and alphavirus nsP3 determine the vector specificity.
In the present study, we investigated the formation of nsP3-granules in insect cells and elucidated the molecular interactions between nsP3 and Rin. Moreover, we studied the effect of Rin silencing on virus replication in mosquito cell culture and on vector competence for CHIKV in mosquitoes. We show that Rin is an important, proviral determinant for CHIKV infection and dissemination in live mosquitoes.
Cells and viruses
Spodoptera frugiperda Sf21 cells were cultured in Grace’s medium (Invitrogen) with 10 % fetal bovine serum (FBS; Invitrogen) and Sf9 cells in Sf900 medium (Invitrogen) with 5 % FBS. Aedes albopictus U4.4 cells and C6/36 cells were cultured in Leibovitz’s medium (Invitrogen) supplemented with 10 % FBS, 2 % tryptose phosphate (Invitrogen) and 1 % non-essential amino acids (Invitrogen). All insect cells were cultured at 27 °C. Vero E6 and HEK293t mammalian cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10 % FBS at 37 °C and 5 % CO2. Infections in cell culture were performed with CHIKV isolate S27 and mosquitoes were infected with CHIKV 06-021 strain.
Oligonucleotides used in this study
Transient expression of nsP3 and Rin
Insect cells were transfected with the indicated expression plasmids using Fectofly I (Polyplus) or ExpreS2 Insect-TR (ExpreS2ion Biotechnologies). Mammalian cells were transfected with lipofectamine 2000 (Invitrogen). Twenty-four hours post transfection the fluorescence of EGFP-nsP3 and/or Rin-mCherry was analysed using a Zeiss Axio Observer Z1m inverted microscope in combination with an X-Cite 120 series lamp.
Rin Knockdown experiments
Linear DNA of Ae. albopictus Rin and firefly luciferase was generated by PCR from pGEM-T easy plasmids using the T7 universal primer (New England Biolabs) and T7-pGEM-Teasy-R and double-stranded (ds) RNA was synthesized in vitro with T7 RNA polymerase (Invitrogen). Knockdown in cell culture was performed by transfecting dsRNA into U4.4 cells grown in 24-wells plates (1 μg of RNA per well) using Fugene (promega). One day later, cells were transfected with plasmid pIB-EGFP-nsP3 to monitor nsP3-granule formation or infected with CHIKV at a multiplicity of infection (MOI) of five. At the indicated times post infection, the medium was removed from the cells and used in end point dilution assays on Vero E6 cells. The remaining cells were lysed in TRIzol (life technologies) reagent and total RNA was isolated. The RNA was DNase treated (Applied Biosystems) and reverse transcribed using random primers. Rin, S7 and genomic CHIKV cDNA were amplified (primers: Rin F2/R2, S7 F/R and nsP1 int F/R2) and detected with real-time PCR platinum SYBR Green (Invitrogen), in a Rotor Gene RG-3000 (Corbett Research).
In parallel experiments, cells were washed with PBS and lysed in SDS-loading buffer [100 mM Tris-Cl (pH 6.8), 4 % (w/v) sodium dodecyl sulfate (SDS), 0.2 % (w/v) bromophenol blue, 20 % (v/v) glycerol and 200 mM β-mercaptoethanol]. Samples were heated at 95 °C for 10 min, clarified by centrifugation for one min at 13 000 r.p.m and loaded on a 12 % SDS-Polyacrylamide gel. After electrophoresis, denatured proteins were transferred to an Immobilonmembrane (Millipore) for analysis by Western blotting. Membranes were blocked in 3 % skimmed milk in PBS with 0.05 % Tween 60 (PBST) for 1 h at room temperature. Membranes were washed three times for 5 min each with PBST and subsequently incubated for one hat room temperature with rabbit polyclonal anti-E2 (diluted 1 : 20000; ) and anti-β-tubulin (diluted 1 : 4000; Abcam) in PBST, respectively. Membranes were washed and treated with alkaline phosphatase conjugated with goat anti-rabbit IgG mAb (Sigma), diluted 1 : 3000 inPBST, for 45 min at room temperature. Membranes were washed twice for 5 min each with PBST and once for 10 min with AP buffer [100 mM NaCl, 5 mM MgCl2, 100 mM Tris/HCl (pH 9.5), 0.1 % Tween 20]. Proteins were detected by nitro blue tetrazolium chloride/BCIP staining (Roche).
