Identification of essential, dispensable and modulatory domains in the core herpesvirus membrane fusion components gB and gH

Herpesviruses are a fascinating group of enveloped DNA viruses, which rely on membrane fusion for infectious entry and direct cell-to-cell spread. Compared with many other enveloped viruses, they utilize a remarkably complex fusion machinery. Three conserved virion proteins, the bona fide fusion protein gB, and the presumably gB activating gH/gL heterodimer constitute the conserved core fusion machinery and are believed to drive membrane fusion in a cascade-like fashion. Activation of this cascade in most alphaherpesviruses is proposed to be triggered by binding of gD to specific host cell receptors. The molecular details of this fusion process, however, remain largely elusive. Yet, a detailed mechanistic knowledge of this process would be greatly beneficial for the development of efficient countermeasures against a variety of diseases. In this thesis, the functional relevance of individual components of the essential gH/gL complex of the alphaherpesvirus PrV has been assessed by two different approaches: by reversion analysis (paper II) and site-directed mutagenesis (papers III-V). In contrast to other herpesviruses, gL-deleted PrV is able to perform limited cell-to-cell spread, providing the unique opportunity to passage the entry-deficient virus in cell culture to select for PrV revertants capable of infecting cells gL-independently. This approach already resulted in an infectious gL-negative PrV mutant (PrV-ΔgLPass), in which the function of gL was compensated by formation of a gDgH hybrid protein. Here, the requirements for gL-independent infectivity of a second independent revertant (PrV-ΔgLPassB4.1), were analyzed. Sequencing of the genes encoding for gB, gH and gD, revealed mutations in each of them. By means of a robust infection-free, transfection-based cell-cell fusion assay (paper I), we identified two amino acid substitutions in the gL-binding domain I of gHB4.1 (L70P, W103R) as sufficient to compensate for lack of gL. Two mutations in gB (G672R, ΔK883) were found to enhance fusogenicity, probably by lowering the energy, required for gB refolding from pre- to postfusion conformation. Coexpression of gHB4.1 and gBB4.1 led to an excess fusion, which was completely suppressed by gDB4.1 in the fusion assays. This was surprising since PrV gD is normally not required for in vitro fusion or direct viral cell-to-cell spread, clearly separating this process from fusion during entry, for which PrV gD is essential. The fusion inhibiting effect of gDB4.1 could be attributed to a single point mutation resulting in an amino acid substitution within the ectodomain (A106V). In conclusion, these results indicated that gL is not central to the fusion process, as its function can be compensated for. As found so far, gL-independent infectivity can be realized by compensatory mutations in gH (as in PrV-ΔgLPass) or in gH plus gB (as in PrV-ΔgLPassB4.1). Excessive fusion induced by gHB4.1 and gBB4.1 was counter-regulated by gDB4.1, indicating that the interplay between these proteins is precisely regulated and further implies that gL and gD, despite being not absolutely essential for the fusion process, have important regulatory functions on gH and/or gB. Both PrV-ΔgLPass mutants had acquired compensatory mutations in gH affecting the predicted gL-binding domain I in gH. By construction of an artificial gH32/98, which lacked the predicted gL-binding domain and was similar to the recently crystallized gH-core fragment present in the gDgH hybrid protein, we identified the N-terminal part of PrV gH as essential for gH function during fusion (paper III). gH32/98 was unable to promote fusion of wild-type gB in fusion assays and led to a total loss of function in the viral context. These results indicated that the gD moiety, present in gDgH, is critical for proper function of the gH-core fragment. We hypothesize that the gD moiety may adopt a stabilizing or modulating influence on the gH structure, which is normally executed by gL and important for interaction of gH with wild-type gB. Remarkably, substitution of wild-type gB by gBB4.1 rescued function of gH32/98 in the cellular and viral contexts. These findings suggest that gBB4.1 has been selected for interaction with “gL-less” gH. In conclusion, these results demonstrated that gL and the gL-binding domain are not strictly required for membrane fusion during virus entry and spread but that compensatory mutations must be present in gB to restore a fully functional fusion machinery. These results strongly support the notion of a functional gH-gB interaction as a prerequisite for membrane fusion. In addition to the N-terminal domain, we identified the transmembrane domain of PrV gH as an essential component of the fusion machinery, while the cytoplasmic domain was demonstrated to play a modulatory but nonessential role (paper IV). Whereas truncation or substitution of the PrV gH TMD by a gpi-anchor or the analogous sequence from PrV gD rendered gH non-functional, the HSV-1 gH TMD was found to functionally substitute for the PrV gH TMD in cell-cell fusion and complementation assays. Since residues in the TMD which are conserved between HSV and PrV gH but absent in PrV gD, are placed on one face of an α-helical wheel plot, we hypothesize that the gH TMD has an intrinsic property to interact with membrane components such as lipids or other molecules