Asparagine (N) linked glycosylation is a protein modification that is widespread in eukaryotes and is essential for protein quality control and trafficking (1). N-Linked glycans contribute to protein structure and stability, receptor targeting, development and immune responses (2) . N-Linked glycosylation occurs in the secretory pathway and is initiated in the lumen of the endoplasmic reticulum (ER) through co-translational transfer of the triantennary Glc3Man9GlcNAc2 glycan from a lipid linked precursor ( Fig. 1 ; see also (2) (3) (4) (5) ). Formation of mature glycoproteins requires the removal of the glucose residues, typically through the action of -glucosidases I and II, which are localized within the rough ER. However, in most cell lines and in human patients genetic disruption of -glucosidase I or II, or inhibition of their activities do not prevent the formation of mature glycoproteins, because of the action of Golgi endo--1,2-mannosidase (MANEA (6)). The action of MANEA provides a glucosidase independent pathway for glycoprotein maturation termed the endomannosidase pathway (7) .
MANEA is present chiefly in cis/medial Golgi (84%) and ER-Golgi intermediate compartment (ERGIC; 15%) (8) and allows trimming of glucosylated mannose residues from Glc1-3Man9GlcNAc2 structures and B and C branch mannose-trimmed variants (see Fig. 1 for definition of N-glycan branches). MANEA action on Glc1-3Man9GlcNAc2 results in cleavage of the glucosylated mannose in the A branch, releasing Glc1-3Man and Man8GlcNAc2; human MANEA acts faster on Glc1Man than on Glc2,3Man glycans (9) . The endomannosidase pathway allows processing of ER-escaped, glucosylated high-mannose glycans on glycoproteins that fold independently of the calnexin/calreticulin (CNX/CRT) folding cycle, allowing them to rejoin the downstream N-glycan maturation pathway. In murine BW6147 cells under normal conditions, flux through the endomannosidase pathway accounts for around 15% of total flux through the secretory pathway (10) ; under conditions of glucosidase blockade through knockout or inhibition, MANEA can support as much as 50% of normal glycoprotein flux, depending on cellular expression levels (10) . MANEA activity accounts for the accumulation of Glc3Man tetrasaccharide in the urine of a neonate suffering from CDG-IIb, a rare genetic disease caused by a deficiency of ER glucosidase I (MOGS) (11) .
Most enveloped viruses (eg coronaviruses, retroviruses, ebolaviruses, hepatitis B virus (HBV), influenza viruses) contain glycoproteins within their envelope (12) . Glycosylation is achieved by coopting host cell glycosylation machinery during replication. Substantial efforts have been deployed in the use of inhibitors of -glucosidases I and II as antivirals (eg Nbutyldeoxynojirimycin, 6-O-butanoylcastanospermine) for the treatment of HIV/AIDS (13), dengue (14, 15) , and hepatitis B (16, 17) (reviewed in refs (18) (19) (20) ). Inhibition of host glycosylation pathways, in particular ER glucosidases I and II and Golgi mannosidase I, interferes with the viral lifecycle by impairing protein folding and quality control, inducing the unfolded protein response, or mistrafficking of viral glycoproteins. These changes can lead to impairment of secretion (17, (21) (22) (23) , fusion (24) (25) (26) or evasion of host immunity (27) . In the case of HBV, when glucosidases are inhibited mature HBV viral glycoproteins are still produced through the alternative processing provided by the endomannosidase pathway (28) .
However, the contribution of the endomannosidase pathway to viral protein assembly under normal conditions remains poorly studied, and the antiviral activity of inhibitors targeting MANEA has not been evaluated.
The Homo sapiens endo--1,2-mannosidase gene, MANEA, is located on chromosome 6. The MANEA gene has one isoform whose protein product, MANEA, is categorized as a member of the glycoside hydrolase (GH) family 99 in the Carbohydrate-Active enZyme (CAZy) database (29) , which also contains bacterial endo--1,2-mannanases from Bacteroides xylanisolvens (BxGH99) and Bacteroides thetaiotaomicron (BtGH99) that act on related structures within yeast mannans, and which share approx. 40% identity (30) (31) (32) . MANEA encodes a protein 462 aa long that consists of a single pass, type II, membrane protein with a transmembrane helix followed by a stem region, followed by the catalytic domain (33) . Here, we reveal the structure of the catalytic domain of human MANEA. Through complexes with substrate we reveal the architecture of the binding groove and identify key catalytic residues.
We report structures with MANEA inhibitors that were designed based on the structure of the glucosylated high mannose N-glycan. Finally, we show that inhibitors of MANEA act as antiviral agents against bovine viral diarrheal virus (BVDV) and dengue virus (DENV), confirming that MANEA is an anti-viral target.
