Ae. aegypti and H. sapiens SUMOylation pathways are highly conserved

Bioinformatic studies had previously identified two Ae. aegypti SUMO orthologue genes (AAEL015064; AAEL013787), along with gene orthologues of SAE1 and SAE2 (AAEL000091 and AAEL010641, respectively), Ubc9 (AAEL007477), and PIAS (AAEL015099) [24,25]. Following the recent re-sequencing and updated annotation of the Ae. aegypti genome (AaegL5.1, available at https://www.vectorbase.org/; [27]), the gene ID of Ubc9 has changed to AAEL027903. In addition, our analysis identified only one SUMO gene (AAEL015064), with sequences relating to the second putative gene (AAEL013787) being re-annotated to that of a calcium ion transporter gene (AAEL019513). Mass spectrometry analysis of Ae. aegypti AF5 cells identified peptides unique to each protein of the Ae. aegypti SUMOylation pathway (SUMO [AAEL015064], SAE1/2, Ubc9, and PIAS), with the exception of the putative AAEL013787 SUMO protein (S1 File). Therefore, AAEL015064 is the only SUMO gene product abundantly expressed in Ae. aegypti AF5 cells (hereafter referred to as AaSUMO).

Alignment of SUMOylation effector proteins (SUMO, SAE1/2, Ubc9, and PIAS) between Ae. aegypti and H. sapiens demonstrated a high degree of protein conservation. AaSAE1, AaSAE2, and AaUbc9 shared 42%, 50%, and 85% amino acid identity with their respective H. sapiens orthologues (Fig 1A). Homology between AaSUMO and HsSUMO1-3 proteins ranged from 47 to 71% identity, with AaSUMO showing the highest degree of similarity to that of HsSUMO3 (Fig 1A). Phylogenetic alignment of SUMO proteins from different species indicated that SUMO orthologues from Ae. aegypti and other mosquito species lack an N-terminal SCM, contrary to the chelicerate Ixodes scapularis and vertebrate SUMO orthologues (Fig 1B and 1C; [25]). Homology modelling confirmed AaSUMO to contain a classical ubiquitin-related modifier tertiary structure with flexible N- and C-terminal regions (Fig 1D; [25,28]). In H. sapiens, PIAS SUMO E3 ligases are known to increase the rate of SUMO modification and poly-SUMO chain formation [29,30]. In contrast to the five PIAS proteins expressed by H. sapiens, only one PIAS protein has been identified in Ae. aegypti [24]. The amino acid identity of AaPIAS ranged from 32 to 37% between HsPIAS orthologues, increasing up to 70% in the catalytic SP-RING domain (Fig 1A). Sequence alignment confirmed the presence of Zn²⁺-coordinating residues within the SP-RING required for SUMO ligase activity (Fig 1E; grey bars) [31]. These data indicate that the function of the SP-RING domain is likely to be conserved between H. sapiens and Ae. aegypti. Overall, our analysis revealed a high degree of protein conservation between Ae. aegypti and H. sapiens SUMOylation pathways. These data suggest a functional conservation of this pathway between mosquitoes and humans, with the notable exception of an absent N-terminal SCM within AaSUMO.

Fig 1

The Aedes aegypti SUMOylation pathway is highly conserved.

(A) Colour-coded pairwise identity matrix showing the percentage amino acid (a.a) identity between orthologue proteins of the Aedes aegypti and Homo sapiens SUMOylation pathways. Each coloured cell represents the percentage identity score between two sequences. Colour key showing the correspondence between percentage identity and the colours displayed shown. (B) Phylogenetic tree and N-terminal amino acid alignment of SUMO proteins from a selection of model organisms (SUMO Uniprot accession number). Hs, Homo sapiens (P55854); Mm, Mus musculus (Q9Z172); Gg, Gallus gallus (Q5ZHQ1); Xl, Xenopus laevis (Q6DK72); Is, Ixodes scapularis (B7PKS5); Cq, Culex quinquefasciatus (B0WVZ6); Aa, Aedes aegypti (Q16EQ3); Aal, Aedes albopictus (A0A182H3N0); Ag, Anopheles gambiae (Q7PNJ2 and Q7PY16 ⁺); Dm, Drosophila melanogaster (O97102). Branch lengths are proportional to sequence divergence with bootstrap values shown. Conserved residues are shown in orange; residues with similarity in green. SUMO consensus motifs (SCM; Ψ-Lys(K)-X-Asp(D)/Glu(E), where Ψ represents a branched hydrophobic amino acid and X represents any amino acid) are higlighted in grey. (C) Amino acid alignment of full length AaSUMO plotted against HsSUMO3. (D) Predicted structure of AaSUMO (blue) plotted against the resolved structure of HsSUMO3 (red; PDB ID: 2MP2; [85]). Position and sequence of SCM in HsSUMO3 highlighted (VKTE; grey). (E) Phylogenetic tree and amino acid alignment of the catalytic SP-RING domain of orthologue PIAS proteins from Ae. aegypti and H. sapiens. Uniprot accession numbers; AaPIAS (Q1DH55), HsPIAS1 (O75925), HsPIAS2α (O75928-2), HsPIAS2β (O75928-1), HsPIAS3 (Q9Y6X2), and HsPIAS4 (Q8N2W9). Branch lengths proportional to sequence divergence with bootstrap values shown. Residues of AaPIAS 340–418, HsPIAS4 311–388, HsPIAS3 312–390, HsPIAS2α 331–409, HsPIAS2β 331–409, and HsPIAS1 320–398 shown. Zn²⁺ binding residues highlighted by grey bars; cysteine (C371) marked with #.