In vivo knock down of Rin was performed in Ae. albopictus mosquitoes originating from la Reunion island (Providence, F11 generation). 500 ng of dsRin or dsLuc RNA was injected directly in the thorax of female mosquitoes (Drummond nanoject II). Two days post injection mosquitoes were either sacrificed and stored at -80 °C or orally infected with an infectious blood meal containing 107 pfu/ml of CHIKV 06-021 strain. Mosquito rearing and preparation of the infectious blood meal was reported previously . Fully engorged females were selected and incubated in climatic chambers (Binder) at 28 °C, with a light: dark cycle of 16 h: 8 h and 70 % relative humidity. Forced salivation were performed 6 days post-infection as described previously . Saliva and mosquitoes were stored at -80 °C pending further analysis.
Frozen mosquitoes were dissected, separating bodies (abdomen and thorax) from the head. Individual mosquito bodies and heads were homogenized in the bullet blender storm (Next Advance) in 100 μl of DMEM Hepes (Gibco)-buffered medium supplemented with 10 % FBS containing penicillin (100 IU/ml), streptomycin (100 μg/ml), fungizone (2,5 μg/ml) and gentamycin (50 μg/ml) and spun down for 90 s at 14,000 rpm in a table top centrifuge. Thirty μl of the supernatant from the mosquito homogenate or the saliva-containing mixture was incubated on a monolayer of Vero cells in a 96-wells plate. After 2-4 h the medium was replaced by 100 μl of fresh cell culture medium, fully supplemented with antibiotics. Wells were scored for virus specific cytopathic effects (CPE) at three days post infection. Viral titres were determined using 10 μl of the supernatant from the mosquito homogenates in an end point dilution assay on Vero E6 cells. Infections were scored by CPE, three days post infection.
Mosquito bodies and heads were scored positive or negative for CHIKV infection and significant differences were calculated using the Fisher’s exact test (P < 0.05). Differences in CHIKV titers (TCID50/ml) in infected mosquito bodies and heads were calculated using the Mann Whitney test (P < 0.05).
CHIKV nsP3 displays granular localization in both insect and mammalian cells
The gene encoding CHIKV nsP3 contains a natural leaky (opal) stop codon, six codons upstream of the nsP3-4 cleavage site. Two isoforms of nsP3 are likely to be expressed from this gene during viral infections. To investigate whether both isoforms would have the same intracellular localization, two additional EGFP-fusions were made, one with nsP3 lacking the C-terminal six amino acids (CHIKV nsP3-DDEL) and one with nsP3 lacking the leaky stop codon (CHIKV nsP3-dUGA) (Fig. 1a). When transiently expressed in insect cells, CHIKV nsP3 and the two isoforms displayed an identical granular localization (Fig. 1c), which shows that the terminal six amino acids of CHIKV nsP3 do not impact its subcellular localization.
The conserved domain of CHIKV nsP3 is sufficient for multimerization but the variable domain is required for the formation of nsP3-granules
In mammalian cells, the C-terminal variable domain was found to be essential for nsP3 granule formation, and upon deletion the localization changed to a filamentous phenotype . To determine which domains within nsP3 are responsible for the formation of nsP3-granules, truncated versions of nsP3 fused with EGFP (Fig. 1a) were expressed in insect cells (Fig. 1d). For these studies we used Sf21 cells because of their superior transfection efficiency as compared to mosquito cells. Removal of the entire C-terminal variable region (nsP3.2) resulted in the formation of filamentous, cytoplasmic structures. (Fig. 1d, left). To investigate whether the macrodomain could be eliminated from nsP3 without affecting its localization, it was deleted from EGFP-fused nsP3 and nsP3.2, yielding truncated mutants nsP3.7 and nsP3.8, respectively (Fig. 1a). When expressed in insect cells, nsP3.7 showed an identical granular phenotype as full-length nsP3, whereas nsP3.8 formed filaments that were very similar to those produced by nsP3.2 (Fig. 1d, middle). We observed similar filaments upon expression of these nsP3.2 or nsP3.8 constructs in mammalian cells, and showed that they did not colocalize with cytoskeleton markers actin or tubulin . To investigate if the C-terminal, variable region alone could cause granule formation, it was N-terminally fused to EGFP (nsP3.10) (Fig. 1a) and expressed in insect cells. The localization of nsP3.10 was diffuse, nuclear-cytoplasmic (Fig. 1d, right), showing that the C-terminal region of CHIKV nsP3 is required, but not sufficient for the formation of nsP3 granules.
