Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIF␣ prolyl hydroxylases is required for optimal parasite proliferation, especially at low O 2 . We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O 2 -dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.Toxoplasma is a worldwide obligate intracellular apicomplexan parasite that infects most nucleated cells of warm-blooded animals (1). Toxoplasmosis, the disease caused by Toxoplasma, is an opportunistic infection in AIDS and other immune-suppressed patients (2). In addition, in utero infections can cause mental retardation, blindness, and death (3). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by converting into slow growing bradyzoites that encyst. Cysts sporadically burst, and the released parasites convert to tachyzoites whose unabated growth, as can occur in immune suppressed hosts, results in cell and tissue damage (4). Currently, no Toxoplasma vaccine exists; anti-toxoplasmosis drugs have severe side effects, and resistance to these drugs is occurring.Recently, disruption of the gene for PhyA, the prolyl 4-hydroxylase that hydroxylates Pro-154 in Skp1, was observed to reduce tachyzoite proliferation in cell culture and fitness in a competition assay (5). Skp1 is an adaptor in the Skp1/Cullin-1/ F-box protein (SCF) 2 class of E3 ubiq...

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. Mutation of proline rich transmembrane protein 2 (PRRT2), a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 (and to a lesser extent, glycine-91) disrupted IFITM3 oligomerization and reduced its antiviral activity against Influenza A virus. An oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent and amphipathic helix-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

Skp1 is a subunit of the SCF (kp1/ullin 1/-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from is hydroxylated by an O-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba , where it regulates SCF assembly and O-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in , generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [H]glucose from UDP-[H]glucose to the trisaccharide form of Skp1 in a -dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 inΔ extracts, suggesting specificity for Skp1. -knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Interferon-induced transmembrane (IFITM) proteins are encoded by many vertebrate species and exhibit antiviral activities against a wide range of viruses. IFITM3, when present in virus-producing cells, reduces the fusion potential of HIV-1 virions, but the mechanism is poorly understood. To define the breadth and mechanistic basis for the antiviral activity of IFITM3, we took advantage of a murine leukemia virus (MLV)-based pseudotyping system. By carefully controlling amounts of IFITM3 and envelope protein (Env) in virus-producing cells, we found that IFITM3 potently inhibits MLV infectivity when Env levels are limiting. Loss of infectivity was associated with defective proteolytic processing of Env and lysosomal degradation of the Env precursor. Ecotropic and xenotropic variants of MLV Env, as well as HIV-1 Env and vesicular stomatitis virus glycoprotein (VSV-G), are sensitive to IFITM3, whereas Ebola glycoprotein is resistant, suggesting that IFITM3 selectively inactivates certain viral glycoproteins. Furthermore, endogenous IFITM3 in human and murine cells negatively regulates MLV Env abundance. However, we found that the negative impact of IFITM3 on virion infectivity is greater than its impact on decreasing Env incorporation, suggesting that IFITM3 may impair Env function, as well as reduce the amount of Env in virions. Finally, we demonstrate that loss of virion infectivity mediated by IFITM3 is reversed by the expression of glycoGag, a murine retrovirus accessory protein previously shown to antagonize the antiviral activity of SERINC proteins. Overall, we show that IFITM3 impairs virion infectivity by regulating Env quantity and function but that enhanced Env expression and glycoGag confer viral resistance to IFITM3. IMPORTANCE The viral envelope glycoprotein, known as “Env” in Retroviridae, is found on the virion surface and facilitates virus entry into cells by mediating cell attachment and fusion. Env is a major structural component of retroviruses and is targeted by all arms of the immune response, including adaptive and innate immunity. Less is known about how cell-intrinsic immunity prevents retrovirus replication at the level of individual cells. Here, we show that cellular IFITM3 and IFITM2 inhibit the fusion potential of retroviral virions by inhibiting Env protein via a two-pronged mechanism. IFITM proteins inhibit Env abundance in cells and also impair its function when levels are low. The posttranslational block of retroviral Env function by IFITM proteins is likely to impede both exogenous and endogenous retrovirus replication. In support of a relevant role for IFITM3 in retrovirus control, the retroviral accessory protein glycoGag counteracts IFITM3 function to promote virus infectivity.