AaPIAS stimulates the efficient formation of poly-AaSUMO chains

As AaSUMO lacks an N-terminal SCM, this protein may not readily support poly-SUMO chain formation, unlike its closest H. sapiens orthologue HsSUMO3 [14]. We tested this hypothesis by incubating together recombinant proteins (S1 Fig) of either the AaSUMOylation pathway (AaSUMO, AaUbc9, and AaSAE1/2) or the HsSUMOylation pathway (HsSUMO3, HsUbc9, and HsSAE1/2) at either 28 or 37°C, the optimal temperatures for mosquito and mammalian cells, respectively. We detected a build-up of AaSUMO-dimers along with high molecular weight (HMW) SUMO conjugates indicative of SAE1/2 auto-SUMOylation (Fig 2A; [32,33]). The formation of HMW AaSUMO conjugates was more comparable to HsSUMO1 than HsSUMO3 at either 28 or 37°C (Fig 2B and 2C). These data indicate that AaSUMO is more biochemically similar to that of HsSUMO1 than HsSUMO3 with respect to supporting the formation of HWM SUMO protein conjugates, consistent with the lack of an internal SCM (Fig 1B; [25]). In order to determine the effect of AaPIAS on poly-AaSUMO chain formation, biochemical assays were repeated in the presence of wild-type (WT) or catalytically inactive (C371A; Fig 1E) AaPIAS. Addition of WT AaPIAS led to a rapid build-up of HMW SUMO conjugates relative to AaPIAS C371A (Fig 2D and 2E). Collectively, these data indicate that AaPIAS stimulates the efficient formation of HMW SUMO conjugates in an SP-RING dependent manner. We next sought to investigate why AaSUMO could not efficiently form chains in the absence of AaPIAS. We hypothesized that this was due to the absence of an SCM (Fig 1B; [25]). We generated a chimeric SUMO protein, in which the N-terminus of AaSUMO (amino acids 1–13) was replaced by the one of HsSUMO3 (amino acids 1–14) to incorporate its SCM. This chimeric SUMO protein was found to support the rapid build-up of HMW SUMO conjugates in the absence of AaPIAS that was dependent upon the Lys acceptor residue (K11) within the SCM (Fig 2F). These data demonstrate that while AaSUMO shares the highest degree of amino acid identity to HsSUMO3, its biochemical activity as a ubiquitin-like protein is more functionally related to that of HsSUMO1 due to the absence of a functional SCM.

Fig 2

AaPIAS is required for efficient poly-AaSUMO chain formation.

Purified Ae. aegypti and H. sapiens SUMO, Ubc9, and SAE1/2 recombinant proteins (50 ng each per reaction, S1 Fig) were incubated in the presence of 5 mM ATP for the indicated time points (minutes, min). (A) Representative western blot showing the accumulation of high molecular weight (HMW) SUMO conjugates over time at 28°C. Free SUMO, SUMO-SUMO dimers, and HMW conjugates highlighted. Molecular mass markers shown. (B/C) Quantitation of the accumulation of HMW SUMO conjugates (as in A; ≥ SUMO dimers) at 28 or 37°C (B and C, respectively). N≥3 independent reactions per condition; values normalized to T = 0 min (T0); mean and standard error of mean plotted. (D/E) Ae. aegypti SUMOylation pathway enzymes were incubated in the presence of WT or catalytically inactive (C371A) AaPIAS (10 ng) for the indicated time points at 28°C. (D) Representative western blot showing the accumulation of HMW SUMO conjugates in the presence of WT AaPIAS. (E) Quantitation of the accumulation of HMW SUMO conjugates (as in D and described in B). (F) Ae. aegypti SUMOylation pathway enzymes (SAE1/2 and Ubc9) were incubated in the presence of chimaeric SUMO (amino acids 1–14 of HsSUMO3 [including WT or mutant (K11R) SCM] in frame with the C-terminus of AaSUMO) for the indicate time points at 28°C. Western blots were quantified for the accumulation of HMW SUMO conjugates (as described in B).

AaSUMO contains eight Lys residues (Fig 1C) which could act as potential acceptor sites for SUMO modification in the presence of AaPIAS. We next investigated which Lys residues within AaSUMO were preferentially modified by AaPIAS. Mass spectrometry analysis was conducted on SUMO-dimers and HMW poly-SUMO chains generated in the presence or absence of either WT or C371A AaPIAS (S2 Fig). Spectral analysis demonstrated that AaSUMO was preferentially conjugated to surface exposed Lys (K) residues K5, K6, K9, K40, and to a lesser extent, K33 (Figs 3A and S3 and S2 Files). Addition of AaPIAS increased the proportion of detectible branched SUMO peptides, consistent with enhanced levels of poly-SUMO chain formation in a SP-RING dependent manner (Fig 2D and 2E) without strong preference for any specific solvent-exposed Lys residue (Figs 3B and 3C and S3). Collectively, these data demonstrate that AaPIAS plays an important role in the SCM-independent formation of HMW AaSUMO conjugates.