as a requirement for promoting membrane fusion. In a final study focusing on the function of gH, we identified the N-glycosylation sites utilized by PrV gH, and determined their individual role in viral infection (paper V). PrV gH was found to be modified by N-glycans at five potential glycosylation sites. N-glycans at PrV specific N77 and the highly conserved site N627 were found to be critical for efficient membrane fusion in the fusion assays, and during viral entry and cell-to-cell spread. N627 was further shown to be crucial for proper gH transport and maturation. In contrast, inactivation of N604, conserved in the Varicellovirus genus, enhanced in vitro fusion activity and viral cell-to-cell spread. These findings demonstrated a role of the N-glycans in proper localization and function of PrV gH.Herpesviren sind eine faszinierende Gruppe umhüllter DNA Viren. Der Eintritt dieser Viren in die Wirtszelle erfolgt durch die Fusion der viralen Hüllmembran mit der zellulären Plasmamembran. Im Vergleich zu anderen umhüllten Viren verwenden Herpesviren eine hoch komplexe Fusionsmaschinerie, die aus den drei konservierten Oberflächen-Glykoproteinen (g)B und dem gH/gL Komplex besteht. Während es sich bei gB um das bona fide Fusionsprotein handelt, übernimmt der heterodimere gH/gL Komplex vermutlich eine regulatorische Rolle. Es wird angenommen, dass es zu einer kaskadenartigen Aktivierung der Glykoproteine während des Fusionsprozesses kommt, die in Alpha-Herpesviren durch die Bindung von gD an einen spezifischen Wirtszellrezeptor eingeleitet wird. Die molekularen Grundlagen des herpesviralen Fusionsprozesses sind jedoch weitestgehend unbekannt. Für die Entwicklung effizienter Maßnahmen gegen eine Vielzahl von Krankheitserregern wäre ein detailliertes mechanistisches Verständnis des Fusionsprozesses jedoch von großer Bedeutung. In dieser Arbeit wurde die Funktion und Relevanz der einzelnen Komponenten der Fusionsmaschinerie des Alpha-Herpesvirus Pseudorabies Virus (PrV) genauer untersucht und charakterisiert. Zunächst wurde ein robuster, infektionsfreier, Transfektions-basierter Zellfusions-Assay etabliert, welcher als Grundlage für die Analyse der Fusionsaktivität und Rolle der einzelnen Komponenten der PrV Fusionsmaschinerie diente (Paper I). In einer ersten Studie wurde die Funktion des gH/gL Komplexes mittels Reversionsanalyse gL-defizienter PrV Mutanten untersucht. Hierbei konnte gezeigt werden, dass gL eine regulatorische, jedoch nicht essentielle Funktion während des Fusionsprozesses übernimmt, da die Abwesenheit von gL durch Mutationen in den anderen Proteinen funktional kompensiert werden konnte (Paper II). In Anwesenheit kompensatorischer Mutationen in gB erwies sich auch die mit gL-interagierende Domäne I des gH Proteins als nicht essentiell für den Fusionsprozess (Paper III). Insgesamt erlaubten die Ergebnisse der beiden Studien den Schluss, dass gL und die gL-bindende Domäne von gH keine essentiellen Komponenten der Fusionsmaschinerie sind. Weiterhin unterstützen die Ergebnisse die Annahme, dass eine funktionelle Interaktion zwischen gH und gB eine Grundvoraussetzung für die Membranfusion darstellt. Im Gegensatz zu gL und der gL-bindenden Domäne des gH konnte die Transmembrandomäne von gH als essentiell identifiziert werden. Die zytoplasmatische Domäne des Proteins hingegen übernimmt eine regulatorische, jedoch nicht essentielle Rolle (Paper IV). In einer anschließenden Studie wurde die Rolle der Asparagin–Glykosylierungen in gH untersucht. Dabei konnte nachgewiesen werden, dass PrV gH fünf funktionelle Glykosylierungsstellen aufweist, die einen Einfluss auf die korrekte Lokalisation, den Transport und die Funktion des PrV gH haben (Paper V). Während gezeigt werden konnte, dass gL und die gL-bindende Domäne von gH nicht essentiell für die Membranfusion sind, konnte kein Surrogat für gH identifiziert werden. So erwiesen sich externe Stimuli wie beispielsweise eine erhöhte Temperatur, ein niedriger pH-Wert und auch die Einführung weiterer fusionssteigernder Mutationen in das gB als unzureichend, um gB zu einem autonomen Fusionsprotein zu transformieren. Dies spricht dafür, dass eine direkte Interaktion zwischen gH und gB notwendig ist, um Fusionen zu induzieren, wirft jedoch auch Fragen dahingehend auf, ob es sich bei gB tatsächlich um das Fusionsprotein handelt. Um dieser Frage nachzugehen wurde die Kristallstruktur der Ektodomäne von PrV gB gelöst, wodurch dieses als Klasse III Fusionsprotein identifiziert werden konnte. Es konnte gezeigt werden, dass jedes Protomer im trimeren gB Protein zwei fusion-loops besitzt mit denen das gB in der Lage ist mit Membranen zu interagieren (Paper VI). Auf Basis der Strukturinformationen konnten durch gezielte Mutagenese der fusion-loops Aminosäuren identifiziert werden, die für die Fusionsaktivität und die Bindung von gB an Membranen essentiell sind. Die strukturellen und funktionellen Daten erlaubten die Erstellung eines Modells der Insertionstiefe der fusion-loops und deren Membran-Verankerung mithilfe flankierender Aminosäurereste. Eine vergleichende Analyse mit Beta- und Gamma-Herpesviren zeigte, dass dieses Model vermutlich auf alle Vertreter der Herpesviridae anwendbar ist. Insgesamt tragen die in dieser Arbeit gewonnen Ergebnisse wesentlich zum derzeitigen Wissen über den Mechanismus der Membranfusion bei Herpesviren bei und liefern entscheidende neue Einblicke in die Erfordernisse und Funktionen der einzelnen Komponenten der Fusionsmaschinerie

