Virus-Wirt-Interaktionen bei Hepatitis-B-Virus-Infektionen: Die Rolle von Gamma2-Adaptin

Mit etwa 350 Millionen chronisch-infizierten Menschen gehört die Hepatitis-B neben der Tuberkulose und AIDS zu den häufigsten Infektionskrankheiten der Welt. Der einzig sichere Schutz vor dem bis zur Leberzirrhose persistierenden Virus bietet eine vorbeugende Impfung. Eine angemessene Therapie chronisch-erkrankter Patienten ist durch die Unkenntnis über viele Bereiche des HBV-Lebenszyklus nur eingeschränkt möglich. Gegenstand dieser Arbeit war vor allem etwas Licht in das Wechselspiel zwischen HBV und der Wirtszelle zu bringen und zelluläre Komponenten und Mechanismen zu identifizieren, die am Sortierungs- und Transportmechanismus viraler Substrukturen zur sogenannten Assembly-Plattform beteiligt sind, um dort die Freisetzung des Virus zu initiieren. Mit der vorliegenden Arbeit habe ich Methoden der Zellbiologie, Mikrobiologie, Molekularbiologie und Virologie vereint, um neue Einblicke in den HBV-Lebenszyklus zu gewinnen. Für die Ausschleusung von HBV wird seither der konstitutive Weg der Sekretion angenommen. In Anlehnung an den Mechanismus umhüllter RNA-Viren kann die Hypothese aufgestellt werden, dass das MVB (Multivesicular Body) im Prozess der HBV-Freisetzung beteiligt sein könnte. Die Freisetzung des Hepatitis-B-Virus aus einer infizierten Zelle ist ein streng organisierter Prozess, der sowohl das HBV-Coreprotein als auch die HBV-Hüllproteine zu benötigen scheint. (max. 5.000 Zeichen)Inhaltszusammenfassung in einer weiteren Sprache deutschenglischfranzösischrussischmehrsprachigsonst.Ausgangspunkt der Arbeit war eine spezifische Interaktion des großen HBV-Hüllproteins (L) mit einem neuen Mitglied der Adaptor-Protein-Komplex-Familie (AP-Komplex), dem g2?Adaptin (Hartmann-Stühler und Prange, 2001), das mutmaßlich an endosomalen Sortierungs- und Transportprozessen beteiligt ist. In Analogie zur Funktionsweise von Adaptin-Molekülen wurde vom g2-Adaptin eine Rolle in der Initiation und Steuerung der Sprossung und Freisetzung von HBV vermutet. Die im Rahmen dieser Arbeit erweiterte Charakterisierung der g2/L-Interaktion zeigte zum einen, dass die Ohrdomäne des Adaptins für die Interaktion essentiell ist und zum anderen, dass die Rekrutierung des Adaptins durch das L-Protein zu cis-Golgi-Strukturen innerhalb kleiner Transportvesikel entlang des Nukleus (perinukleär) erfolgt. Erste Ergebnisse dieser Arbeit deuteten bereits an, dass das virale Coreprotein mit demselben Adaptorprotein zu interagieren vermag. Für die g2/Core-Interaktion erwies sich in Folgearbeiten die Kopfregion des g2-Adaptins als die entscheidende Bindungsdomäne (Rost et al., 2006). Sowohl eine funktionelle Inaktivierung des g2-Adaptins durch RNA-Interferenz als auch eine g2-Adaptin- Überexpression störten die Virusmontage in späten Phasen der Morphogenese. Während ein g2-Adaptin-Überschuss die HBV-Produktion indirekt durch die Induktion dysfunktioneller endosomaler Kompartimente blockierte, verhinderte der siRNA-induzierte g2-Adaptin-Verlust die späte Ausschleusung des Virus aus der Zelle. Das Silencing von g2-Adaptin zeigte dabei weder einen Einfluss auf endosomale Strukturen noch auf die Freisetzung subviraler Partikel (Viren ohne Genom). Demzufolge scheinen sich die Mechanismen der Produktion von subviralen und viralen HBV-Partikeln bezüglich ihrer Anforderungen an Zellfunktionen und Transportwegen deutlich voneinander zu unterscheiden. Es konnten erste Hinweise darauf gefunden werden, dass die Virusmontage an endosomalen Kompartimenten (zum Beispiel dem MVB) erfolgen könnte, was durch bereits weitergeführte Untersuchungen bestätigt werden konnte (Lambert et al., J Virol, epub ahead of print). Darüber hinaus ist inzwischen bekannt, dass das Hepatitis-B-Virus für den Zusammenbau und die Freisetzung nicht nur das Adaptor-verwandte g2-Adaptin, sondern auch die endosomale Ubiquitin Ligase Nedd4, wahrscheinlich in Zusammenhang mit Ubiquitin selbst (Rost et al., 2006), benötigt. Eine Untersuchung dieser weiterführenden Aspekte war jedoch nicht mehr Inhalt dieser Arbeit. Insgesamt weisen die Daten darauf hin, dass die Sortierung und der Transport der Substrukturen des Hepatitis-B-Virus durch den MVB-Komplex zu erfolgen scheint. Dabei könnte das g2-Adaptin mit Hilfe einer Reihe von Kofaktoren (wie zum Beispiel Nedd4, Ubiquitin, etc.) eine entscheidende Rolle an der Sortierung und Anbindung des viralen L- und Coreproteins an die Assembly-Plattform spielen. Doch der genaue Mechanismus, welcher große Ähnlichkeit mit dem umhüllter RNA-Viren (wie zum Beispiel HIV-1) aufweist, bleibt noch ungeklärt. Ob weitere zelluläre Kofaktoren an der HBV-Sprossung und Freisetzung beteiligt sind, bleibt ebenfalls unbekannt und bedarf weiterer Untersuchungen.

ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise—in fact they decline—during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295–306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.

Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

Characterization of a Fourth Adaptor-related Protein Complex

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (ε, β4, μ4, and ς4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to ∼10–20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The μ4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B/GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.

Vps41p Function in the Alkaline Phosphatase Pathway Requires Homo-oligomerization and Interaction with AP-3 through Two Distinct Domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein–dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

Structural variety of arginine-rich RNA-binding peptides.

Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an alpha-helical conformation. Here we show that related peptides from splicing factors, viral coat proteins, and bacteriophage antiterminators (the N proteins) also have propensities to form alpha-helices and that the N peptides require helical conformations to bind to their cognate RNAs. In contrast, introducing proline mutations into the arginine-rich domain of the human immunodeficiency virus Tat protein abolishes its potential to form an alpha-helix but does not affect RNA-binding affinity in vitro or in vivo. Based on results from several peptide-RNA model systems, we suggest that helical peptides may be used to recognize RNA structures having particularly wide major grooves, such as those found near loops or large bulges, and that nonhelical or extended peptides may be used to recognize less accessible grooves.

Phospholipase D is present on Golgi-enriched membranes and its activation by ADP ribosylation factor is sensitive to brefeldin A.

ADP ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that regulates the binding of coat proteins to membranes and is required for several stages of vesicular transport. ARF also stimulates phospholipase D (PLD) activity, which can alter the lipid content of membranes by conversion of phospholipids into phosphatidic acid. Abundant PLD activity was found in Golgi-enriched membranes from several cell lines. Golgi PLD activity was greatly stimulated by ARF and GTP analogs and this stimulation could be inhibited by brefeldin A (BFA), a drug that blocks binding of ARF to Golgi membranes. Furthermore, in Golgi membranes from BFA-resistant PtK1 cells, basal PLD activity was high and not stimulated by exogenous ARF or GTP analogs. Thus, ARF activates PLD on the Golgi complex, suggesting a possible link between transport events and the underlying architecture of the lipid bilayer.

Potyvirus causando mosaico em plantas daninhas nas culturas do milho e do sorgo.

2016

Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented.

Whitefly Transmission and Efficient ssDNA Accumulation of Bean Golden Mosaic Geminivirus Require Functional Coat Protein

Bean golden mosaic geminivirus (BGMV) has a bipartite genome composed of two circular ssDNA components (DNA-A and DNA-B) and is transmitted by the whitefly, Bemisia tabaci. DNA-A encodes the viral replication proteins and the coat protein. To determine the role of BGMV coat protein systemic infection and whitefly transmission, two deletions and a restriction fragment inversion were introduced into the BGMV coat protein gene. All three coat protein mutants produced systemic infections when coinoculated with DNA-B onto Phaseolus vulgaris using electric discharge particle acceleration "particle gun." However, they were not sap transmissible and coat protein was not detected in mutant-infected plants. In addition, none of the mutants were transmitted by whiteflies. With all three mutants, ssDNA accumulation of DNA-A and DNA-B was reduced 25- to 50-fold and 3- to 10-fold, respectively, as compared to that of wild-type DNA. No effect on dsDNA-A accumulation was detected and there was 2- to 5-fold increase in dsDNA-B accumulation. Recombinants between the mutated DNA-A and DNA-B forms were identified when the inoculated coat protein mutant was linearized in the common region.

Viable chimaeric viruses confirm the biological importance of sequence specific maize streak virus movement protein and coat protein interactions

Background. A variety of interactions between up to three different movement proteins (MPs), the coat protein (CP) and genomic DNA mediate the inter- and intra-cellular movement of geminiviruses in the genus Begomovirus. Although movement of viruses in the genus Mastrevirus is less well characterized, direct interactions between a single MP and the CP of these viruses is also clearly involved in both intra- and intercellular trafficking of virus genomic DNA. However, it is currently unknown how specific these MP-CP interactions are, nor how disruption of these interactions might impact on virus viability. Results. Using chimaeric genomes of two strains of Maize streak virus (MSV) we adopted a genetic approach to investigate the gross biological effects of interfering with interactions between virus MP and CP homologues derived from genetically distinct MSV isolates. MP and CP genes were reciprocally exchanged, individually and in pairs, between maize (MSV-Kom)- and Setaria sp. (MSV-Set)-adapted isolates sharing 78% genome-wide sequence identity. All chimaeras were infectious in Zea mays c.v. Jubilee and were characterized in terms of symptomatology and infection efficiency. Compared with their parental viruses, all the chimaeras were attenuated in symptom severity, infection efficiency, and the rate at which symptoms appeared. The exchange of individual MP and CP genes resulted in lower infection efficiency and reduced symptom severity in comparison with exchanges of matched MP-CP pairs. Conclusion. Specific interactions between the mastrevirus MP and CP genes themselves and/or their expression products are important determinants of infection efficiency, rate of symptom development and symptom severity. © 2008 van der Walt et al; licensee BioMed Central Ltd.

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene. © 1988.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.