Fig 3

Internal lysine acceptor residues for AaSUMO modification.

(A) Summary of mass spectrometry data (S2 Fig) associated with branched SUMO peptides identified on SUMO Lys (K5, K6, K9, K19, K31, K33, K34, and K40) acceptor residues from in vitro reaction mixtures containing AaSAE1/2, AaUbc9, and AaSUMO. Data aggregated from all samples. MaxQuant Andromeda (peptide assignment score) values shown. (B) Total branched peptide intensity for all Lys acceptor residues from in vitro reaction mixtures (as in A) incubated in the presence or absence of WT or catalytically inactive (C371A, CA) AaPIAS (as shown). (C) Slice-specific (dimeric SUMO, SUMOx2; HWM SUMO conjugates, polySUMO) peptide intensity data of Lys-specific acceptor residues. Data presented as a percentage of the total intensity of all branched peptides found in each gel slice. Representative spectra can be found in S7–S14 Figs. Data summary presented in S2 File. N = 3.

AaSUMO is differentially expressed in Ae. aegypti tissues

To determine if AaSUMO was expressed in tissues relevant to arbovirus infection, we determined the relative expression levels of SUMO transcripts in multiple mosquito tissues from female mosquitoes; including salivary glands, digestive tract, ovaries, carcass (remaining abdominal tissue), and perfused haemocytes (mosquito immune cells). RNA was extracted and transcript expression levels analysed by RT-qPCR. SUMO transcripts were detected in all tissues and haemocytes, with significantly higher levels of SUMO expression observed in haemocytes and ovaries relative to the carcass (Figs 4A and S4).

Fig 4

AaSUMO is differentially expressed in Ae. aegypti tissues.

(A) RNA was extracted from perfused haemocytes, dissected salivary glands, digestive tracts, ovaries, and carcasses (pools of 25 digestive tracts, ovaries or carcasses, 60 salivary glands, or pooled perfused haemocytes from 70 females per replicate experiment, N = 3 independent experiments). cDNA was synthesised and RT-qPCR was conducted to examine mRNA expression of SUMO. Data were analyzed as described by Taylor et al. (78). SUMO expression values were normalized to ribosomal S7 as a reference transcript, with a RQ geometric mean (geomean) of 1 for carcass. SD shown. Log2-normalized expression values were used for statistical analyses; one-way ANOVA (F_4,10 = 17.99, p = 0.0001) followed by a Dunnett multiple comparison test relative to carcass (p values shown). (B to L) Representative confocal microscopy images showing the expression and sub-cellular localization of SUMO (red) in Ae. aegypti cells and tissues as detected by immunofluorescence assay. (B,C) AF5 cells; (D) perfused prohaemocytes; (E) perfused granulocytes; (F,G) salivary glands and surrounding fat body; (H) midgut; (I) oviduct; (J) ovarioles surrounded by the ovarian sheath; and (K,L) primary previtellogenic follicle and germarium inside ovarioles. Nuclei are stained by DAPI (blue) and F-actin is stained by Phalloidin 488 (green). Bar = 20 μm. Arrows indicate SUMO accumulation around the nucleus of the future oocyte (white) and at the perinuclear region of the nurse cells (yellow).

We next examined the expression pattern of AaSUMO protein in Ae. aegypti. Immunofluorescent confocal microscopy analysis was conducted using polyclonal antibodies raised to HsSUMO2/3, which detect both unconjugated and conjugated AaSUMO protein (Fig 2A and 2D). Microscopy assays were conducted on cultured Ae. aegypti-derived AF5 cells, perfused haemocytes, salivary glands, digestive tract, and ovaries from Ae. aegypti NBF females (Fig 4). Controls without primary antibody were also analysed under the same conditions to ensure signal specificity to AaSUMO (S5 Fig). In AF5 cells, AaSUMO was predominantly detected as nuclear punctate structures (Fig 4B and 4C). Consistent with our RT-qPCR results, AaSUMO expression was detected in perfused haemocytes and in every Ae. aegypti tissue analysed. In haemocytes, AaSUMO expression was restricted to the nucleus in prohaemocytes (Fig 4D), which have a high nuclear/cytoplasmic ratio [34], and differentiated granulocytes (Fig 4E), which are bigger cells with filopodia and a low nuclear/cytoplasmic ratio [34]. Very low levels of AaSUMO expression were detected in the lobes of salivary glands (Fig 4F), with higher levels of AaSUMO detected in surrounding fat body cells (Fig 4G). In the midgut, AaSUMO was expressed in the nucleus of enterocytes (recognizable by their large nuclei) and in smaller enteroendocrine and/or stem cells (Fig 4H). In the ovaries, AaSUMO was found to be highly expressed in the nuclei of the epithelial cells of the oviduct (Fig 4I). Similar staining was found in the ovarian sheath surrounding the ovarioles (Fig 4J). In the ovarioles, AaSUMO was expressed within the primary previtellogenic follicle and in the germarium (Fig 4K and 4L). In the primary follicle, AaSUMO was detected in the cytoplasm of the follicular cells surrounding the follicle. A strong circular signal of AaSUMO protein was detected close to the nucleus of the oocyte (Fig 4K and 4L; white arrows) and at perinuclear regions of nurse cells (Fig 4K; yellow arrows). In the germarium, AaSUMO was detected in the nucleus and cytoplasm of germline stem cells and follicle stem cells, in the cystoblasts as well as in the germline and follicular cells of the developing secondary follicle. Overall, our findings shows that AaSUMO is differentially expressed in multiple tissues and cells known to support arbovirus replication, including the gut, ovaries, and haemocytes.

