1. Microbiology and Infectious Disease
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Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir

  1. Alex C Stabell
  2. Nicholas R Meyerson
  3. Rebekah C Gullberg
  4. Alison R Gilchrist
  5. Kristofor J Webb
  6. William M Old
  7. Rushika Perera
  8. Sara L Sawyer  Is a corresponding author
  1. University of Colorado Boulder, United States
  2. Colorado State University, United States
Research Article
Cite this article as: eLife 2018;7:e31919 doi: 10.7554/eLife.31919
6 figures, 1 table, 2 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
Dengue virus (DENV2) can cleave human but not nonhuman primate STING.

(A) A phylogeny of select primate species, showing the three main simian clades: apes, Old World monkeys, and New World monkeys (Perelman et al., 2011). The primate species from which STING is tested in this study are shown with purple arrows. Possible primate reservoir hosts for sylvatic dengue viruses, based on virus isolation from sentinel monkeys, or antibody detection, are shown in red (Africa) and green (Asia). The current evidence for these primate reservoir hosts is reviewed in the discussion section. (B) 293T cells were cotransfected with plasmids encoding STING-HA, and the NS2B3-Flag protease complex with or without the S135 inactivating mutation. Whole cell lysate isolated 24 hr post transfection was run on a protein gel and immunoblotted with anti-Flag or anti-HA antibodies. The encoded NS2B-NS3-Flag polyprotein auto-processes into the NS2B3 protease complex if the protease is active, as seen in the anti-Flag blot where in some samples the NS3-Flag protein has been liberated through cleavage. We sometimes see lower bands underneath the full-length mouse STING, but conclude that they are endogenous degradation products since they are equal in intensity in the presence of the active or dead protease.

https://doi.org/10.7554/eLife.31919.003
Figure 1—figure supplement 1
Many primate species reside in areas where dengue viruses are endemic in humans.
https://doi.org/10.7554/eLife.31919.004
Figure 2 with 2 supplements
STING residue 78 determines susceptibility to NS2B3 cleavage in human versus chimpanzee STING comparisons.

(A) A domain diagram of human STING is shown, as defined in (Wu et al., 2014). An alignment of human and chimpanzee STING in the region of the newly identified cleavage determinant (78/79) and the one previously determined (95/96) (Aguirre et al., 2012; Yu et al., 2012). (B) Site-directed mutagenesis was performed on either human or chimpanzee STING at position 78, substituting the residue at this position in human (R) with that in chimpanzee (W) and vice versa. Plasmids encoding the NSB3 protease complex and STING were cotransfected into 293T cells, and 48 hr later lysates were collected and analyzed by anti-FLAG western blot. In this experiment, both the protease and STING are tagged with FLAG. Data presented are representative of at least two experiments. (C) (bottom) 293T cells were transfected with plasmids expressing the DENV2 NS2B3 protease and wildtype (wt) or mutated (78W or 78R) STING. IRF3 and phosphorylated IRF3 (pIRF3) were detected by western blot in lysates harvested 48 hr later. (top) The identical experiment, but performed in biological triplicate and with the addition of plasmids encoding a firefly luciferase gene driven by the interferon beta (IFNb) promoter, and a renilla luciferase gene driven by a CMV promoter. The relative luciferase activity (Y-axis) was calculated by normalizing the luciferase signal to the renilla signal in each replicate. A Welch’s T-test was used to compare the levels of luciferase produced in the presence of active versus dead protease. Data is representative of at least two experiments.

https://doi.org/10.7554/eLife.31919.005
Figure 2—figure supplement 1
Generation of STING knockout cells using CRISPR-Cas9, and stable re-complementation of these lines.

(A) A depiction of the insertion induced in the first exon of STING in A549 cells. Below, a Sanger sequencing chromatogram from a this genomic locus shows the homozygous insertion. (B) Both wildtype (wt) A549 cells and A549 cells that were knocked out for STING were probed by western blot for Actin or STING. An antibody recognizing the endogenous STING was used (anti-STING; Abcam 92605). (C) STING-KO A549 cells were re-complimented with HA-tagged versions of STING using lentiviral transduction. Cell lines were generated to express either wildtype (wt) or mutant STING from human or chimpanzee. A mutation was made in human STING to render it cleavage-resistant (78W). A mutation was made in chimpanzee STING to make it cleavage-susceptible (78R). Western blots where performed with antibodies against GAPDH or STING.

https://doi.org/10.7554/eLife.31919.006
Figure 2—figure supplement 2
Wildtype and mutated human STING both colocalize with the ER-resident protein BiP.

