Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins – major histocompatibility complex II (MHCII), CD86 and CD97 – from the surface of antigen-presenting cells. SteD specifically localises at the trans -Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

Plant trans-golgi network/early endosome pH regulation requires cation chloride cotransporter (CCC1)

Plant cells maintain a low luminal pH in the Trans-Golgi-Network/Early Endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified - a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis Cation Chloride Cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function.

Influenza A Virus Infection Activates NLRP3 Inflammasome through Trans-Golgi Network Dispersion

The NLRP3 inflammasome consists of NLRP3, ASC, and pro-caspase-1 and is an important arm of the innate immune response against influenza A virus (IAV) infection. Upon infection, the inflammasome is activated, resulting in the production of IL-1β and IL-18, which recruits other immune cells to the site of infection. It has been suggested that in the presence of stress molecules such as nigericin, the trans-Golgi network (TGN) disperses into small puncta-like structures where NLRP3 is recruited and activated. Here, we investigated whether IAV infection could lead to TGN dispersion, whether dispersed TGN (dTGN) is responsible for NLRP3 inflammasome activation, and which viral protein is involved in this process. We showed that the IAV causes dTGN formation, which serves as one of the mechanisms of NLRP3 inflammasome activation in response to IAV infection. Furthermore, we generated a series of mutant IAVs that carry mutations in the M2 protein. We demonstrated the M2 proton channel activity, specifically His37 and Trp41 are pivotal for the dispersion of TGN, NLRP3 conformational change, and IL-1β induction. The results revealed a novel mechanism behind the activation and regulation of the NLRP3 inflammasome in IAV infection.

Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.

First person – Yixing Wu and Ying Bai

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Yixing Wu and Ying Bai are co-first authors on ‘ Palmitoylated small GTPase ARL15 is translocated within Golgi network during adipogenesis’, published in BiO. Yixing is a research fellow in the lab of Frances Wiseman at UCL Queen Square Institute of Neurology, London, UK, investigating Down's syndrome and Alzheimer's disease-related endo-lysosomal pathways and cathepsin deficits. Ying is a postdoc in the lab of Roger D. Cox at MRC Harwell Institute, Didcot, UK, investigating how fat cells are formed, and genes that are involved in regulating body fat distribution.

V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission

Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel-Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant-negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.

Effects of Varicella-Zoster Virus Glycoprotein E Carboxyl-Terminal Mutation on mRNA Vaccine Efficacy

Glycoprotein E (gE) is one of the most abundant glycoproteins in varicella-zoster virus and plays pivotal roles in virus replication and transmission between ganglia cells. Its extracellular domain has been successfully used as an antigen in subunit zoster vaccines. The intracellular C-terminal domain was reported to be decisive for gE trafficking between the endoplasmic reticulum, trans-Golgi network and endosomes and could influence virus spread and virus titers. Considering that the trafficking and distribution of mRNA vaccine-translated gE may be different from those of gE translated against the background of the viral genome (e.g., most gE in virus-infected cells exists as heterodimers with another glycoprotein, gI,), which may influence the immunogenicity of gE-based mRNA vaccines, we compared the humoral and cellular immunity induced by LNP-encapsulated mRNA sequences encoding the whole length of gE, the extracellular domain of gE and a C-terminal double mutant of gE (mutant Y569A with original motif AYRV, which targets gE to TGN, and mutants S593A, S595A, T596A and T598A with the original motif SSTT) that were reported to enhance virus spread and elevate virus titers. The results showed that while the humoral and cellular immunity induced by all of the mRNA vaccines was comparable to or better than that induced by the AS01B-adjuvanted subunit vaccines, the C-terminal double mutant of gE showed stable advantages in all of the indicators tested, including gE-specific IgG titers and T cell responses, and could be adopted as a candidate for both safer varicella vaccines and effective zoster vaccines.

Duplication of NRAMP3 gene in poplars generated two homologous transporters with distinct functions

ABSTRACTTransition metals are essential for a wealth of metabolic reactions, but their concentrations need to be tightly controlled across cells and cell compartments, as metal excess or imbalance has deleterious effects. Metal homeostasis is achieved by a combination of metal transport across membranes and metal binding to a variety of molecules. Gene duplication is a key process in evolution, as emergence of advantageous mutations on one of the copies can confer a new function. Here, we report that the poplar genome contains two paralogues encoding NRAMP3 metal transporters localized in tandem. All Populus species analyzed had two copies of NRAMP3, whereas only one could be identified in Salix species indicating that duplication occurred when the two genera separated. Both copies are under purifying selection and encode functional transporters, as shown by expression in the yeast heterologous expression system. However, genetic complementation revealed that only one of the paralogues has retained the original function in release of metals stored in the vacuole previously characterized in A. thaliana. Confocal imaging showed that the other copy has acquired a distinct localization to the Trans Golgi Network (TGN). Expression in poplar indicated that the copy of NRAMP3 localized on the TGN has a novel function in the control of cell-to-cell transport of manganese. This work provides a clear case of neo-functionalization through change in the subcellular localization of a metal transporter as well as evidence for the involvement of the secretory pathway in cell-to-cell transport of manganese.