The Ae. aegypti SUMOylation pathway has broad antiviral activity against arbovirus infection

We next assessed the biological significance of the Ae. aegypti SUMOylation pathway during arbovirus infection. AF5 cells were treated with gene-specific (AaSUMO, AaUbc9, AaPIAS) or negative control (lacZ) dsRNA to transiently deplete SUMOylation effector proteins. As a positive control, AF5 cells were treated with a dsRNA targeting an effector of the exo-RNA interference (RNAi) pathway (ago2), which has known antiviral activity against SFV [35,36], BUNV [37], but not ZIKV [36,38,39]. Transfected cells were infected (MOI 0.05 PFU/cell) with luciferase reporter viruses from three independent arboviral families: BUNV (Orthobunyavirus, Bunyaviridae); SFV (Alphavirus, Togaviridae); or ZIKV (Flavivirus, Flaviviridae). These reporter viruses have been previously used to analyse the antiviral action of Ago2 and shown to be as genetically stable as their respective wild-type viruses (SFV [36,40–42]; BUNV [37]; ZIKV [43]). Transcript levels of SUMO, Ubc9, PIAS, and ago2 were decreased from 50 to 80% relative to dsLacZ-treated control cells (Fig 5A, 5D and 5G), validating the efficiency of dsRNA depletion. As expected, depletion of Ago2 led to a significant increase in BUNV and SFV, but not ZIKV, replication relative to infected dsLacZ control cells (Fig 5B, 5E and 5H, respectively). Importantly, depletion of AaSUMOylation pathway effectors (AaSUMO, AaUbc9, and AaPIAS) led to significantly higher levels of BUNV, SFV, and ZIKV replication independently of the effector protein targeted for depletion (Fig 5B, 5E and 5H, respectively). These data indicate that multiple component proteins of the AaSUMOylation pathway contribute to the suppression of arbovirus replication; with BUNV and SFV being the most susceptible to AaSUMOylation-mediated restriction. Significant increases in BUNV, SFV, and ZIKV intracellular RNA levels could also be observed in cells depleted for AaSUMOylation proteins (Fig 5C, 5F and 5I, respectively); with SFV demonstrating the greatest increase in viral RNA (vRNA) levels (up to 10-fold relative to dsLacZ-treated control cells). It remains to be determined why depletion of AaUbc9 or AaPIAS had no significant effect on the intracellular levels of BUNV RNA. However, this may reflect the relative level of effector protein depletion between replicate experiments (Fig 5A; Ubc9). In the majority of cases, disruption of the AaSUMOylation pathway led to increases in both arbovirus replication (Fig 5B, 5E and 5H) and intracellular vRNA levels (Fig 5C, 5F and 5I). Collectively, these data indicate that the AaSUMOylation pathway has a broad antiviral action against arboviruses from multiple families in Ae. aegypti cells.

Fig 5

AaSUMOylation effector proteins suppress arbovirus replication in Ae. aegypti cells.

AF5 cells were treated with dsRNA targeting SUMO, Ubc9, PIAS, LacZ (non-target negative control), or ago2 (positive control) for 72 hours prior to infection (MOI 0.05 PFU/cell) with BUNV, SFV, or ZIKV luciferase reporter viruses. Cells were harvested at 48 hours post-infection for luciferase and RT-qPCR analysis. (A,D,G) RT-qPCR analysis of mRNA levels of ago2, SUMO, Ubc9, or PIAS within infected AF5 cells. N = 5 independent biological replicates; values normalized to ribosomal S7 and expressed relative to dsLacZ-treated control samples; RQ mean and SD plotted. (B, E, H) Luciferase readings from BUNV, SFV, or ZIKV infected samples. N = 15 independent biological replicates per condition; values expressed relative to dsLacZ-treated control samples (set to 1, dotted line); mean and SD plotted. (C, F, I) Viral RNA (vRNA) levels from BUNV, SFV, or ZIKV infected samples. N = 5 independent biological replicates; values expressed relative to dsLacZ-treated control samples (set to 1, dotted line); RQ mean and SD plotted. Statistical analysis, one sample (two-tailed) t test to a hypothetical mean of 1 (dsLacZ control), significant probability (P) values (≤ 0.05) shown; ns = not significant.