Immunofluorescence imaging was performed on STING and the ER-resident protein BiP. A549 cells knocked out for STING were reconstituted with either wildtype human STING (top) or human STING 78W (cleavage resistant) by retroviral transduction (as shown in Figure 2—figure supplement 1). These cells were fixed in a 4% paraformaldehyde solution. Antibodies against the HA epitope tag (STING) or BiP were incubated with the fixed cells. Cells were imaged at 40x magnification on the Nikon A1R confocal microscope.

https://doi.org/10.7554/eLife.31919.007
Figure 3 with 1 supplement
Cleavage of STING at position 78/79 promotes virus replication.

The endogenous copies of STING in A549 cells were knocked out using the Cas9 nuclease (see Figure 2—figure supplement 1). These cells were re-complemented by retroviral transduction with no gene (pLPCX-empty), wildtype human STING, cleavage-resistant human STING (human 78W), wildtype chimpanzee STING, or cleavage-susceptible chimpanzee STING (chimp 78R). These cell lines were infected at MOI of 0.3 with dengue virus 2 (DENV2 16681). After 24 and 48 hr the virus supernatant was removed and titrated on BHK21 cells. At the same time, cells were collected in RIPA buffer, lysed, and run on a gel for western blotting using antibodies against STING, dengue virus NS3, and GAPDH (loading control). A Tukey's multiple comparisons test indicated significant differences in infectious virus in the presence of each mutant STING compared to wildtype STING, as shown (****=p < 0.0001), after significant one-way ANOVA. Data are representative of at least two independent experiments.

https://doi.org/10.7554/eLife.31919.008
Figure 3—figure supplement 1
STING is cleaved during dengue virus infection.

The indicated A549 cells lines (wild-type, STING knockout, or re-complemented) were plated in F-12K media with 10% FBS. Dengue virus 2 (16681) was diluted in F-12K media with 2% FBS, added to cells at an MOI of 5, and allowed to attach to cells for 1 hr at room temperature. Uninfected wells were incubated with F-12K media with 2% FBS not containing virus. F-12K media with 10% FBS was added to cells and they were maintained at 37°C with 5% CO2. After 20 hr the media in all wells was replaced with F-12K media with 2% FBS containing 20 μM proteasome inhibitor MG-132 (Sigma-Aldrich M7449). 4 hr following MG-132 treatment (24 hr following infection) the cells were collected and lysed in RIPA buffer supplemented with protease inhibitor (Roche, 4693159001). Protein concentration was calculated using the Bradford method. 15% 37.5:1 Acrylamide/Bisacrylamide gels were used to separate 30 μg of whole cell lysate for each sample. Protein was transferred overnight at 30 volts onto a nitrocellulose membrane. Blocking was performed with a 10% milk solution in tris-buffered saline supplemented with 0.1% TWEEN20. Primary antibodies were used against HA (3F10 clone Sigma 11867423001) and β-actin (Santa Cruz Biotechnology Sc47778).

https://doi.org/10.7554/eLife.31919.009
Residues 78 and 79 of STING define a dengue virus cleavage determinant in both primate and mouse STING.

(A) A phylogeny and multiple sequence alignment of STING from various primate species and mouse. Shown in green is the 78/79 motif in STING that is mutated in panel B. Shown in red is the motif changed in mouse STING, only, in panel B. (B) Site directed mutagenesis was performed on human, rhesus macaque, marmoset, or mouse STING at sites 78/79 or 93–96 (mouse only). 293T cells were cotransfected with mammalian expression plasmids encoding STING along with wildtype or mutant NS2B3. 24 hr after transfection, whole-cell lysate was harvested and probed for FLAG or HA by western blot. Data presented are representative of at least two experiments.

https://doi.org/10.7554/eLife.31919.010
Figure 5 with 1 supplement
The 78/79 cleavage determinant in STING is targeted by proteases encoded by all endemic human dengue viruses, and by at least one sylvatic dengue virus.

(A) 293T cells were cotransfected with plasmids encoding NS2B3 from DENV1-4 along with human STING with or without a mutation at site 78. Western blotting was performed on lysate harvested 24 hr post transfection to detect NS2B3 (anti-FLAG) or STING (anti-HA). Data presented are representative of at least two experiments. (B) 293T cells were cotransfected with plasmids encoding the indicated STING and the NS2B3 from a sylvatic isolate of dengue virus (DakAr-141069). 24 hr post transfection, lysates where harvested, run on a gel, and western blotting was performed with an anti-HA antibody to detect STING. All data presented are representative of at least two experiments.

https://doi.org/10.7554/eLife.31919.011
Figure 5—figure supplement 1
Alignment of the NS2B3 protease from sylvatic (top) versus human (bottom) dengue viruses.
https://doi.org/10.7554/eLife.31919.012
The dengue virus cleavage determinant in STING of various species.