NsP3 granules co-localize with Rasputin, the insect homolog of mammalian G3BP
The subcellular localization of Rin was studied by transient expression in insect cells as C-terminal fusion with mCherry, in a similar fashion to a previously described and functional G3BP-EGFP fusion . When expressed in insect cells, Rin was evenly distributed throughout the cytoplasm (Fig.2b, left). However, when Rin was co-expressed with nsP3, which localized to small nsP3 granules (Fig. 2b, right), both proteins displayed strong co-localization and formed much larger granules (Fig. 2c, top). These nsP3- and Rin-positive granules or aggregates were larger and more asymmetrical than normal nsP3-granules. In this experiment, Rin was transiently (over) expressed, which may explain the large size of the granules. In contrast, when mCherry-Rin was co-expressed with the C-terminal truncated, filamentous mutants EGFP-nsP3.8 (Fig. 2c, bottom) or EGF-nsP3.2 (not shown), Rin did not co-localize with the filaments formed by these mutants and retained its diffuse, cytoplasmic localization (Fig. 2c, bottom). In conclusion, the C-terminal hypervariable domain of nsP3 is important for the interaction with Rin in insect cells.
The C-terminal TFGD repeats in the variable domain of CHIKV nsP3 interact with Rasputin
Next, we mutated both domains of the two conserved TFGD repeats separately or together resulting in pIB-EGFP-nsP3-FG479AA, pIB-EGFP-nsP3-FG497AA, and pIB-EGFP-nsP3-FG479AA/FG497AA. The single and double EGFP-nsP3 TFGD mutants were transiently expressed in insect cells together with Rin-mCherry. Both the single FG479AA and FG497AA mutants still sequestered Rin into nsP3-granules (Fig. 3c, top and middle panels). The double TFGD mutant, however, displayed a completely diffuse intracellular distribution of Rin (Fig. 3c, bottom panel) but retained a normal granular distribution similar to wildtype EGFP-nsP3 (Fig. 1b).
These results indicate that deletion of the SH3-domain binding motif does abrogate the formation of nsP3 granules, but the formation of nsP3-granules and the interaction with Rin is retained when conserved amino acids within this domain are substituted for alanines. However, the formation of CHIKV nsP3-Rin-granules is also abrogated when both the C-terminal conserved TFGD repeats are mutated, suggesting that these motifs are involved in the nsP3-Rin interaction.
The NTF2-like domain of mosquito Rasputin interacts with CHIKV nsP3
Effect of Rasputin silencing on the formation of CHIKV nsP3-granules
Rasputin silencing reduces the CHIKV infection rate in Aedes albopictus without affecting CHIKV infection in cell culture
On day six post infection, mosquito saliva was obtained and later the mosquito heads were separated from their thorax and abdomen, to be able to distinguish between infections that were transmissible, fully disseminated or limited to the mosquito body, respectively. Mosquito saliva or homogenate from either heads or bodies were incubated on Vero cells to determine the presence of CHIKV. CHIKV infected 75 % of the mosquitoes that were injected with dsLuc RNA (Fig. 6c). In these mosquitoes the infection had also disseminated into the head of the mosquito. In addition, it resulted in two mosquitoes with infectious saliva (Fig. 6d, red symbols). Injection with dsRin significantly reduced (P < 0.05) the number of infected mosquito bodies and heads to 40 % (Fig. 6c). No mosquitoes had infectious saliva. Furthermore, Rin silencing also reduced the viral titers in the infected mosquitoes, with a significant >20-fold reduction in the mosquito heads (P < 0.05, Fig. 6d). Together, these results show a significant effect of Rin silencing on the infection rate and dissemination of CHIKV in Ae. albopictus.