Cell culture

Ae. aegypti cell lines A20 and AF5 [40,72,73] were maintained in Thermo Fisher Scientific Nunclon Delta surface (25 cm²) tissue culture flasks with Leibovitz’s L-15 media (Gibco) supplemented with 10% (w/v) Tryptose phosphate broth (TPB; Gibco) and 10% (w/v) Foetal Bovine Serum (FBS; Gibco). BHK-21 cells (obtained from Roger D. Everett [MRC-UoG CVR]) were cultured in GMEM (Gibco), supplemented with 10% TPB and 10% FBS, Vero-E6 cells (obtained from Claire Donald [MRC-UoG CVR]) were cultured in DMEM (Gibco) supplemented with 10% FBS. A549-Npro cells (obtained from Claire Donald [MRC-UoG CVR]) were also cultured in DMEM (Gibco), supplemented with 10% FBS and 0.5 μg/ml puromycin. A549-Npro, BHK-21 and Vero-E6 cells were grown at 37°C with 5% CO₂, A20 and AF5 cells were cultured at 28°C.

Virus stocks

Semliki Forest virus (SFV) with a firefly luciferase reporter (SFV4-FFluc described in [74]) was a kind gift from Dr. Esther Schnettler (MRC-UoG CVR) and grown and titrated in BHK-21 cells as previously described [42]. Stocks of BUNV encoding a NanoLuciferase reporter protein (BUNV-NLuc described in [37]) was a kind gift from Dr. Xiaohong Shi (MRC-UoG CVR) and grown and titrated in BHK-21 cells as previously described [75]. ZIKV encoding a NanoLuciferase protein (ZIKV-NLuc described in [43]) was produced and kindly gifted by Jamie Royle (MRC-UoG CVR) grown and titrated in Vero-E6 and A549-Npro cells, respectively as previously described [43,76].

Plasmids

AaSAE1/2 were amplified from A20 cDNA using gene-specific primers (S1 Table). CDS were cloned into a pACYCDuet-1 dual expression vector (Sigma-Aldrich; Merck KGaA) using EcoRI/NotI (SAE2) and NdeI/XhoI (SAE1) unique sites. A Strep.II tag (5’-TGGAGCCACCCGCAGTTCGAAAAG-3’) was synthesised (Dundee Cell Products) and inserted to replace 6xHis tag sequences between NcoI-EcoRI in frame with AaSAE2. WT SUMO chimaera (amino acids 1–14 of HsSUMO3 and 14–91 of AaSUMO) and mutant SUMO chimaera (K11R) CDS were synthesised (GENEWIZ), amplified with the primers ‘pET28-AaSUMO R’ and ‘pET28-SUMO chimaera F’ (S1 Table), and inserted into pET28a (Novagen) using NdeI/XhoI restriction sites. A CDS for AaSUMO (cloned from A20 cells; a kind gift from Dr. Sue Jacobs, Pirbright Institute) was cloned into pET28a using primers ‘pET28-AaSUMO F’ and ‘pET28-AaSUMO R’ (S1 Table) using NdeI/XhoI restriction sites. WT and mutant (C371A) AaPIAS CDS were synthesised (GENEWIZ), amplified by PCR with the primers ‘AaPIAS F’ and ‘AaPIAS R’ (S1 Table), and cloned into a pET45b (Sigma-Aldrich; Merck KGaA) using AgeI/NotI restriction sites. AaUbc9 was cloned from A20 cDNA using primers ‘pET45-AaUbc9 F’ and ‘pET45-AaUbc9 R’ (S1 Table) and cloned into pET45b using AgeI/XhoI restriction sites. Bacterial expression vectors for HsSAE1/2, HsSUMO1, HsSUMO2, HsSUMO3, and HsUbc9 are described in [14]. All restriction enzymes were purchased from New England Biolabs. All final constructs were confirmed by Sanger sequencing (Source Bioscience).

Protein expression and purification

BL21 (DE3) cells were transformed with a bacterial expression plasmid containing the CDS of interest under the appropriate antibiotic selection. Single colonies were grown in 10 ml of Luria Bertani Broth (LB) O/N at 37°C in an orbital shaker (200 RPM). 5 ml was added to 200 ml of LB and allowed to grow for a further 2 hours at 37°C, 200 RPM. Protein expression was induced with 0.1 mM isopropyl-thio-β-D-galactoside. Induction occurred in an orbital shaker at 30°C, 200 RPM for 4–5 hours. Bacteria were pelleted by centrifugation at 3500 x g for 10 min at 4°C. Unless otherwise stated, the bacterial pellets were resuspended in 25 ml 6xHis Purification Buffer (50 mM Tris (pH7.5), 250 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol, 0.1% Triton-X-100, 2.5 mM MgCl₂, 30 mM imidazole and a protease inhibitor cocktail (Roche, pH 7.5). Bacteria were lysed by probe-sonication (45 pulses, 30 Amps) and centrifuged (13,000 x g for 10 min). Bacteria containing pACYC-Strep.II-AaSAE2/1 were resuspended in 2 ml Strep Purification Buffer (50 mM Tris pH 7.0, 50 mM NaCl, 1 mM MgCl₂, and a protease inhibitor cocktail (Roche, pH 7.5). Samples were then lysed by digital sonicator (7 repeats at 50% intensity for 30 sec, at 4°C) and centrifuged (14,000 x g for 10 min). Supernatants containing recombinant proteins were filtered through a 0.45 μm filter, bound to 250 μl (w/v) of equilibrated Ni-NTA (Qiagen) or Strep-tactin Superflow agarose beads (Novagen) in the corresponding purification buffers. Samples were tumbled end-over-end for 90 min at room temperature (RT), beads sedimented centrifugation (1500 x g for 5 min), washed twice in 10 ml of purification buffer and three times in 1 ml of purification buffer. 6xHis tag proteins were eluted in 750 μl of 6xHis Purification Buffer containing 350 mM (final concentration) imidazole at pH 7.5 prior to dialysis in Dialysis Buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5% glycerol, 2 mM MgCl₂ and 5 mM β-mercaptoethanol, 0.1% Triton-X-100) for 4 hours. Strep.II-AaSAE2/1 was eluted in 600 μl Strep Purification Buffer with 2.5 mM D-desthiobiotin (pH 8.5) prior to dialysis in Dialysis Buffer for 4 hours. Proteins were visualized by SDS-PAGE (4–12% gradient PAGE; Invitrogen) alongside purified commercial BSA standards (BioRad) by Coomassie brilliant blue staining. Protein concentration was determined on the Li-Cor Odyssey Imaging System.