In order to obtain soluble MANEA GH99 protein for structure-function studies to inform inhibitor design, attempts were made to express the gene. A truncated gene for MANEA, consisting of the catalytic domain beyond the stem domain, i.e. residues 98-462 (hereafter MANEA-Δ97), was synthesized in codon-optimized form. Expression trials in E. coli BL21(DE3) cells gave only insoluble protein, but we were inspired by a report (34) of soluble expression using cold-shock promotors (pCold-I vector) and co-expression of GroEL chaperones. Optimal yields of soluble soluble MANEA-Δ97 were obtained using Terrific Broth supplemented with glycerol and 20 mM MgCl2 affording approx. 2-3 mg L -1 (Supplemental Figure 1A , see Experimental procedures). The recombinant protein was purified using the encoded N-terminal His6-tag and was stable with a Tm of 50 ℃ (Supplemental Figure 1B) .
Treatment of GlcMan9GlcNAc2 with recombinant MANEA-Δ97 released α-Glc-1,3-Man and Man8GlcNAc2 (Supplemental Figure 1C) . Enzyme kinetics were measured using α-Glc-1,3-α-Man-1,2-α-Man-1,2-α-Man-OMe (GlcMan3OMe) as substrate using a coupled assay in which the product α-1,2-Man-ManOMe is hydrolysed by α-mannosidase and the resultant mannose quantified using a D-mannose/D-fructose/D-glucose detection kit (Megazyme, Inc.) (31) . MANEA-Δ97 hydrolysed GlcMan3OMe with a kcat of 27.7 ± 1.0 min -1 and KM of 426 ± 33 μm (kcat/KM of 65 mM -1 min -1 ) (Supplemental Figure 1D ). The catalytic efficiency is similar to that displayed by BtGH99 on the epimeric Man4OMe substrate (KM = 2.6 mM, kcat = 180 min -1 , kcat/KM = 69 mM -1 min -1 ) (31) . The E404Q variant of MANEA-Δ97 was inactive on this substrate, consistent with the proposed mechanism (35).
GlcDMJ is a cell-permeable inhibitor of MANEA (36, 37) and is comprised of the wellknown mannosidase iminosugar inhibitor deoxymannojirimycin (DMJ) modified with a glucosyl residue at the 3-position to enhance specificity and binding to MANEA by mimicking the substrate and benefitting from substrate-enzyme contacts in the -2 subsite. The related compound GlcIFG (38) , which is also cell-permeable (39) , was developed through a similar approach applied to the azasugar isofagomine (IFG). Similarly, ManIFG was synthesized to match the substrate stereochemistry of yeast mannan and is a superior inhibitor of bacterial endo--1,2-mannanases (31) . As glucosylated mannans are substrates for -glucosidase II, GlcDMJ and GlcIFG may potentially be degraded in cell-based systems (40) , leading to loss of inhibition. Examination of the 3D X-ray structure of -glucosidase II in complex with Nbutyldeoxynojirimycin reveals binding of the sugar-shaped heterocycle involves a pocket-like active site that will not readily accommodate additional substituents (41) . Conversely, it is known that MANEA can cleave Glc1-3Man substrates in which a substituent is tolerated at the 3-position of the glucosylated mannose. Therefore, we synthesized CyMe-GlcIFG, which bears a bulky cyclohexylmethyl substituent at the same position, in the expectation that it should be impervious to -glucosidase II activity, yet still bind to MANEA.
Isothermal titration calorimetry revealed that GlcIFG binds to MANEA-Δ97 with Kd = 19.6 ± 5.6 nM and ManIFG with Kd = 170 ± 32 nM. Whilst we were encouraged by preliminary studies on a bacterial MANEA homolog that showed that CyMeGlc-IFG bound better (Kd = 284 nM) than GlcIFG alone (Kd = 625 nM), on the human enzyme CyMe-GlcIFG bound less tightly that GlcIFG with Kd = 929 ± 52 nM (Supplemental Figure 1E) , consistent with structural features observed subsequently (see below). GlcDMJ, previously reported to have IC50 values of 1.7 to 5 M (36, 40), was the weakest binding of the four compounds, with a Kd value of 1020 ± 36 nM. MANEA thus exhibits a preference for inhibitors that match the stereochemistry of the substrate -2/-1 residues (GlcIFG versus ManIFG), which is the reverse of the preference of bacterial endo--1,2-mannanases for the same inhibitors (i.e. for BtGH99:
GlcIFG Kd = 625 nM; ManIFG Kd = 140 nM (31)). Consistent with previous findings in bacterial GH99 enzymes, IFG is preferred to DMJ in the -1 subsite.