Discussion and conclusions
In this study we investigated the localization of CHIKV nsP3 and its interaction with mosquito Rin in insect cells and live mosquitoes. Our results show that the intracellular distribution of CHIKV nsP3 is conserved in cells from mammalian and insect origin. In mosquito cells, CHIKV nsP3 forms cytoplasmic granules, which are highly similar to the nsP3-G3BP granules that inhibit the formation of SGs in mammalian cells . Removal of the variable domain results in the formation of filaments. Both the granular or filamentous nsP3 structures form independent of the N-terminal macrodomain. This indicates that multimerization of nsP3 is attributed to the central conserved domain. How nsP3 multimerizes into these two diverse cytoplasmic phenotypes is unknown, however, removal of the C-terminal variable domain may cause a conformational change or affect interactions with host factors which allows nsP3 to form long cytoplasmic filaments. Whereas Rin is clearly sequestered into nsP3-granules and transient overexpression of Rin may increase the size of the nsP3-granules, silencing of Rin and reducing its co-localization with nsP3 by mutagenesis shows that Rin is not required for the formation of nsP3-granules (Figs. 3, 4 and 5). Elucidation of the exact structural composition of these nsP3 granules and filaments, however, needs further experimentation.
In mammalian cells, nsP3-granule formation and the inhibition of SGs is lost when the conserved SH3-domain binding motif is removed from the variable domain of nsP3 . Similarly, nsP3-d398/406 was diffuse throughout insect cells. Amino acid substitutions within the SH3-domain binding motif, however, did not affect the formation of nsP3-granules or the sequestration of mosquito Rin into granules. Apparently, the amino acid substitutions were not sufficient to abrogate the interaction between nsP3 and Rin. Alternatively, deletion of the complete SH3-domain binding motif may have disrupted the folding of nsP3, rendering a dysfunctional protein that can no longer execute its normal function. Indeed, deletion of the entire SH3-domain binding motif from a CHIKV replicon yielded a replication-negative phenotype .
G3BP and nsP3 were also shown to interact via two conserved repeats in the C-terminal variable domain of nsP3 . When we replaced the phenylalanine and glycine from either one of the nsP3 C-terminal TFGD repeat with alanines there was no apparent change in the co-localization of nsP3 and Rin. However, the interaction between nsP3 and Rin was completely lost when both TFGD repeats were mutated (Fig. 3c). This suggests a direct interaction between these amino acid repeats and Rin, and shows that both repeats are redundant for the interaction with Rin. Ae. albopictus Rin was isolated from U4.4 cells. Sequence analysis revealed that the N-terminal NTF2-like domain has high homology with other NTF2-like domains including human G3BP (Fig. 4a). The three-dimensional crystal structures of the NTF2-like domains from Drosophila Rin and human G3BP have recently been resolved, and contain a binding pocket for FxFG containing peptides [44, 45]. The NTF2-like domain from Ae. albopictus Rin was modelled onto that of Drosophila, showing high resemblance (Fig. 4b). As expected from this model, point mutations in the binding pocket of the Rin NTF2-like domain (position F34) greatly reduced the interaction between nsP3 and Rin (Fig. 4c). Although Rin still partly localized to nsP3-granules, this result does provide evidence of an interaction between CHIKV nsP3 and the NTF2-like FxFG binding pocket of Rin. A recent study has confirmed this interaction between homologous sites in SFV nsP3 and mammalian G3BP . Additional interactions were predicted between FxFG peptides and residues in the NTF2-like binding pocket of G3BP , which could explain the strongly reduced, but not completely abolished, interaction of mutated Rin with nsP3.