Biochemical assays

Master mixes of Ae. aegypti or H. Sapiens recombinant SUMO pathway component proteins were aliquoted into individual tubes containing SUMO, Ubc9, and SAE1/2 (50 ng each/reaction), and if required, WT or mutant (C371A) PIAS (10 ng/reaction). The reaction was initiated by addition of SAE1/2 and incubated at 37 or 28°C for the desired length of time. The reaction was terminated by the addition of 1.5xBM (Laemmli buffer supplemented with 2.6 M Urea and 50 mM Dithiothretol). This sample mixture was boiled at 95°C for 10 min prior to SDS-PAGE and western blotting. Biochemical assays for Mass spectrometry analysis were performed with 5 μg SUMO, 500 ng Ubc9, 500 ng SAE1/2, and 50 ng WT or C371A PIAS, and incubated at 28°C for 1 hour. Reactions were terminated by the addition of NuPAGE Sample Reducing Agent (Invitrogen).

Western blots

Samples were resolved on a NuPAGE-Novex 4–12% Bis-Tris Gradient gel (Invitrogen) with 1X NuPage MES SDS running buffer (Invitrogen). PAGE gels were transferred onto an Amersham protran 0.2 μm nitrocellulose blotting membrane (GE Healthcare) in transfer buffer (1x NuPage transfer buffer and 10% Methanol) at 30 V for 60 min. The membranes were blocked in blocking buffer (5% FBS in PBS) for 1 hour. The mouse anti-SUMO2/3 primary antibody (Cat. No. Ab81371; Abcam) diluted 1:1,000 in blocking buffer was incubated on the blotting membrane for 1 hour or O/N at 4°C. The membrane was washed 3 times for 5 min in PBST (0.1% Tween-20). A DyLight800 fluorescent goat anti-mouse secondary antibody (SA535521; Thermo Fisher Scientific) diluted 1:10,000 was incubated with the membrane for 1 hour. All steps were conducted at room temperature unless stated otherwise. Membranes were imaged and quantified using a LiCor Odyssey Imaging System.

dsRNA production

Total RNA was extracted from Ae.aegypti-derived A20 cells using an RNAeasy Plus Kit (Qiagen), following manufacturer’s instructions. cDNA was synthesised with TaqMan Reverse Transcription Reagents kit (Life Technologies) with Random Hexamer primers according to the manufacturer’s instructions. To synthesise dsRNA, cDNA was amplified with gene-specific primers (S1 Table) incorporating the T7 RNA polymerase promoter sequence at the 5’ end. PCR was conducted using Phusion polymerase (New England Biolabs), PCR products were excised from the gel using QIAquick Gel Extraction Kit (Qiagen), and sequenced to confirm identity by Sanger sequencing (Source Bioscience). dsRNA were produced using the MEGAscript RNAi kit (Ambion) according to the manufacturer’s instructions. The dsRNA were quantified using a Nanodrop spectrophotometer.

dsRNA transfection and infection

AF5 cells (2.5x10⁵ / well) were seeded in 24-well plates and left in the incubator overnight (O/N) at 28°C prior to the transfection of 300 ng dsRNA with 2 μl DharmaFECT2 (Dharmacon) per well, following manufacturer’s instructions. Cells were incubated for 72 hours prior to infection (MOI of 0.05 PFU per cell). After 48 hours, cells were harvested for RT-qPCR (viral load and depletion efficiency) or luciferase analysis.

Luciferase assay

Luciferase expression was determined using the Luciferase Assay System Kit (Promega) for SFV-Ffluc and the Nano-Glo Luciferase Assay System Kit (Promega) for BUNV-NLuc and ZIKV-NLuc. Luciferase activities were determined on a GloMax 20/20 Luminometer following the manufacturer’s instructions. A minimum of 5 independent biological repeats were conducted in triplicate for each experimental condition.

