Membran-assoziierte Protein-Protein-Interaktionen des Herpes simplex-Virus 1

Herpesviren umfassen eine große Gruppe von human- sowie tierpathogenen Erregern. Auf Grund ihrer hohen Durchseuchungsrate und Fähigkeit zur Etablierung einer latenten Infektion stellen humane Herpesviren vor allem für immunsupprimierte Patienten eine ernsthafte Bedrohung dar. Deshalb ist eine umfassende Aufklärung des viralen Replikationszyklus für die Entwicklung von antiviralen Therapiestrategien zwingend erforderlich. Besonders die membran-assoziierten Vorgänge der Virionmorphogenese - primäre Umhüllung an der inneren Kernmembran mit darauffolgendem Verlust der Virushülle an der äußeren Kernmembran sowie sekundäre Umhüllung an cytoplasmatischen Membranen - sind nur unvollständig entschlüsselt. Um das komplexe Zusammenspiel der viralen Proteine während des Replikationszyklus an den verschiedenen zellulären Membranen aufzudecken, wurde im Rahmen dieser Arbeit eine genomweite Analyse der Protein-Protein-Interaktion (PPI) der durch Herpes simplex-Virus 1 (HSV-1) kodierten Membranproteine durchgeführt. Außerdem lieferte die Identifizierung von PPI zwischen dem HSV-1 Proteom und Untereinheiten der zellulären ESCRT-Maschinerie (endosomal sortingcomplex required for transport) weitere Belege für die Ausbeutung von Wirtsfaktoren durch das Virus zur Knospung der Partikel. Zur Detektion der genomweiten PPI sowohl intraviral als auch zwischen Virus und Wirt wurde das Hefe-2-Hybridsystem (Y2H) im Hochdurchsatz angewandt. Beide Datensätze konnten eine Vielzahl neuer PPI aufdecken und somit eine solide Grundlage für Interaktionsnetzwerke und zukünftige funktionale Studien schaffen. Auch wurde duch das breite Interaktionsspektrum des Virus mit den z.T. funktionell redundanten ESCRT-Proteinen erneut veranschaulicht, wie die Nutzung flexibler Strategien zur Stabilität des HSV-1 beiträgt. Anhand der Y2H-Analysen wurde ein virales Membranprotein als interessanter Kandidat zur funktionalen Charakterisierung ausgewählt. Glykoprotein M (gM/UL10) von HSV-1 ist ein Typ-III Transmembranprotein, das während der Infektion in verschiedenen Membrankompartimenten lokalisiert. Obwohl evolutionär konserviert, ist es zumindest für HSV-1 nicht-essenziell und seine molekulare Funktion unklar. Auch die funktionale Relevanz einiger potenzieller trafficking Motive von gM ist noch nicht aufgeklärt. In dieser Studie konnte gezeigt werden, dass das targeting von gM zum trans-Golgi Netzwerk (TGN) unabhängig von anderen viralen Faktoren sowie seinen potenziellen C terminalen trafficking Motiven erfolgt und keiner Homooligomerisierung bedarf. Erstaunlicherweise führt die Deletion der C-terminalen Domäne von gM (gMΔC) zu seiner Retention im ER, wohingegen der Vorwärtstransport durch eine kurze, nicht-verwandte Sequenz wiederhergestellt wurde. Demzufolge enthält die C-terminale Domäne von gM wahrscheinlich keine Sequenzinformation für das targeting vom ER zum Golgi-Apparat, jedoch scheint die Faltung und Integrität des Proteins dafür von Bedeutung zu sein. Im Kontext der Virusinfektion führte die Deletion der C-terminalen Domäne von gM (HSV-1 gMΔC) zur Akkumulation von nicht-umhüllten Partikeln im Cytoplasma, verminderter Freisetzung von Viruspartikeln und in ihrer Infektiosität beeinträchtigten reifen Virionen. Alle Effekte wurden durch eine Revertante wieder aufgehoben und sind demnach spezifisch. Im Gegensatz dazu zeigten zwei zusätzliche Mutanten, HSV-1 ΔgM mit einem frühzeitigen Stoppcodon an Position 3 von UL10 und gMΔac ohne potenzielle trafficking Motive, Wildtyp-ähnliche Wachstumskinetiken. Daraus lässt sich schließen, dass zwar gM entbehrlich ist, gMΔC jedoch einen dominant-negativen Effekt ausübt. Daher wird eine Beteiligung der N-terminalen Bereiche von gM (Aminosäuren 1-361) an der Rekrutierung von viralen und/oder zellulären Faktoren zum Ort der sekundären Umhüllung postuliert. Diese Daten enthüllen neue unbekannte Eigenschaften von HSV-1 gM.Herpesviruses comprise a large group of pathogens infecting both humans and animals. Due to their high frequency of infection and ability to establish latent infections, human herpesviruses are a serious threat especially for immunosuppressed patients. Therefore, the comprehensive understanding of the herpesviral replication cycle is mandatory for the development of antiviral therapeutic strategies. The membrane-associated steps in virion morphogenesis, such as primary envelopment and subsequent loss of this envelope at both nuclear leaflets as well as secondary envelopment at cytoplasmic membranes, are of particular interest as only partially elucidated. To reveal the complex interplay of viral proteins during replication at diverse cellular membranes, a genome-wide analysis of protein-protein-interactions (PPI) regarding membrane proteins encoded by Herpes simplex virus-1 (HSV-1) was carried out in this study. In addition, identification of PPI among HSV-1 proteome and subunits of the cellular ESCRT-machinery (endosomal sorting complex required for transport) provided further evidence for viral hijacking of host factors to perform viral budding. In order to detect both genome-wide intraviral as well as virus-host PPI, the yeast-2-hybridsystem (Y2H) was applied in a high-throughput fashion. Either dataset uncovered a multitude of new PPI, serving as a solid foundation for interaction networks and future functional studies. Also, the broad spectrum of viral interactions with ESCRT-proteins that are to some extent of functional redundance illustrates once again how the use of flexible strategies contributes to the stability of HSV-1. On the basis of this Y2H-analysis, a viral membrane protein was selected as interesting candidate for functional characterisation. Glycoprotein M (gM/UL10) of HSV-1 is a type-III transmembrane protein reported to be present at various membrane compartments during infection. Although evolutionarily conserved, HSV-1 gM is non-essential and its molecular function unclear. Also the functional relevance of several potential trafficking motifs encoded by gM has yet to be elucidated. This study could show that targeting of gM to the trans-Golgi network (TGN) occurs independent of other viral factors, of the potential C-terminal trafficking motifs, and does not involve homo-oligomerization. Surprisingly, deletion of the C-terminal domain of gM (gMΔC) caused its ER retention, whereas forward transport was rescued by a short unrelated tail. Hence, the C-terminal domain of gM is unlikely to contain sequence information for ER to TGN targeting, but protein folding and integrity seem to be of importance. In the context of viral infection, the deletion of the C-terminal domain of gM (HSV-1 gMΔC) led to accumulation of unenveloped cytoplasmic particles, reduced egress of viral particles and mature virions compromised in infectivity. These effects were specific, as not observed for the revertant virus strain. On the contrary, two additional viral mutants, HSV-1 ∆gM carrying a premature stop codon at position 3 of the UL10 gene as well as the gM∆ac virus missing all predicted trafficking motifs, displayed wildtype-like growth kinetics. In conclusion, gM is fully dispensable but gM∆C exhibits a dominant effect. Thus, a role for the N-terminal domains of gM (residues 1-361) in the recruitment of viral and/or cellular components to the site of final envelopment is postulated. These data elucidate novel unknown characteristics of HSV-1 gM

Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M

The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors

Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M

The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors

A beta-herpesvirus with fluorescent capsids to study transport in living cells.

Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion

Trip to immunity: resistant cytomegalovirus infection in a lung transplant recipient

We report the case of a young female lung transplant recipient with difficult-to-treat cytomegalovirus (CMV) disease. While treatment with intravenous (IV) ganciclovir failed due to antiviral drug resistance, a trial with foscarnet resulted in severe side effects. In addition, the patient received IV CMV-specific immune globulins as adjunctive therapy and leflunomide as experimental therapy. In this context, CMV-specific immune monitoring was performed and was successfully implemented in management decisions. The patient was screened for acquisition of an adaptive immune response, and antiviral prophylaxis and therapy was tailored according to results. This report highlights the impact of CMV-specific immune monitoring on individualized therapy for appropriate prophylaxis and management of CMV infection and diseases

Fluorescent virus particles are spread-competent.

<p>(A) Confluent M2-10B4 cells were infected with S-GFP-SCP labeled virus on cover-slips in 24-wells with 100 PFU per well and overlaid with methyl-cellulose. 4 dpi cells were fixed and processed for immunofluorescence. MCP-specific antiserum was used to detect virus producing cells as well as single virus particles while GFP fluorescence was visualized directly. Cell nuclei were counterstained with TO-PRO-3. Inserts depict single virus particles surrounding a cell nucleus (circles) not showing any evidence for being on the late stage of infection and producing infectious particles. Scale bars indicate 20 µm in the upper row and 5 µm in the lower row.</p

Mutants carrying tagged SCP are viable.

<p>(A) WT (WT) as well as mutant viruses coding for either HA- (S-HA-SCP) or HA-GFP-tagged wt SCP (S-GFP-SCP) were titrated on MEF cells. 4 days post infection (dpi) cells were fixed with PFA and processed for immunofluorescence against the HA-epitope. The scale bars represent 100 µm. (B) Multistep growth curve of mutant viruses used in this study in comparison to WT virus. C) Comparison of plaque diameters of simultaneously titrated WT, HA-, GFP-, and mCherry-tagged virus 4 dpi on MEF cells. (D) Genome to PFU ratio of GFP-tagged and WT virus. The ratio between genome content and titer for two independently prepared and purified virus stocks per virus was determined by titration and quantitative PCR in triplicates.</p