Rasputin silencing during oral, in vivo infections resulted in a marked decrease in the percentage of CHIKV infected mosquitoes in concert with strongly reduced viral titers in the mosquito heads (Fig. 6). Interestingly, in vitro Rin silencing did not affect CHIKV infection in cultured mosquito cells (Fig. 7), which is in agreement with in vitro studies with SINV and siRNA-mediated G3BP1/2 silencing in mammalian cells . However, simultaneous G3BP1/2 silencing reduced early CHIKV replication in 293/ACE2 cells . These observations suggest that Rin may be involved in the initial establishment of a productive infection and/or affects CHIKV infections in specific mosquito tissues, e.g. the midgut. It also is an indication that results obtained in cell lines are not always a good proxy for results obtained in vivo. Indeed, midgut barriers have been described in arthropods that limit arbovirus replication and/or dissemination through the organism [49, 50]. The interaction between nsP3 and Rin may play a significant role in modulating the midgut antiviral responses. Interestingly, exchanging the nsP3 genes of CHIKV and ONNV made CHIKV infectious for An. gambiae . Moreover, replacing only the C-terminal end of CHIKV nsP3, which is required for Rin interaction, with that of ONNV was sufficient to orally infect An. gambiae with CHIKV. This fragment encompasses the variable domain of CHIK nsP3, suggesting a strong role for the C-terminal domain of nsP3 in facilitating oral infection in specific vector species.
The decreased infectivity of CHIKV in Rin depleted mosquitoes suggests a proviral role for Rin. In Drosophila, Rin is involved in Ras and Rho-mediated signaling, cell proliferation and oogenesis and has been suggested to form RNase inhibitor complexes [51–53], which could protect CHIKV RNA replication during the initial infection in the mosquito midgut. Clearly, the molecular details of the nsP3-Rin interaction in mosquitoes should be examined in follow up studies to uncover the exact mechanism.
CHIKV nsP3 co-localizes with G3BP homologue Rasputin in cytoplasmic granules.
Two TFGD repeats within the C-term hypervariable domain of nsP3 interact with the NTF2-like domain of Rasputin.
Rasputin silencing does not disrupt the granular localization of nsP3 and has no profound effects on viral replication in vitro.
Rasputin silencing in vivo significantly reduces the CHIKV infection rate and dissemination in live Aedes albopictus mosquitoes.
We thank Els Roode for technical support concerning the BSL3 laboratory in Wageningen. We acknowledge Marie Vazeille for injecting mosquitoes and Anubis Vega Rua and Laurence Mousson for their assistance in forced salivation.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Schwartz O, Albert ML. Biology and pathogenesis of chikungunya virus. Nat Rev Microbiol. 2010;8:491–500. doi:10.1038/nrmicro2368. Nature Publishing Group.View ArticlePubMedGoogle Scholar
- Thiboutot MM, Kannan S, Kawalekar OU, Shedlock DJ, Khan AS, Sarangan G, et al. Chikungunya: a potentially emerging epidemic? PLoS Negl Trop Dis. 2010;4:e623. doi:10.1371/journal.pntd.0000623.PubMed CentralView ArticlePubMedGoogle Scholar
- Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, et al. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet. 2007/12/07 ed. 2007;370: 1840–1846. doi:10.1016/S0140-6736(07)61779-6
- Grandadam M, Caro V, Plumet S, Thiberge JM, Souares Y, Failloux AB, et al. Chikungunya virus, southeastern France. Emerg Infect Dis. 2011/05/03 ed. 2011;17:910–913.Google Scholar
- ECDC_Epidemiological update- autochthonous cases of chikungunya fever in France.pdf [Internet]. Available: www.ecdc.europa.eu.