Mosquito rearing

Ae. aegypti mosquitoes (Liverpool strain, gift from Pr. Eileen Devaney, University of Glasgow) were reared at 28°C and 80% humidity with a 12:12 light photoperiod. Larvae were reared in water with some dry pieces of cat food pieces from larvae hatching to pupal stage. Emerging adult mosquitoes were transferred in cages with unlimited access to 10% w/v sucrose solution. For rearing, female mosquitoes were fed on rabbit blood supplemented with heparin (Orygen Antibodies Ltd) using a Hemotek (Hemotek Ltd., UK) at 37°C.

Dissection of mosquito tissues and perfusion of haemocytes

Tissues were dissected from 5 day-old females in RNase free 0.05% PBS-Tween 20 (PBST) (v/v). For RNA extraction, tissues were stored at -80°C. For immunostainings, tissues were stored in PBS-T 0.05% on ice before fixation. Haemocytes were collected by perfusion. The last segment of the abdomen was cut and mosquitoes were then injected in the thorax with 0.01% PBS-T. For RT-qPCR experiments, PBS-T diluted haemocytes were collected in tubes on ice, further centrifuge at 2,000 RPM for 15 min at 4°C. The supernatant was removed before the addition of TRIzol reagent (Thermo Fisher Scientific). For immunostainings, haemocytes were perfused on slides (ibidi) which were incubated for 30 min at 28°C before removal of PBS-T for fixation and staining.

RNA extraction and reverse transcription (RT)-quantitative PCR

Total RNA was isolated from cells using the RNAeasy Plus Kit (Qiagen), following manufacturer’s instructions. cDNA was synthesised with TaqMan Reverse Transcription Reagents kit (Life Technologies) with Random Hexamer primers, as per manufacturer’s instructions. Samples were analysed in triplicate using SYBR Green Master mix (Applied Biosystems) with gene-specific primers (S1 Table). Quantitative PCR (qPCR) was conducted on an Applied Biosystems 7500 Fast-Real-Time PCR system using MicroAmp plates and Optical Adhesive covers (Applied Biosystems). Relative mRNA expression levels were determined using the 2^−ΔΔCt (cycle threshold) method [77], normalised to the S7 ribosomal gene and presented as relative values to that of the dsLacZ control. Data presented for the knockdown efficiency and the viral loads is the mean of 5 independent biological repeats. RQ means and standard deviations are presented.

Dissected mosquito tissues (pools of 25 digestive tracts, ovaries or carcasses, pools of 60 salivary glands, and pools of perfused haemocytes from 70 females per independent replicate) were homogenised in TRIzol (Thermo Fisher Scientific) with glass beads using the Precellys 24 homogeniser (Bertin Instruments). RNA from tissues and haemocytes were further extracted using the Trizol method according to the manufacturer’s instructions except that 1-Bromo-3-chloropropane (Sigma) was used instead of chloroform and DNase (TURBO DNase, Ambion) treatment was performed. Reverse-transcriptions (RT) and RT negative controls were performed using the MMLV retro-transcriptase (Promega) from 25 ng/μl of RNA according to the manufacturer’s instructions. cDNAs were aliquoted and stored at -20°C until qPCR. qPCR was performed using Fast SYBR Green master mix (Applied Biosystems) following the manufacturer’s instructions using the 7500 Fast machine (Applied Biosystems). Primers used are listed in S1 Table. Results were analysed with the 7500 Software v2.0.6. Data were analyzed as described by Taylor et al. [78] to obtain normalized expression values, relative to ribosomal S7 transcript expression, with a geometric mean (geomean) of 1 for the carcass condition. Log2-normalized expression values were used for statistical analyses.

Immunofluorescence assays

Salivary glands and ovaries (n≥10 of each per replicate) were fixed at room temperature for 20 min in 4% (w/v) paraformaldehyde (PFA) diluted in PBS-T 0.05%. AF5 cells (3x10⁴ cells/well, incubated O/N at 28°C on ibidi slides), perfused haemocytes (from a pool of 5 females per replicate) and guts (n≥10 per replicate) were fixed the same way with the exception that the PFA was diluted using PBS. Fixed haemocytes/tissues (except ovaries) were washed (three times, 15 min each at 4°C) in PBS-T 0.05%, before being blocked for a minimum of 30 min in blocking solution (0.25% PBS-T 5% FBS (v/v), 5% BSA (w/v), 0.25% Triton X-100 (v/v) at 4°C. Unlike other tissues, ovaries were dilacerated and washed in 1% PBS-T before being blocked in blocking solution containing 0.5% Tween and 0.5% Triton. AF5 cells, haemocytes, and tissues were incubated at 4°C overnight with a mouse anti-SUMO2/3 antibody (Cat. No. Ab81371; Abcam) diluted 1:1000 in blocking solution. For each experiment, a negative control without primary antibody was performed. Samples were washed (five times, 15 min each at 4°C) in 0.05% PBS-T (0.5% PBS-T for ovaries) and incubated with an Alexa Fluor 568 goat anti-mouse IgG (H+L) diluted 1:1,000, DAPI 1X (405 nm) and Phalloidin 1X (488 nm) in blocking solution for 2 hours at RT. Four washes in 0.05% PBS-T (0.5% PBS-T for ovaries) were done and tissues were mounted between a slide and coverslip (24 mm x 24 mm) with an imaging spacer (1 well, diam. x thickness 13 mm x 0.12 mm Grace Bio-Labs SecureSeal imaging spacer, Sigma-Aldrich) using ibidi Mounting Medium (ibidi). Mounting media was used to replace the PBS-T in haemocytes. Images were acquired on a Zeiss LSM 710 inverted confocal microscope, equipped with a 40X or 63X oil-immersion objective and processed with Fiji/ImageJ and Adobe Photoshop. Two independent replicates were performed.