Association of cytokines with endothelium dependent flow mediated vasodilation (FMD) of systemic arteries in patients with non-ischemic cardiomyopathy

<p>Abstract</p> <p>Background</p> <p>Aim of this study was to elucidate the relation between localised inflammatory heart disease and endothelial dysfunction in the peripheral circulation, considering circulating cytokines as a potential link.</p> <p>Methods</p> <p>In 38 patients with non-ischemic heart disease, myocardial biopsies were examined for myocardial inflammation (immunohistology) and virus persistence (PCR). Cytokines (sIL-4, IFN-g, IFN-b, IFN-a, sIL-12p7, TNF-a) were measured by ELISA in venous serum. Endothelial function of the radial artery was examined by ultrasound, measuring diameter changes in response to reactive hyperemia (FMD), compared to glyceroltrinitrate (GTN-MD). Patients with EF < 35% were excluded.</p> <p>Results</p> <p>Age 44 ± 14 years, 19 male, 19 female, EF 63.5[16]%. FMD 4.38 [4.82]%. 30 patients had myocardial inflammation (8 not), 23 virus persistence (15 not). FMD correlated significantly with sIL-12p7 (p = 0.024, r = -0.365), but not with other cytokines. sIL-12p7 levels were significantly higher in patients with severely impaired FMD (n = 17), compared with normal FMD (n = 21): 10.70 [10.72] vs. 4.33 [7.81] pg/ml (p = 0.002). Endothelium independent vasodilation (GTN-MD 23.67 [8.21]%) was not impaired.</p> <p>Conclusion</p> <p>Endothelial dysfunction of peripheral arteries in patients with non-ischemic cardiomyopathy is associated with elevated serum concentrations of sIL-12p7, but not of other cytokines. Circulating sIL-12p7 may partly explain, that endothelial dysfunction is not restricted to the coronary circulation, but involves systemic arteries.</p

The prefusion structure of herpes simplex virus glycoprotein B.

Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins

Hepatitis A: New information on an old virus

An outbreak of hepatitis A virus (HAV) infection in a neonatal intensive care unit (NICU) provided the opportunity to examine the duration of HAV excretion in infants and the mechanisms by which HAV epidemics are propagated in NICUs. The outbreak affected 13 NICU infants (20%), 22 NICU nurses (24%), 8 other staff caring for NICU infants, and 4 household contacts; 2 seropositive infants (primary cases) received blood transfusions from a donor with HAV infection. Risk factors for infection among nurses were care for a primary infant-case (relative risk [RR], 3.2), drinking beverages in the unit (odds ratio [OR], ∞), and not wearing gloves when taping an intravenous line (OR, 13.7). Among infants, risk factors were care by a nurse who cared for a primary infant-case during the same shift (RR, 6.1). Serial stool samples from infantcases were tested for HAV antigen (HAV-Ag) by enzyme immunoassay and HAV RNA by nucleic acid amplification using the polymerase chain reaction. Infantcases excreted HAV-Ag ( n = 2) and HAV RNA ( n = 3) 4–5 months after they were identified as being infected. Breaks in infection control procedures and possibly prolonged HAV shedding in infants propagated the epidemic in a critical care setting.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38377/1/1840150537_ftp.pd

The Diagnostic Utility of Anti-cyclic Citrullinated Peptide Antibodies, Matrix Metalloproteinase-3, Rheumatoid Factor, Erythrocyte Sedimentation Rate, and C-reactive Protein in Patients with Erosive and Non-erosive Rheumatoid Arthritis

Objective: To compare the diagnostic utility of laboratory variables, including matrix metalloproteinase-3 (MMP-3), anti-cyclic citrullinated peptide (CCP) antibodies, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) in patients with erosive and non-erosive rheumatoid arthritis (RA)

The pre-fusion structure of Herpes simplex virus glycoprotein B

Cell entry of enveloped viruses requires specialized viral proteins which mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the Herpes simplex virus (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the post-fusion form, thereby precluding structural elucidation of the pharmacologically relevant pre-fusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB, allowed the structural determination of its membrane-embedded pre-fusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a striking conservation of conformational domain rearrangements during fusion between HSV-1 gB and the Vesicular Stomatitis Virus glycoprotein G (VSV-G) despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins. Rey, M. Topf, K