- Powers AM. Risks to the Americas associated with the continued expansion of chikungunya virus. J Gen Virol. 2015;96:1–5. doi:10.1099/vir.0.070136-0.View ArticlePubMedGoogle Scholar
- Countries and territories where chikungunya cases have been reported [Internet]. 2014 p. 2014. Available: http://www.cdc.gov/chikungunya
- Metz SW, Gardner J, Geertsema C, Le TT, Goh L, Vlak JM, et al. Effective chikungunya virus-like particle vaccine produced in insect cells. PLoS Negl Trop Dis. 2013;7:e2124. doi:10.1371/journal.pntd.0002124.PubMed CentralView ArticlePubMedGoogle Scholar
- Metz SW, van den Doel P, Geertsema C, Osterhaus AD, Vlak JM, Martina BE, et al. Chikungunya virus-like particles are more immunogenic in a lethal AG129 mouse model compared to glycoprotein E1 or E2 subunits. Vaccine. 2013;9:6092–6. doi:10.1016/j.vaccine.2013.09.045.View ArticleGoogle Scholar
- Powers AM. Chikungunya virus control: Is a vaccine on the horizon? Lancet Elsevier Ltd; 2014;384:2008–9. doi:10.1016/S0140-6736(14)61290-3.View ArticleGoogle Scholar
- Hallengärd D, Lum F, Kümmerer BM, Lulla A, Lulla V, García-arriaza J, et al. Prime-Boost Immunization Strategies against Chikungunya Virus. J Virol. 2014;88:13333–43. doi:10.1128/JVI.01926-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Weaver SC, Osorio JE, Livengood J a, Chen R, Stinchcomb DT. Chikungunya virus and prospects for a vaccine. Expert Rev Vaccines. 2012;11:1087–101. doi:10.1586/erv.12.84.PubMed CentralView ArticlePubMedGoogle Scholar
- Pijlman GP. Enveloped virus-like particles as vaccines against pathogenic arboviruses. Biotech J. 10. doi:10.1002/biot.201400427
- Chang LJ, Dowd K a, Mendoza FH, Saunders JG, Sitar S, Plummer SH, et al. Safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: A phase 1 dose-escalation trial. Lancet. 2014;384:2046–52. doi:10.1016/S0140-6736(14)61185-5. Elsevier Ltd.View ArticlePubMedGoogle Scholar
- Solignat M, Gay B, Higgs S, Briant L, Devaux C. Replication cycle of chikungunya: a re-emerging arbovirus. Virology. Elsevier Inc.; 2009;393: 183–97. doi:10.1016/j.virol.2009.07.024
- Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. 1994;58:491–562.Google Scholar
- Frolova EI, Gorchakov R, Pereboeva L, Atasheva S, Frolov I. Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol. 2010;84:11679–95. doi:10.1128/JVI.01441-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Fros JJ, van der Maten E, Vlak JM, Pijlman GP. The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling. J Virol. 2013;87:10394–400. doi:10.1128/JVI.00884-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Fros JJ, Liu WJ, Prow NA, Geertsema C, Ligtenberg M, Vanlandingham DL, et al. Chikungunya virus nonstructural protein 2 inhibits type I/II interferon-stimulated JAK-STAT signaling. J Virol. 2010/08/06 ed. American Society for Microbiol (ASM). 2010;84:10877–87. doi:10.1128/JVI.00949-10.Google Scholar
- Akhrymuk I, Kulemzin SV, Frolova EI. Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II. J Virol. 2012;86:7180–91. doi:10.1128/JVI.00541-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Fros JJ, Major LD, Scholte FEM, Gardner J, van Hemert MJ, Suhrbier A, et al. Chikungunya virus nsP2-mediated host shut-off disables the unfolded protein response. J Gen Virol. 2014; doi:10.1099/vir.0.071845-0
- Vihinen H, Saarinen J. Phosphorylation site analysis of Semliki forest virus nonstructural protein 3. J Biol Chem. 2000;275:27775.PubMedGoogle Scholar
- Li GP, LaStarza MW, Hardy WR, Strauss JH, Rice M. Phosphorylation of Sindbis Virus nsP3 in vivo and in vitro. Virology. 1990;427:416–27.View ArticleGoogle Scholar
- Lastarza MW, Lemm JA, Rice CM. Genetic Analysis of the nsP3 Region of Sindbis Virus : Evidence for Roles in Minus-Strand and Subgenomic RNA. Synthesis. 1994;68:5781–91.Google Scholar
- Malet H, Coutard B, Jamal S, Dutartre H, Papageorgiou N, Neuvonen M, et al. The crystal structures of Chikungunya and Venezuelan equine encephalitis virus nsP3 macro domains define a conserved adenosine binding pocket. J Virol. 2009;83:6534–45. doi:10.1128/JVI.00189-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Aaskov J, Jones A, Choi W, Lowry K, Stewart E. Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus. Virology. Elsevier Inc.; 2011;410: 353–9. doi:10.1016/j.virol.2010.11.025.
- Shin G, Yost SA, Miller MT, Elrod EJ, Grakoui A. Structural and functional insights into alphavirus polyprotein processing and pathogenesis. 2012; 1–6. doi:10.1073/pnas.1210418109/-/DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.1210418109.