Shotgun proteomic analysis to identify expressed SUMO pathway proteins

AF5 cell extracts were harvested in 1.5XBM and proteins fractionated by 4–12% Bis-tris SDS-PAGE gel running in MES buffer. Each lane was excised into 12 slices and tryptic peptides extracted as described previously [79]. All 132 peptide samples were analyzed by LC-MS/MS on a Q Exactive mass spectrometer (Thermo Scientific) coupled to an EASY-nLC 1000 liquid chromatography system via an EASY-Spray ion source (Thermo Scientific) running a 75 μm x 500 mm EASY-Spray column at 45°C. Peptides were fractionated over a 90 minute gradient and data were acquired in the data-dependent mode. Full scan spectra (m/z 300–1800) were acquired with resolution R = 70,000 at m/z 200 (after accumulation to a target value of 1,000,000 with maximum injection time of 20 ms). The 10 most intense ions were fragmented by HCD and measured with a resolution of R = 17,500 at m/z 200 (target value of 500,000, maximum injection time of 60 ms) and intensity threshold of 2.1x10⁴. Peptide match was set to ‘preferred’ and a 40 second dynamic exclusion list was applied. The resultant 132 raw MS data files were processed using MaxQuant (v 1.6.1.0) with the built-in Andromeda peptide search engine [80]. The Ae. aegypti uniprot protome (downloaded August 2017) was searched. Enzyme specificity was set to trypsin-P. Cysteine carbamidomethylation was selected as a fixed modification with methionine oxidation and protein N-terminal acetylation as variable modifications. Initial maximum allowed mass deviation was set to 20 parts per million (ppm) for peptide masses and 0.5 Da for MS/MS peaks. The minimum peptide length was set to 7 amino acids and maximum size 4600 Da. A maximum of two missed cleavages were considered. A false discovery rate (FDR) of 1% was required at both the protein and peptide levels. The ‘match between runs’ option was applied with a time window of two minutes matching between samples from the same gel slice position across different lanes, or a slice above or below. 5462 protein groups were identified after removal of decoy proteins, putative contaminants and those only identified by modified peptide. Peptide level data for SUMO, Ubc9, SAE1, SAE2 and PIAS proteins are shown in S1 File.

Shotgun proteomic analysis of in vitro SUMO conjugation reactions to identify sites of polymerisation

Three in vitro SUMO conjugation reactions were prepared in triplicate: without any E3, with WT PIAS, or with C371A mutant PIAS (see above). Proteins were fractionated by 10% Bis-tris SDS-PAGE gel running in MOPS buffer. Each lane was excised into upper and lower slices to gain information on SUMO-SUMO dimers, and high molecular weight polymers. Tryptic peptides were extracted as described above. Half of each tryptic peptide sample was further subjected to in solution digestion with GluC to shorten the C-terminal SUMO adduct attached to substrate peptides, hence increasing likelihood of detection of as many SUMO conjugation sites as possible. Two mass spectrometry runs were performed for tryptic peptides and one for the doubly digested Trypsin-GluC peptides. Instrument setting were essentially as described above except for the second trypic peptide run and the only Try-GluC run for which a top1 method was applied with a 1000ms fill time. These settings improve MS/MS spectra for large low abundance peptides. To automatically detect branched peptides a concatenated database approach was taken, similar to previously described [81]. Briefly, a sequence file in fasta format was generated whereby the C-terminus of SUMO was concatenated to all conceivable peptides yielded by protease digestion of SUMO, Ubc9, SAE1 and SAE2. This concatenated sequence database contains individual ‘proteins’ that mimic the SUMO-substrate branched peptides allowing MaxQuant to automatically search for them in the MS data. MaxQuant was set up as described above except the concatenated peptide fasta file was used along with a file containing all proteins present in the in vitro reactions, GluC and Try/P (6 missed cleavages) or just Try/P alone (4 missed cleavages) were defined as proteases, and max peptide mass was set to 10000. Representative spectra can be found in S7–S14 Figs and data summary can be found in S2 File.

Bioinformatics and statistical analysis

Amino acid alignment was conducted using the online software T-COFFEE (http://tcoffee.crg.cat/) [82]. These results were then plotted and exported with BOXSHADE (https://embnet.vital-it.ch/software/BOX_form.html). Models of AaSUMO were conducted by obtaining the annotated amino acid sequence from UniProt, and predicting the tertiary structure with Phyre2 and plotting the structure in Chimera 1.10.1 [83,84]. Statistical analysis and graphs were produced using GraphPad Prism 7.02. Statistical tests are indicated in figure legends.