(A) An alignment of the eight amino acid region in STING surrounding residues 78R/79G, the newly identified dengue virus cleavage determinant (downward arrow at top). Deviations from the human motif are highlighted. The green boxes indicate STING orthologs sequenced as part of this study. The brown boxes indicate STING sequences obtained from Genbank. Apes are shown at the top of the tree (pink type), monkeys at the bottom (green type). Depicted below are sequences from the same region of STING from two animal models for dengue virus (mouse, pig [Cassetti et al., 2010]), several small rodent species which encode the correct cleavage motif at 78/79 (naked mole rat, common chinchilla, desert woodrat), and one that does not (13 lined ground squirrel). Genbank accession numbers of sequences shown: mouse (XP_017173483), pig (XP_005661761), 13-lined ground squirrel (XM_005327275), naked mole rat (JAO02071), chinchilla (XP_005382124), and desert woodrat (OBS58238). (B) STING-HA genes were synthesized for the rodent species discussed in panel A. Cleavage assays were performed by co-transfecting plasmids encoding the dengue protease (dead or active) as well as each STING, and then performing immunoblotting as described in the methods. The data presented are representative of at least two independent experiments.

https://doi.org/10.7554/eLife.31919.013

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
gene (Homo sapiens)STING; TMEM173NAGENBANK:NM_198282GENBANK:MF622062
gene (Homo sapiens)STING; TMEM173this studyGENBANK:MF622062
gene (Pan troglodytes)STING; TMEM173NAGENBANK:XM_016953921
gene (Pan paniscus)STING; TMEM173this studyGENBANK:MF616339
gene (Gorilla gorilla)STING; TMEM173NAGENBANK:XM_0040426
gene (Pongo abelii)STING; TMEM173NAGENBANK:XM_002815952
gene (Hylobates agilis)STING; TMEM173this studyGENBANK:MF616342
gene (Symphalangus
syndactylus)
STING; TMEM173this studyGENBANK:MF616343
gene (Nomascus
leucogenys)
STING; TMEM173this studyGENBANK:MF616344
gene (Hylobates lar)STING; TMEM173this studyGENBANK:MF616341
gene (Rhinopithecus
roxellana)
STING; TMEM173NAGENBANK:XM_010388119
gene (Rhinopithecus
bieti)
STING; TMEM173NAGENBANK:XM_017895026
gene (Trachypithecus
francoisi)
STING; TMEM173this studyGENBANK:MF616352
gene (Colobus
guereza)
STING; TMEM173this studyGENBANK:MF616351
gene (Chlorocebus
sabaeus)
STING; TMEM173NAGENBANK:XM_008014636
gene (Cercopithecus
wolfi)
STING; TMEM173this studyGENBANK:MF616350
gene (Miopithecus
talapoin)
STING; TMEM173this studyGENBANK:MF616349
gene (Macaca
nemestrina)
STING; TMEM173NAGENBANK:XM_011716377
gene (Macaca mulatta)STING; TMEM173NAGENBANK:XM_015141010
gene (Macaca mulatta)STING; TMEM173this studyGENBANK:MF622060
gene (Macaca
fascicularis)
STING; TMEM173this studyGENBANK:MF616346
gene (Papio papio)STING; TMEM173this studyGENBANK:MF616348
gene (Lophocebus
albigena)
STING; TMEM173this studyGENBANK:MF616347
gene (Cercocebus atys)STING; TMEM173NAGENBANK:XM_012090448
gene (Mandrillus
leucophaeus)
STING; TMEM173NAGENBANK:XM_011997224
gene (Aloutta sara)STING; TMEM173this studyGENBANK:MF616355
gene (Callicebus
cupreus)
STING; TMEM173this studyGENBANK:MF616354
gene (Callithrix jacchus)STING; TMEM173NAGENBANK:XM_00898588
gene (Callithrix jacchus)STING; TMEM173this studyGENBANK:MF622061
gene (Cebus capucinus
imitator)
STING; TMEM173NAGENBANK:XM_017536735
gene (Samiri boliviensis)STING; TMEM173NAGENBANK:XM_003933913