Three-dimensional structure of human MANEA sheds light on eukaryotic enzyme specificity Crystals of human MANEA-Δ97, Figure 3A , were obtained in several crystal forms (see Experimental). The initial crystal form, in space group P21212, diffracted to around 2.25 Å resolution, and structure solution by molecular replacement (using the bacterial BxGH99 as a search model) was successful leading to structures with R/Rfree 18/22% (Supplemental Table   1 ). Unfortunately, this crystal form suffered from occlusion of the active centre by the His6-tag and a metal-ion assumed to be Ni 2+ . A second crystal form, P43212 gave data of lower resolution (3 Å) but in this case we could build a loop (131-141) absent in the first crystal form; however, this crystal form could not be reliably reproduced. A third crystal form in space group A key difference between the substrates for the human and bacterial GH99 enzymes are the -2 sugar residues, which is glucose in high-mannose N-glycans, and mannose in yeast mannan. These differences are achieved by differences in recognition between the human and bacterial GH99 enzymes in the -2 subsite and its environs. In bacterial endo--1,2-mannanases Trp126 (numbering for BxGH99 (38) ) forms a hydrophobic interaction with the C2 that was believed to be responsible for the selectivity of bacterial enzymes for ManMan versus GlcMan substrates. In MANEA, the equivalent residue is Tyr189, which makes a water-mediated interaction with O2 of Glc in the -2 subsite, 
glycans at a lower rate than monoglucosylated N-glycans (9) . In the GlcIFG complex of MANEA, the -2 loop is closed over the active site, and in this conformation, triglucosylated N-glycans would be unable to bind to human MANEA. However, the other observed conformations show flexibility in the loop that could allow these extended structures to bind.
Of note, bovine MANEA was observed not to process triglucosylated N-glycans (9) (44) . Therefore, we digested BVDV envelope glycoproteins E1/E2 with endo H. Increased sensitivity of E1/E2 proteins to endo H cleavage was observed both in the case of GlcIFG and the glucosidase I/II inhibitor NAP-DNJ, indicating an increased prevalence of high mannose N-glycans. The increased sensitivity for GlcIFG was greater than for NAP-DNJ, most likely reflecting the higher concentration of the former. Treatment with a combination of GlcIFG and NAP-DNJ gave an even higher sensitivity to endo H treatment, consistent with more effective cessation of N-glycan processing by blocking the glucosidase and endomannosidase pathways. We next examined BVDV replication in the presence of a combination of GlcIFG and NAP-DNJ ( Figure 4C) . These data showed that the combination of GlcIFG and NAP-DNJ gave a greater reduction than either agent alone suggesting an additive antiviral effect from inhibiting both the -glucosidase I/II and endomannosidase pathways. The addition of NAP-DNJ not only inhibits calnexin mediated folding and quality control but requires viral glycans to undergo processing by MANEA, hence potentiating the antiviral effect.
To further define the antiviral potential of MANEA inhibitors, we extended these studies to DENV. The MANEA inhibitors GlcIFG, GlcDMJ, CyMe-GlcIFG and ManIFG were used to study levels of viral particle formation and infectivity. Viral particle formation, as assessed by secreted viral RNA, was unaffected by treatment with MANEA inhibitors ( Figure   4D ). However, treatment with GlcIFG, CyMe-GlcIFG and GlcDMJ caused a reduction in plaque number, showing that differences in glycosylation resulting from MANEA inhibition impair DENV infectivity. The greatest effect was seen for GlcDMJ, which reduced the number of plaque forming units by 6-fold; ManIFG had no effect. The antiviral effect observed is at a relatively high concentration but iminosugars are known to have difficulty in gaining access to the secretory pathway (21) . This proof of principle demonstrates GlcDMJ is a potential broadspectrum antiviral with changes in glycosylation reducing infectivity of the progeny. As it is the weakest-binding inhibitor, factors other than affinity, such as cell permeability, may be responsible for potency of antiviral effects.
