Mosquito Rasputin interacts with chikungunya virus nsP3 and determines the infection rate in Aedes albopictus

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

CHIKV nsP3 forms granules in insect cells. a Schematic representation of CHIKV nsP3 variants used in this study. Representing either the full length protein N-terminally fused to EGFP (nsP3), nsP3 isoforms with adapted C-terminal amino acids (nsP3, nsP3-DDEL and nsP3-dUAG) or truncated nsP3 variants (nsP3.2, 3.7, 3.8 and 3.10). Asterisk indicates opal stop codon between nsP3 and nsP4. b Intracellular distribution of wild type CHIKV nsP3 in cultured insect and mammalian cells. Mosquito and lepidopteran cells were transfected with plasmids that express the nsP3 variants from OpIE2 insect promoters. In mammalian cells expression of the nsP3 variants was driven by a cytomegalovirus promoter. c Intracellular distribution of nsP3-DDEL and nsP3-dAUG in insect cells. d Intracellular distribution of the truncated nsP3 variants

Fig. 2

CHIKV nsP3 sequesters mosquito Rasputin into cytoplasmic granules. a Schematic representation of Ae.albopictus-Rin-mCherry. NTF2; nuclear transport factor 2-like domain, RRM: RNA recognition motif, RGG; arginine-glycine rich box. b Ae.albopictus-Rin-mCherry or EGFP-nsP3 were transiently expressed in SF21 insect cells and display diffuse and granular intracellular distributions, respectively. c Ae.albopictus-Rin-mCherry and EGFP-nsP3 (top) or EGFP-nsP3.8 (bottom) were co-transfected into insect cells. Magnified images are presented

Fig. 3

The C-terminal TFGD repeats of nsP3 interact with mosquito Rasputin. a Schematic representation of nsP3 and the conserved domains within the C-terminal variable domain. Deletions and mutations are indicated in the amino acid sequence. Insect cells (Sf21) were co-transfected with Rin-mCherry and either one of the EGFP-nsP3 variants. b SH3-domain binding motif, nsP3-d398/406, nsP3-P398A or nsP3-PPR401AAA. c C-terminal repeats, EGFP-nsP3-FG479AA, EGFP-nsP3-FG497AA or EGFP-nsP3-FG479AA/FG497AA

Fig. 4

nsP3 interacts with the NTF2-like domain of mosquito Rasputin. a Protein alignment of the Rin/G3BP NTF2-like domains from Ae. albopictus (KP641128), Ae. aegypti, (XP_001651045), Culex quincefasciatus (XP_001861860), Anopheles gambiae (XP_001688309), Anopheles sinensis (KFB40464), Drosophila melanogaster (AF231031), Ixodes ricinus (GANP01009274), and Homo sapiens (CAG38772). Genbank accession numbers in brackets. Alignment made with CLUSTALX and modified using Genedoc. b Structural modeling of Ae. albopictus-Rin modeled onto D. melanogaster Rin. Modeling was performed using the Phyre2 server (www.sbg.bio.ic.ac.uk/phyre2), results were visualized with PyMOL (www.pymol.org). Left, Ae. albopictus Rin (deep blue) is depicted together with D. melanogaster Rin (cyan). Right, the FxFG binding pocket is shown and phenylalanine 34 is highlighted in red. c Sf21 insect cells were co-transfected with EGFP-nsP3 and either Rin-mCherry (wild type), Rin-mCherry F34A or Rin-mCherry F34W

Fig. 5

Formation of nsP3-granules is independent of mosquito Rasputin. a. Rin silencing was determined by semi-quantitative RT-PCR on Ae. albopictus Rin mRNA, relative for the internal control S7. Bars represent relative Rin mRNA expression normalized to dsLuc transfected samples. Error bars indicate standard deviation of duplicate samples from a single representative experiment, which is presented in B. b. U4.4 mosquito cells were transfected with dsRNA against Ae. albopictus Rin or luciferase. Twenty-four hours later these cells were transfected with wild type EGFP-nsP3, displaying a granular localization

Fig. 6

In vivo Rin silencing reduces the CHIKV infection rate in Aedes albopictus mosquitoes. a Schematic representation of the experiment. Mosquitoes were injected with 500 ng of dsRin or dsLuc two days prior to blood feeding. On day 0, a subset of mosquitoes was sacrificed to determine the level of Rin depletion. Remaining mosquitoes were orally infected with CHIKV (10⁷pfu/ml) and sacrificed six days post infection. b Total RNA was isolated and Rin silencing was determined by semi-quantitative RT-PCR on Ae. albopicutus Rin mRNA, normalized for the internal control S7. Bars represent mean Rin mRNA values normalized to dsLuc injected mosquitoes. Error bars indicate one standard error of the mean (n = 5). c Heads and bodies from the blood fed mosquitoes were separated, homogenized and the presence of CHIKV was determined by incubating the homogenate on Vero E6 cells. Bars represent the percentage of CHIKV positive mosquito bodies and heads from both dsLuc and dsRin injected mosquitoes. Asterisk indicates significant difference (P < 0.05 Fisher’s exact test). d From all the CHIKV positive mosquito heads and bodies the viral titers (TCID⁵⁰/ml) were determined. Data points represent one individual mosquito head or body. Asterisk indicates significant difference between dsLuc and dsRin injected mosquitoes (P < 0.05, Mann Whitney test) and red data points indicate mosquitoes with positive saliva

Fig. 7

CHIKV infection in vitro is not affected by Rin depletion. In four independent experiments, U4.4 mosquito cells were transfected with dsRNA against Ae. albopictus Rin or luciferase. Twenty-four hours later these cells were infected with CHIKV (MOI 5) and total RNA was isolated at 16 or 24 hpi. a Rin silencing was determined by semi-quantitative RT-PCR on Ae. albopictus Rin mRNA, normalized for the internal control S7. b CHIKV genomic RNA was quantified with primers that anneal to the nsP1 gene. c CHIKV structural protein expression was determined by immunoblot staining against CHIKV E2 and host cell β-tubulin. Protein sizes indicated in kDa. Results of duplicate experiments are shown. d At the indicated hpi medium was harvested and the CHIKV titer (TCID₅₀/ml) was determined in end point dilution essays. A,B,D. Bars represent the mean of four independent experiments, which have been normalized to the respective value of dsLuc transfected samples in each individual experiment. Error bars represent one standard error of the mean