gene (Saimiri sciureus)STING; TMEM173this studyGENBANK:MF616353
gene (Mus musculus)STING; TMEM173NAGENBANK:NM_001289591
gene (Sus scrofa)STING; TMEM173NAGENBANK:XP_005661761
gene (Heterocephalus
glaber)
NAGENBANK:JAO02071
gene (Chinchilla lanigera)NAGENBANK:XP_005382124
gene (Neotoma lepida)NAGENBANK:OBS58238
gene (Dengue viurs 2)NS2B3NAGENBANK:M29095
cell line (Homo sapiens)293T cellsATCCCRL-3216
cell line (Homo sapiens)A549 cellsATCCCCL-185
antibodyRat anti-HA-HRP (3F10)Sigma11867423001
antibodyMouse anti-Flag (M2)SigmaF3165
antibodyRabbit anti-pIRF3abcamab76493
antibodyRabbit anti-IRF3Santa Cruz
Biotech
sc-9082
antibodyRabbit anti-GAPDHCell
Signaling
14C10
antibodyRabbit anti-STINGabcamab92605
antibodyMouse anti-Actin (C4)Santa Cruz
Biotech
Sc47778
recombinant DNA reagentDENV2 NS2B3 WT
(plasmid)
PMID: 1642612Progenitors: DENV2 NGC
(GENBANK:M29095), pCR3.1
recombinant DNA reagentDENV2 NS2B3
S135A (plasmid)
PMID: 1642612Progenitors: DENV2 NS2B3 WT
pCR3.1 plasmid, SDM
recombinant DNA reagentDENV1 (Hawaii)
cDNA
this paperProgenitors: World Reference
Center for Emerging Viruses
and Arboviruses (WRCEVA)
Catalog number NR-4287
recombinant DNA reagentDENV2 (New
Guinea C) cDNA
this paperProgenitors: World Reference
Center for Emerging Viruses
and Arboviruses (WRCEVA)
Catalog number NR-4288
recombinant DNA reagentDENV3 (Philippines/
H87/1956) cDNA
this paperProgenitors: World Reference
Center for Emerging Viruses and
Arboviruses (WRCEVA)
Catalog number NR-2771
recombinant DNA reagentDENV4 (H241) cDNAthis paperProgenitors: World Reference
Center for Emerging Viruses
and Arboviruses (WRCEVA)
Catalog number NR-4289
recombinant DNA reagentDENV1 (Hawaii)
NS2B3 WT (plasmid)
this paperProgenitors: DENV1 (Hawaii)
cDNA, pCR3.1
recombinant DNA reagentDENV2 (New Guinea C)
NS2B3 WT (plasmid)
this paperProgenitors: DENV2 (New Guinea C)
cDNA, pCR3.1
recombinant DNA reagentDENV3 (Philippines/
H87/1956) NS2B3
WT (plasmid)
this paperProgenitors: DENV3
(Philippines/H87/1956)
cDNA, pCR3.1
recombinant DNA reagentDENV4 (H241)
NS2B3 WT (plasmid)
this paperProgenitors: DENV4 (H241)
cDNA, pCR3.1
recombinant DNA reagentSylvatic (DakAr-141069)
Dengue NS2B3
Protease (WT)
this paperProgenitors: DakAr-141069 NS2B3
sequence (GenBank EF105389)
recombinant DNA reagentSylvatic (DakAr-141069)
Dengue NS2B3
Protease (S135A)
this paperProgenitors: Sylvatic
(DakAr-141069) Dengue NS2B3
Protease (WT) SDM product
recombinant DNA reagenthuman cDNAthis paperProgenitors: A549 cell line
(ATCC CCL-185)
recombinant DNA reagentchimpanzee cDNAthis paperProgenitors: Coriell PR00748
recombinant DNA reagentrhesus
macaque cDNA
this paperProgenitors: Mm265-95
recombinant DNA reagentmarmoset cDNAthis paperProgenitors: Coriell PR07404
recombinant DNA reagentmouse cDNAthis paperProgenitors: RNA extracted
from mouse liver
recombinant DNA reagenthuman
STING-HA (plasmid)
this paperProgenitors: human cDNA,
pcDNA3.1 plasmid
recombinant DNA reagenthuman
STING-HA (plasmid)
this paperProgenitors: human cDNA,
pLPCX plasmid
recombinant DNA reagenthuman
STING(R78W)-HA
(plasmid)
this paperProgenitors: human STING-HA
pcDNA3.1 SDM product
recombinant DNA reagenthuman
STING(R78W)-HA
(plasmid)
this paperProgenitors: human STING-HA
pLPCX SDM product
recombinant DNA reagenthuman
STING(R79D)-HA
(plasmid)
this paperProgenitors: human STING-HA
pcDNA3.1 SDM product
recombinant DNA reagenthuman