We report the 3D structure of Golgi endo--1,2-mannosidase, MANEA, a key eukaryotic N-glycosylation pathway glycosidase. This data provides a structural rationale to understand the change in specificity of this enzyme for mono-, di-and triglucosylated high mannose N-glycans. We also show the potential for MANEA inhibitors to alter N-glycan structures of viral envelope glycoproteins and reduce viral infectivity. MANEA processes Glc1-3Man9GlcNAc2 structures and provides a pathway for glycoprotein maturation that is independent of the classical -glucosidase I/II dependent pathways. Treatment of HBV with miglustat (N-butyldeoxynojirimycin, an inhibitor of -glucosidase II) impaired viral DNA secretion and led to aberrant N-glycans on M glycoprotein, but other viral glycoproteins displayed mature glycans that arose through the endomannosidase pathway (28) . Conversely, GlcDMJ treatment of VSV led to changes in G protein glycosylation (44) . These prior studies and the present work demonstrate that different viral glycoproteins have varying degrees of dependence on the -glucosidase I/II and endomannosidase pathways for maturation, and that inhibition of MANEA alone can alter infectivity of two encapsulated viruses. The human MANEA 3D structure, alongside demonstrated anti-viral activity of disaccharide imino and aza sugar inhibitors, provides a foundation for future inhibitor and drug development work.
Given the devastating consequences of global outbreaks of viral disease, the present work highlights the potential for MANEA as a new target for host-directed antiviral agents exploiting viral glycoprotein biosynthesis.
Full methods are provided as Supplemental Information.
Briefly, recombinant MANEA-Δ97 featuring an N-terminal His tag was expressed in E. coli from a pColdI system co-expressing the groEL and groES genes encoding the GroEL/ES chaperone system. Recombinant MANEA-Δ97 was purified by metal-ion affinity and cationexchange chromatography. Extensive crystal screening identified five different crystal forms, with a condition including 1 mᴍ TEW (tellurium-centered Anderson-Evans polyoxotungstate [TeW6O24] 6-) allowing ligand binding studies without an occluded active site. Structure solution by molecular replacement, and refinement featured programs from the CCP4 suite (45) . Structures, and observed data, have been deposited on the Protein Data Bank. MANEA activity was determined by mass spectrometry using GlcMan9GlcNAc2 as substrate with subsequent permethylation and analysis by MALDI-MS. Michael-Menten kinetics was performed using a coupled assay with GlcMan3OMe as substrate (31) . Ligand binding thermodynamics were determined by isothermal titration calorimetry.
MDBK cells were infected with BVDV at a multiplicity of infection (MOI) of 1, followed by incubation with different concentrations of GlcIFG (0-1 mM) and in combination with NAP-DNJ for 24 h. Cell culture medium was harvested and serial dilutions were made and used to infect naive MDBK cells. Cells were incubated for 24 h, then washed and fixed with 4% (v/v) paraformaldehyde in PBS for 30 min. Following washing and blocking, cells were permeabilised and incubated with MAb103/105 (1:500 dilution in 5% milk-PBS; Animal Health Veterinary Laboratories Agency, Weybridge, UK) for 1 h. Incubation with anti-mouse FITC-conjugated secondary antibody (1:500 dilution in 5% milk-PBS; Sigma) and staining with 4′,6-diamidino-2-phenylindole allowed counting of fluorescent foci and calculation of virus titers (fluorescent focus units (FFU)/ml). The samples obtained after treatment for 24 h were treated with Endo H and separated by SDS-PAGE under non-reducing conditions. The membrane produced following transfer was probed with MAb214 primary antibody and then visualized with anti-mouse horseradish peroxidase secondary antibody (1:1000; Dako) for 1 h.
Huh7.5 cells were infected for 2 h with 50 µL of DENV2 strain 16681 at an MOI of 0.1. The inoculum was removed and replaced with 200 µL of growth media with drug at concentrations as indicated in triplicate, and the infected cells were incubated with drug for 2 d. The supernatant was harvested. DENV RNA in cell culture supernatants was isolated according to the manufacturer's protocol for Direct-zol RNA MiniPrep Kit (Zymo Research) and assayed by qRT-PCR. Samples were read in technical duplicate and compared to a standard curve generated from high-titer viral RNA isolated from C6/36-grown DENV2. 95% confidence intervals were determined based on biological and technical variation and graphed using Prism 6 (GraphPad Software, Inc). The infectious DENV titres in supernatants collected were evaluated by plaque assay. tetradecasaccharide (branches labelled A/B/C) from the dolichol-precursor occurs cotranslationally to Asn residues within the consensus sequence Asn-Xxx-Ser/Thr. Trimming of glucose residues can be achieved through the classical pathway involving sequential action of -glucosidases I and II. Alternatively, Golgi endo--1,2-mannosidase (MANEA) provides a glucosidase independent pathway for glycoprotein maturation, through cleaving the glucosesubstituted mannose residues. ER mannosidase I resides in quality control vesicles (QCVs) (46) . ER associated degradation of terminally misfolded glycoproteins is omitted. 