- Gorchakov R, Garmashova N, Frolova E, Frolov I. Different types of nsP3-containing protein complexes in Sindbis virus-infected cells. J Virol. 2008/08/08 ed. 2008;82: 10088–10101. doi:10.1128/JVI.01011-08
- Cristea IM, Carroll J-WN, Rout MP, Rice CM, Chait BT, MacDonald MR. Tracking and elucidating alphavirus-host protein interactions. J Biol Chem. 2006;281:30269–78. doi:10.1074/jbc.M603980200.View ArticlePubMedGoogle Scholar
- Irvine K, Stirling R, Hume D, Kennedy D. Rasputin, more promiscuous than ever: a review of G3BP. Int J Dev Biol. 2004;48:1065–77. doi:10.1387/ijdb.041893ki.View ArticlePubMedGoogle Scholar
- Khong A, Jan E. Modulation of stress granules and P bodies during dicistrovirus infection. J Virol. 2011;85:1439–51. doi:10.1128/JVI.02220-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Buchan JR, Parker R. Eukaryotic Stress Granules : The Ins and Out of Translation What are Stress Granules ? Mol Cell. 2009;36:932–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Fros JJ, Domeradzka NE, Baggen J, Geertsema C, Flipse J, Vlak JM, et al. Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci. J Virol. 2012;86:10873–9. doi:10.1128/JVI.01506-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Panas MD, Varjak M, Lulla A, Eng KE, Merits A, Karlsson Hedestam GB, et al. Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection. Mol Biol Cell. 2012;23:4701–12. doi:10.1091/mbc.E12-08-0619.PubMed CentralView ArticlePubMedGoogle Scholar
- Vega-Rúa A, Zouache K, Girod R, Failloux A-B, Lourenço-de-Oliveira R. High level of vector competence of Aedes aegypti and Aedes albopictus from ten American countries as a crucial factor in the spread of Chikungunya virus. J Virol. 2014;88:6294–306. doi:10.1128/JVI.00370-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Zouache K, Fontaine A, Vega-Rua A, Mousson L, Thiberge J-M, Lourenco-De-Oliveira R, et al. Three-way interactions between mosquito population, viral strain and temperature underlying chikungunya virus transmission potential. Proc Biol Sci. 2014;281. doi:10.1098/rspb.2014.1078
- McFarlane M, Arias-Goeta C, Martin E, O’Hara Z, Lulla A, Mousson L, et al. Characterization of Aedes aegypti Innate-Immune Pathways that Limit Chikungunya Virus Replication. PLoS Negl Trop Dis. 2014;8:e2994. doi:10.1371/journal.pntd.0002994.PubMed CentralView ArticlePubMedGoogle Scholar
- Blair CD. Mosquito RNAi is the major innate immune pathway controlling arbovirus infection and transmission. Future Micrbiol. 2012;6:265–77. doi:10.2217/fmb.11.11.Mosquito.View ArticleGoogle Scholar
- Saxton-Shaw KD, Ledermann JP, Borland EM, Stovall JL, Mossel EC, Singh AJ, et al. O’nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3. PLoS Negl Trop Dis. 2013;7:e1931. doi:10.1371/journal.pntd.0001931.PubMed CentralView ArticlePubMedGoogle Scholar
- Metz SW, Geertsema C, Martina BE, Andrade P, Heldens JG, van Oers MM, et al. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells. Virol J. 2011/07/19 ed. 2011;8: 353. doi:10.1186/1743-422X-8-353
- Frolova E, Gorchakov R, Garmashova N, Atasheva S, Vergara LA, Frolov I. Formation of nsP3-specific protein complexes during Sindbis virus replication. J Virol. 2006/03/31 ed. 2006;80: 4122–4134. doi:80/8/4122 [pii] 10.1128/JVI.80.8.4122-4134.2006
- Varjak M, Zusinaite E, Merits A. Novel functions of the alphavirus nonstructural protein nsP3 C-terminal region. J Virol. 2010;84:2352–64. doi:10.1128/JVI.01540-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Panas MD, Ahola T, McInerney GM. The C-terminal repeat domains of nsP3 from the Old World alphaviruses bind directly to G3BP. J Virol. 2014;88:5888–93. doi:10.1128/JVI.00439-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Vognsen T, Møller IR, Kristensen O. Crystal structures of the human G3BP1 NTF2-like domain visualize FxFG Nup repeat specificity. PLoS One. 2013;8:e80947. doi:10.1371/journal.pone.0080947.PubMed CentralView ArticlePubMedGoogle Scholar
