siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

A Therapeutic Antiviral Antibody Inhibits the Anterograde Directed Neuron-to-Cell Spread of Herpes Simplex Virus and Protects against Ocular Disease.

Herpes simplex virus (HSV) is a leading cause of blindness and viral encephalitis in the developed world. Upon reactivation from sensory neurons, HSV returns via axonal transport to peripheral tissues where it causes, e.g., severe, potentially blinding ocular diseases. In the present study we investigated whether the HSV-1/2 glycoprotein B-specific antibody mAb 2c or its humanized counterpart mAb hu2c can protect from ocular disease in a mouse model of HSV-1-induced acute retinal necrosis (ARN). In this model the viral spread from the initially infected to the contralateral eye resembles the routes taken in humans upon HSV reactivation. Systemic antibody treatment prior or early after infection effectively protected the mice from the development of ARN. These observations suggest that the antibody potently neutralized the infection and inhibited the viral transmission, since there was almost no virus detectable in the contralateral eyes and trigeminal ganglia of antibody treated mice. Besides of neutralizing free virus or limiting the infection via activating the complement or cellular effector functions, blocking of the anterograde directed neuron-to-cell spread of HSV represents a viable mode of action how mAb 2c protected the mice from ARN. We proved this hypothesis using a microfluidic chamber system. Neurons and epithelial cells were cultured in two separate compartments where the neurons sent axons via connecting microgrooves to the epithelial cells. Neurons were infected with a reporter HSV-1 strain expressing mCherry, and the co-culture was treated with neutralizing antibodies. In contrast to commercial polyclonal human HSV-neutralizing immunoglobulins, mAb 2c effectively blocked the anterograde directed neuron-to-cell transmission of the virus. Our data suggest that the humanized HSV-1/2-gB antibody protects mice from ocular disease by blocking the neuronal spread of HSV. Therefore, mAb hu2c may become a potent novel therapeutic option for severe ocular HSV infections.

Human Cytomegalovirus-Induced Degradation of CYTIP Modulates Dendritic Cell Adhesion and Migration.

As potent antigen-presenting cells, dendritic cells (DCs) are essential for the initiation of effective antiviral immune responses. Viruses and especially herpesviruses, which are able to establish lifelong persistence, exploit several immune evasion mechanisms targeting DC biology. Our group has previously shown that the α-herpesvirus herpes simplex virus type 1 inhibits mature DC (mDC) migration by inducing adhesion via degrading the cellular protein CYTIP (cytohesin-1 interacting protein), an important negative regulator of ß2-integrin activity. In the present study, we extended our analysis to the ß-herpesvirus human cytomegalovirus (HCMV), to investigate whether other herpesviridae also induce such modulations. Indeed, HCMV impairs mDC transwell migration capability following a CCL19-chemokine gradient, despite equivalent expression levels of the cognate chemokine receptor CCR7 at the corresponding time points post-infection. Remarkably, HCMV infection potently induced ß2-integrin activity on mDCs. Furthermore, directly HCMV-infected mDCs, exhibiting viral gene expression, strongly adhere to fibronectin and ICAM-1, in contrast to mDCs lacking infection or viral gene expression. Interestingly, HCMV-positive mDCs display a proteasome-dependent degradation of CYTIP. Contrasting the migration toward CCL19, elevated expression levels of the chemokine receptor CXCR4 in HCMV-infected mDCs were associated with functional CXCL12-chemotaxis under the herein used conditions. In summary, our results show that HCMV shapes mDC adhesion to compromise migration toward CCL19, but retaining CXCL12 responsiveness. Thus, we hypothesize that a preferred migration pattern toward the bone marrow, but not to secondary lymphoid organs, could ultimately cause a failure in the induction of potent antiviral immune responses.

Crystal Structure of the Extracellular Domain of the Human Dendritic Cell Surface Marker CD83.

CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7Å solved in three different crystal forms. Interestingly, ß-strands C', C″, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse.

The Major Immediate-Early Protein IE2 of Human Cytomegalovirus Is Sufficient to Induce Proteasomal Degradation of CD83 on Mature Dendritic Cells.

Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation.

Role of L-Particles during Herpes Simplex Virus Infection.

Infection of eukaryotic cells with α-herpesviruses results in the formation and secretion of infectious heavy particles (virions; H-particles) and non-infectious light particles (L-particles). Herpes simplex virus type 1 (HSV-1) H-particles consist of a genome-containing capsid surrounded by tegument proteins and a glycoprotein-rich lipid bilayer. Non-infectious L-particles are composed mainly of envelope and tegument proteins and are devoid of capsids and viral DNA. L-particles were first described in the early nineties and from then on investigated for their formation and role during virus infection. The development and secretion of L-particles occur simultaneously to the assembly of complete viral particles. HSV-1 L-particles are assembled by budding of condensed tegument into Golgi-delivered vesicles and are capable of delivering their functional content to non-infected cells. Thereby, HSV-1 L-particles contribute to viral pathogenesis within the infected host by enhancing virion infectivity and providing immune evasion functions. In this review we discuss the emergence of HSV-1 L-particles during virus replication and their biological functions described thus far.

L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation.

UNLABELLED: Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE: HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.

CD83 and GRASP55 interact in human dendritic cells.

CD83 is one of the best known surface markers for mature human dendritic cells (DCs). The full-length 45 kDa type-I membrane-bound form (mbCD83) is strongly glycosylated upon DCs maturation. As co-stimulatory properties of CD83 are attributed to mbCD83 surface expression is required for efficient T-cell stimulation by mature DCs. By yeast two-hybrid screening, we were able to identify GRASP55 as interaction partner of CD83. DCs maturation induces endogenous CD83 protein expression with simultaneous regulation of CD83 glycosylation, interaction and co-localization with GRASP55 and CD83 surface exposure. GRASP55 is especially known for its role in maintaining Golgi architecture, but also plays a role in Golgi transport of specific cargo proteins bearing a C-terminal valine residue. Here we additionally demonstrate that binding of CD83 and GRASP55 rely on the C-terminal TELV-motif of CD83. Mutation of this TELV-motif not only disrupted binding to GRASP55, but also altered the glycosylation pattern of CD83 and reduced its membrane expression. Here we show for the first time that GRASP55 interacts with CD83 shortly after induction of DC maturation and that this interaction plays a role in CD83 glycosylation as well as in surface expression of CD83 on DCs.

Herpes simplex virus type 1 ICP0 induces CD83 degradation in mature dendritic cells independent of its E3 ubiquitin ligase function.

Mature dendritic cells (mDCs) are the most potent antigen-presenting cells known today, as they are the only antigen-presenting cells able to induce naïve T-cells. Therefore, they play a crucial role during the induction of effective antiviral immune responses. Interestingly, the surface molecule CD83 expressed on mDCs is targeted by several viruses. As CD83 has been shown to exert co-stimulatory functions on mDCs, its downmodulation represents a viral immune escape mechanism. Mechanistically, it has been shown that herpes simplex virus type 1 infection leads to proteasomal degradation of CD83, resulting in a strongly diminished T-cell stimulatory capacity of the infected mDC. Previous data suggest that the viral immediate-early protein ICP0 (infected-cell protein 0) plays an important role in this process. In the present study, we showed that ICP0 is sufficient to induce CD83 degradation in the absence of any other viral factor. However, the mechanism of ICP0-mediated CD83 degradation is not yet understood. Here, we provide evidence that ubiquitination of lysine residues is, despite the published E3 ubiquitin ligase activity of ICP0, not necessary for CD83 degradation. This finding was underlined by the observation that expression of an ICP0 mutant lacking the E3 ubiquitin ligase domain in mDCs still induced CD83 degradation. Finally, inhibition of E1 activating enzyme using the specific inhibitor 4[4-(5-nitro-furan-2-ylmethylene)-3.5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester did not prevent CD83 degradation. Taken together, our data provide strong evidence that ICP0 alone induces CD83 degradation independent of its E3 ubiquitin ligase function and of the ubiquitin machinery.