Mirko Kummer

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome.

ABSTRACT Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.

Herpes simplex virus type I (HSV‐1) replicates in mature dendritic cells but can only be transferred in a cell–cell contact‐dependent manner

HSV‐1 is a very successful representative of the α‐herpesvirus family, and ∼90% of the population is seropositive for this particular virus. Although the pathogen usually causes the well‐known mild lesions on the lips, also, severe infections of the eye or the brain can be observed in rare cases. It is well known, that this virus can efficiently infect the most potent APCs, i.e., the DCs, in their immature and mature state. Although the infection of the iDC has been shown to be productive, infection of mMDDCs is believed to be abortive in the early phase of the viral replication cycle. In line with these findings, no virus particles can be detected in the supernatant of HSV‐1‐infected mMDDC. In this study, however, we show for the first time that this pathogen completes its replication cycle in mMDDCs. We detected the presence of viral gene transcripts of all three phases of the replication cycle, as well as of late viral proteins, and even the generation of small amounts of progeny virus. Although we could confirm the findings that these particles are not released into the supernatant, surprisingly, the newly generated viral particles can be passed on to Vero cells, as well as to primary keratinocytes in a cell–cell contact‐dependent manner. Finally, we provide evidence that the viral gE is involved in the transfer of infectious virus from mMDDCs to other permissive cells.

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.

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

ABSTRACT 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 hosts 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.

Eukaryotic expression of functionally active recombinant soluble CD83 from HEK 293T cells

The cell surface protein CD83 belongs to the immunoglobulin super family and is highly expressed on mature dendritic cells (DCs). A membrane bound and a soluble form of CD83 (sCD83) have been described. Previously, the isolation of a purified recombinant sCD83 molecule from bacterial cultures using high pressure liquid chromatography was reported. This recombinant protein reduced DC-mediated T cell proliferation in vitro and displayed an inhibitory effect in the experimental autoimmune encephalomyelitis (EAE) model. When purifying sCD83 from bacteria, however, a lipopolysaccharide fraction is frequently co-isolated with the recombinant sCD83 protein. Moreover, the subsequent separation of sCD83 from contaminating LPS is usually accompanied by a considerable loss of soluble CD83. A further disadvantage of soluble CD83 expression in prokaryotic cells is the lack of functional glycosylation. To overcome these problems, we developed an alternative strategy to express sCD83 in eukaryotic human embryonic kidney (HEK) 293 T cells. Using this system, we showed that recombinant sCD83 was LPS-free and effectively glycosylated with all three asparagine residues at least partially involved. The functionality of the expressed sCD83 protein was examined using the mixed lymphocyte reaction (MLR) assay, demonstrating a reduced DC-mediated T cell proliferation as previously reported for the sCD83 protein purified from E. coli. Thus, a new protocol for efficient eukaryotic expression and purification of sCD83 was established, which might have several advantages compared to prokaryotic expression systems.

Herpes simplex virus type I infection of mature dendritic cells leads to reduced LMP7-mRNA-expression levels.

Mature dendritic cells (mDCs) are the most potent antigen presenting cells within the human immune system known today. However, several viruses, including herpes simplex virus type 1 (HSV-1) have developed numerous immune escape mechanisms, such as the avoidance of peptide presentation through the major histocompatibility complex (MHC) class I to CD8(+) cytotoxic T-cells. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome, a multicatalytic protease complex. Upon exposure to IFN-gamma, the constitutive proteasome is partially replaced by the immunoproteasome, which contains the IFN-gamma-inducible subunits LMP2, MECL1 and LMP7. In this study, we report the downregulation of LMP7 on mRNA level in HSV-1 infected mDCs. Interestingly, this reduction was not vhs-mediated since using a virus strain lacking the vhs gene we obtained similar results. However, on protein level, LMP7-expression was not affected, which is probably due the high stability of the LMP7 protein. Also the incorporation of LMP7 into the immunoproteasome was not affected by HSV-1. However, for the in vivo situation, in which DC reside for a prolonged time period in peripheral tissues, the reduced LMP7-mRNA level could be of biological importance, since the virus could escape/hide from immune system of the host and establish latency processes.

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.

HSV-1 upregulates the ARE-binding protein tristetraprolin in a STAT1- and p38-dependent manner in mature dendritic cells.

Dendritic cells are the sentinels of the immune system and as such represent the first-line of defense against incoming pathogens. Upon encounter with harmful antigens, these antigen-presenting cells start to mature and migrate towards the draining lymph nodes to display the antigen to T-lymphocytes, thereby eliciting the immune response of the host. Viruses, including human herpesvirus type I (HSV-1), seek to avoid such immune reactions. Therefore, they developed an arsenal of immune evasion strategies, some of which have been described earlier by our group and others. The secretion of tumor necrosis factor (TNF) represents a typical defense line of the host and it has been shown that this cytokine contributes to the inhibition of viral replication and augments the proliferation of cytotoxic T-lymphocytes. Here we report, that upon infection of mature dendritic cells, HSV-1 very strongly induces the expression of the AU-rich elements (ARE)-binding protein tristetraprolin (TTP), an mRNA-destabilizing protein. One of the best described targets of TTP is the TNF mRNA. This induction is dependent on the phosphorylation of both signal transducer and activator of transcription (STAT1) and p38 in a collaborative manner. By repressing this phosphorylation with specific inhibitors, we were able to reduce TTP mRNA levels. At the same time TNF mRNA levels were increased, suggesting that TNF mRNA is indeed a target of TTP in this setting. In summary, these data underline that HSV-1 induces TTP transcription in order to reduce TNF levels generated by infected mature dendritic cell.

What Goes Around, Comes Around - HSV-1 Replication in Monocyte-Derived Dendritic Cells

HSV-1 is a very successful human pathogen, known for its high sero-prevalence and the ability to infect a wide range of different cell types, including dendritic cells (DCs). As very potent antigen-presenting cells DCs play an important role in the induction of antiviral immune responses and therefore represent a strategic target for viral-mediated immune escape mechanisms. It is known that HSV-1 completes its gene expression profile in immature as well as in mature DCs, while lytic infection is only found in immature DCs (iDCs). Notably, HSV-1 infected mature DCs (mDCs) fail to release infectious progeny virions into the supernatant. Apart from HSV-1 dissemination via extracellular routes cell-to-cell spread counteracts a yet unknown mechanism by which the virus is trapped in mDCs and not released into the supernatant. The dissemination in a cell–cell contact-dependent manner enables HSV-1 to infect bystander cells without the exposure toward the extracellular environment. This supports the virus to successfully infect the host and establish latency. In this review the mechanism of HSV-1 replication in iDCs and mDCs and its immunological as well as virological implications, will be discussed.