- Vognsen T, Kristensen O. Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster. Biochem Biophys Res Commun. Elsevier Inc.; 2012;420: 188–92. doi:10.1016/j.bbrc.2012.02.140
- Panas MD, Schulte T, Thaa B, Sandalova T, Kedersha N, Achour A, et al. Viral and Cellular Proteins Containing FGDF Motifs Bind G3BP to Block Stress Granule Formation. PLoS Pathog. 2015;11:e1004659. doi:10.1371/journal.ppat.1004659.PubMed CentralView ArticlePubMedGoogle Scholar
- Cristea IM, Rozjabek H, Molloy KR, Karki S, White LL, Rice CM, et al. Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus replication. J Virol. 2010;84:6720–32. doi:10.1128/JVI.01983-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Scholte FEM, Tas A, Albulescu IC, Žusinaite E, Merits A, Snijder EJ, et al. Stress granule components G3BP1 and G3BP2 play a proviral role early in chikungunya virus replication. J Virol. 2015; JVI.03612–14. doi:10.1128/JVI.03612-14
- Mellor PS. Replication of arboviruses in insect vectors. J Comp Pathol. 2000/10/24 ed. 2000;123: 231–247. doi:10.1053/jcpa.2000.0434
- Hardy JL, Houk EJ, Kramer LD, Reeves WC. Intrinsic factors affecting vector competence of mosquitoes for arboviruses. Annu Rev Entomol. 1983/01/01 ed. 1983;28: 229–262. doi:10.1146/annurev.en.28.010183.001305
- Baumgartner R, Stocker H, Hafen E. The RNA-binding proteins FMR1, rasputin and caprin act together with the UBA protein lingerer to restrict tissue growth in Drosophila melanogaster. PLoS Genet. 2013;9:e1003598. doi:10.1371/journal.pgen.1003598.PubMed CentralView ArticlePubMedGoogle Scholar
- Costa A, Pazman C, Sinsimer KS, Wong LC, McLeod I, Yates J, et al. Rasputin functions as a positive regulator of orb in Drosophila oogenesis. PLoS One. 2013;8:e72864. doi:10.1371/journal.pone.0072864.PubMed CentralView ArticlePubMedGoogle Scholar
- Pazman C, Mayes CA, Fanto M, Haynes SR, Mlodzik M. Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in Ras- and Rho-mediated signaling. Development. 2000;1725:1715–25.Google Scholar
- Reineke LC, Lloyd RE. The stress granule protein G3BP1 recruits PKR to promote multiple innate immune antiviral responses. J Virol. 2014; doi:10.1128/JVI.02791-14
- White JP, Lloyd RE. Regulation of stress granules in virus systems. 2013;20: 175–183. doi:10.1016/j.tim.2012.02.001.Regulation
- White JP, Cardenas AM, Marissen WE, Lloyd RE. Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase. Cell Host Microbe. 2007;2:295–305. doi:10.1016/j.chom.2007.08.006.View ArticlePubMedGoogle Scholar
- Emara MM, Brinton M. Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly. Proc Natl Acad Sci U S A. 2007;104:9041–6. doi:10.1073/pnas.0703348104.PubMed CentralView ArticlePubMedGoogle Scholar
- Yi Z, Pan T, Wu X, Song W, Wang S, Xu Y, et al. Hepatitis C virus co-opts Ras-GTPase-activating protein-binding protein 1 for its genome replication. J Virol. 2011;85:6996–7004. doi:10.1128/JVI.00013-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Baird NL, York J, Nunberg JH. Arenavirus infection induces discrete cytosolic structures for RNA replication. J Virol. 2012;86:11301–10. doi:10.1128/JVI.01635-12.PubMed CentralView ArticlePubMedGoogle Scholar
- Li W, Li Y, Kedersha N, Anderson P, Emara M, Swiderek KM, et al. Cell Proteins TIA-1 and TIAR Interact with the 3 Ј Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication. 2002;76: 11989–12000. doi:10.1128/JVI.76.23.11989