STING(R78Q)-HA
(plasmid)
this paperProgenitors: human STING-HA
pcDNA3.1 SDM product
recombinant DNA reagentchimpanzee
STING-HA
(plasmid)
this paperProgenitors: chimpanzee cDNA,
pcDNA3.1 plasmid
recombinant DNA reagentchimpanzee
STING-HA (plasmid)
this paperProgenitors: chimpanzee cDNA,
pLPCX plasmid
recombinant DNA reagentchimpanzee
STING(W78R)-HA
(plasmid)
this paperProgenitors: chimpanzee
STING-HA pcDNA3.1 SDM product
recombinant DNA reagentchimpanzee
STING(W78R)-HA
(plasmid)
this paperProgenitors: chimpanzee
STING-HA pLPCX SDM product
recombinant DNA reagentrhesus macaque
STING-HA (plasmid)
this paperProgenitors: rhesus macaque
cDNA, pcDNA3.1 plasmid
recombinant DNA reagentrhesus macaque
STING(D79G)-HA
(plasmid)
this paperProgenitors: rhesus macaque
STING-HA pcDNA3.1 SDM product
recombinant DNA reagentmarmoset STING-HA
(plasmid)
this paperProgenitors: marmoset
cDNA, pcDNA3.1 plasmid
recombinant DNA reagentmarmoset
STING(Q78R)-HA
(plasmid)
this paperProgenitors: marmoset
STING-HA pcDNA3.1 SDM product
recombinant DNA reagentmouse STING-HA
(plasmid)
this paperProgenitors: mouse cDNA,
pcDNA3.1 plasmid
recombinant DNA reagentmouse
STING(Q78R)-HA
(plasmid)
this paperProgenitors: mouse STING-HA
pcDNA3.1 SDM product
recombinant DNA reagentmouse
STING(93LRRG96)-HA
(plasmid)
this paperProgenitors: mouse STING-HA
pcDNA3.1 SDM product
recombinant DNA reagenthuman STING-3xFLAG
(plasmid)
this paperProgenitors: human cDNA,
pLPCX plasmid
recombinant DNA reagenthuman
STING(R78W)-3xFLAG
(plasmid)
this paperProgenitors: human
STING-3xFLAG pLPCX SDM product
recombinant DNA reagenthuman
STING(G230A)-3xFLAG
(plasmid)
this paperProgenitors: human
STING-3xFLAG pLPCX SDM product
recombinant DNA reagenthuman STING
(R78W, G230A)-3xFLAG
(plasmid)
this paperProgenitors: human
STING-3xFLAG pLPCX SDM product
recombinant DNA reagentchimpanzee
STING-3xFLAG
(plasmid)
this paperProgenitors: chimpanzee
cDNA, pLPCX plasmid
recombinant DNA reagentchimpanzee STING
(W78R)-3xFLAG
(plasmid)
this paperProgenitors: chimpanzee
STING-3xFLAG pLPCX SDM product
recombinant DNA reagentchimpanzee
STING(A230G)-3xFLAG
(plasmid)
this paperProgenitors: chimpanzee
STING-3xFLAG pLPCX SDM product
recombinant DNA reagentchimpanzee
STING(W78R,
A230G)-3xFLAG
(plasmid)
this paperProgenitors: chimpanzee
STING-3xFLAG pLPCX SDM product
recombinant DNA reagentIFN-ß1-luc (plasmid)PMID:
21512573
recombinant DNA reagentpRL-CMV (plasmid)Promega:
AF025843
Progenitors: pRL-null
commercial assay or kitDual-Glo Luciferase
Assay System
PromegaCat#E2920
commercial assay or kitSuperscript III
First-Strand
Synthesis System
Thermo
Scientific
Cat#18080051
software, algorithmMEGA7http://www.megasoftware.net/
software, algorithmImageJ version 1.43uhttp://rsb.info.nih.gov/ij
software, algorithmPython 2.7.11https://www.python.org
software, algorithmSequencherhttps://www.genecodes.com

Data availability

The following data sets were generated
  1. 1
    outbreak_mapping
    1. Stabell A
    (2018)
    Available at Dryad Digital Repository under a CC0 Public Domain Dedication.
The following previously published data sets were used
  1. 1
    Dengue_Occurrence_12122013.xlsx
    1. Messina JP
    2. Brady OJ
    3. Pigott DM
    4. Brownstein JS
    5. Hoen AG
    6. Hay SI
    (2014)
    Available at figshare under a CC0 Public Domain licence.

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