No abstract

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

150 words) 29 30The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 31 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and 32 virions through a poorly characterized mechanism. We identified a GxxxG motif in many CD225 33 proteins, including IFITM3 and proline rich transmembrane protein 2 (PRRT2). Mutation of 34 PRRT2, a regulator of neurotransmitter release, at glycine-305 was previously linked to 35 paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the 36 homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG 37 motif. Mutation of glycine-95 in IFITM3 disrupted its oligomerization and reduced its antiviral 38 activity against Influenza A and HIV-1. The oligomerization-defective variant was used to reveal 39 that IFITM3 promotes membrane rigidity in a glycine-95-dependent manner. Furthermore, a 40 compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the 41 IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 42oligomers inhibit virus-cell fusion by promoting membrane rigidity. 43 44Introduction 45 46The intrinsic protection of cells from virus infection represents an early and essential 47 aspect of antiviral innate immunity. Cytokines including interferons signal the presence of 48 invading viruses and induce an "antiviral state" via the expression of hundreds of antiviral genes 49 [1, 2]. This arsenal of antiviral proteins converges on many steps of the virus life cycle in order 50to collectively inhibit infection of cells and prevent virus spread. In addition, certain "front-line" 51 antiviral proteins impose a constant barrier to infection because they are expressed constitutively 52 and are further upregulated by interferons. The interferon-induced transmembrane (IFITM) 53 proteins are the earliest acting restriction factors known, inhibiting the entry of diverse viruses 54 into cells by restricting fusion pore formation during virus-cell membrane fusion [3-6]. Among 55 the growing list of viruses shown to be inhibited by IFITM proteins in cell culture and in vivo are 56 orthomyxoviruses, flaviviruses, filoviruses, alphaviruses, and coronaviruses [3]. IFITM3 is a 57 potent inhibitor of Influenza A virus (IAV) infection in cell culture and in vivo, and 58consequently, it is the most studied member of the IFITM family [7][8][9]. While the precise 59 mechanism by which IFITM3 reduces virus-cell fusion remains unresolved, evidence suggests 60 that it does so by altering the properties of lipid membranes. 61Two models have been proposed to explain how IFITM3 inhibits virus fusion. In the first, 62IFITM3 plays an indirect role by interacting with VAMP-associated protein A (VAPA) and 63inhibiting lipid transport between the endoplasmic reticulum and endosomes, resulting in an 64accumulation of endosomal cholesterol [10]. High cholesterol content may inhibit the fusion of 65 virus-containing vesicles with the limiting me...

37SecA is an evolutionarily conserved and essential ATPase that is required for the 38 translocation of a subset of proteins across the cytoplasmic membrane in bacteria. SecA can 39 recognise proteins that are destined for translocation as they are still being synthesised in 40 order to deliver them to the membrane-bound Sec machinery. However, the mechanism of 41 cotranslational substrate recognition is not well defined. In E. coli, SecA contains a relatively 42 long C-terminal tail (CTT), which consists of a small metal-binding domain (MBD) that is 43 attached to the C-terminus of the catalytic core by a flexible linker (FLD). In this study, we 44 investigated the role of the CTT in the interaction of SecA with the ribosome and nascent 45 polypeptides. Previous work indicates that the CTT is required for interaction with the 46 molecular chaperone SecB. However, phylogenetic analysis suggested that the CTT (and the 47 MBD in particular) has an additional function. Binding experiments indicated that the CTT 48 interacts with 70S ribosomes, and disruption of the entire CTT moderately reduced the 49 affinity of SecA for ribosomes. However, disruption of the MBD alone significantly 50 increased the affinity of SecA for ribosomes and inhibited the interaction of SecA with 51 substrate protein, suggesting that the FLD affects the conformation of SecA. 52 Photocrosslinking and mass spectrometry indicated that the FLD is bound at the site where 53 SecA binds to substrate proteins. Structural analysis by x-ray crystallography and small-angle 54 x-ray scattering (SAXS) provided insight into how the CTT influences the structure of SecA 55 in solution. Finally, site-specific crosslinking experiments indicated that binding to nascent 56 substrate protein affects the conformation of SecA. Taken together, our results suggest that 57 the CTT regulates the ability of SecA to interact with substrate proteins.58 105 involved in binding of SecA to ribosomes, which we confirmed using ribosome 106 cosedimentation and chemical crosslinking approaches. Strikingly, disruption of the MBD 107 alone or the entire CTT had opposing effects on multiple different activities of SecA, 108 6 suggesting that the CTT affects conformation of the catalytic core. Mass spectrometry, x-ray 109 crystallography, and small-angle x-ray scattering experiments indicated that the FLD is 110 bound in the substrate binding groove and affects the conformation of the PPXD. Finally, site 111 specific chemical crosslinking suggested that binding of the MBD to the ribosome allows 112 full-length SecA to interact with nascent substrate proteins. Taken together, our results 113 provide insight into the molecular mechanism underlying nascent substrate recognition by 114 SecA. 115 116 Results 117 Evolutionary distribution of the MBD of SecA. To investigate the evolutionary 118 distribution of the CTT, we analysed the sequences of 156 SecA proteins from bacterial 119 species in 155 phylogenetic families using ClustalOmega (McWilliam et al., 2013). The 120 phylogenetic tr...