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.

Analysis of the interaction of Ebola virus glycoprotein with DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) and its homologue DC-SIGNR.

BACKGROUND: The lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) augments Ebola virus (EBOV) infection. However, it its unclear whether DC-SIGN promotes only EBOV attachment (attachment factor function, nonessential) or actively facilitates EBOV entry (receptor function, essential). METHODS: We investigated whether DC-SIGN on B cell lines and dendritic cells acts as an EBOV attachment factor or receptor. RESULTS: Engineered DC-SIGN expression rendered some B cell lines susceptible to EBOV glycoprotein (EBOV GP)-driven infection, whereas others remained refractory, suggesting that cellular factors other than DC-SIGN are also required for susceptibility to EBOV infection. Augmentation of entry was independent of efficient DC-SIGN internalization and might not involve lectin-mediated endocytic uptake of virions. Therefore, DC-SIGN is unlikely to function as an EBOV receptor on B cell lines; instead, it might concentrate virions onto cells, thereby allowing entry into cell lines expressing low levels of endogenous receptor(s). Indeed, artificial concentration of virions onto cells mirrored DC-SIGN expression, confirming that optimization of viral attachment is sufficient for EBOV GP-driven entry into some B cell lines. Finally, EBOV infection of dendritic cells was only partially dependent on mannose-specific lectins, such as DC-SIGN, suggesting an important contribution of other factors. CONCLUSIONS: Our results indicate that DC-SIGN is not an EBOV receptor but, rather, is an attachment-promoting factor that boosts entry into B cell lines susceptible to low levels of EBOV GP-mediated infection.

Infection of mature dendritic cells with herpes simplex virus type 1 interferes with the interferon signaling pathway.

It has been shown that herpes simplex virus type 1 (HSV-1) blocks specific immune responses by various mechanisms. Cell lines infected with HSV-1 for instance show a severe impairment of the interferon-gamma (IFN-gamma)-induced signal transducer and activator of transcription 1 (STAT1) signaling pathway. Thus, we examined the influence of HSV-1 infection on IFN-gamma signal transduction in mature dendritic cells (mDCs). In this study, we report the down-regulation of the IFN-gamma receptor alpha chain (IFNGR1) at the mRNA level in HSV-1 infected mDCs. As a consequence, the expression of the IFNGR1 subunit on the cell surface of the infected cell was strongly reduced. Furthermore, we were able to show the inhibition of STAT1 phosphorylation following HSV-1 infection in mDCs, while protein levels of STAT1 remained constant. As a direct downstream effect of STAT1 phosphorylation, the activation of the transcription factor IRF-1 was also clearly inhibited and could no longer be induced by the addition of IFN-gamma. Additional experiments using a virus strain lacking the vhs gene suggested that the mutant virus is more sensitive to IFN-gamma as STAT1 signaling was inhibited to a lesser extent. Infection with a UV-inactivated, replication incompetent virus did not influence the STAT1 signaling pathway at all. In conclusion, we show that HSV-1 blocks IFN-gamma signaling in mDCs, which requires viral gene expression and involves the viral protein vhs.

DC-SIGN and CLEC-2 mediate human immunodeficiency virus type 1 capture by platelets.

Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.

Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration.

Herpes simplex virus type 1 (HSV-1) is able to establish latency in infected individuals. In order to characterize potential new immune-escape mechanisms, mature dendritic cells (DCs) were infected with HSV-1 and total cellular RNA was isolated from infected and mock-infected populations at different time points. RNA profiling on Affymetrix Human Genome U133A arrays demonstrated a dramatic downregulation of the migration-mediating surface molecules CCR7 and CXCR4, an observation that was further confirmed by RT-PCR and fluorescence-activated cell sorting analyses. Furthermore, migration assays revealed that, upon infection of mature DCs, CCR7- and CXCR4-mediated migration towards the corresponding CCL19 and CXCL12 chemokine gradients was strongly reduced. It is noteworthy that the infection of immature DCs with HSV-1 prior to maturation led to a failure of CCR7 and CXCR4 upregulation during DC maturation and, as a consequence, also induced a block in their migratory capacity. Additional migration assays with a Deltavhs mutant virus lacking the virion host shutoff (vhs) gene, which is known to degrade cellular mRNAs, suggested a vhs-independent mechanism. These results indicate that HSV-1-infected mature DCs are limited in their capacity to migrate to secondary lymphoid organs, the areas of antigen presentation and T-cell stimulation, thus inhibiting an antiviral immune response. This represents a novel, previously unrecognized mechanism for HSV-1 to escape the human immune system.

DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus.

The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

A naturally occurring variation in the proline-rich region does not attenuate human immunodeficiency virus type 1 nef function.

We analyzed human immunodeficiency virus type 1 (HIV-1) Nef variants to further evaluate the functional relevance of the R71T substitution previously proposed to attenuate viral replication (Fackler et al., Curr. Biol. 11:1294-1299, 2001). Our results demonstrate that this variation in the proline-rich region does not significantly affect the functional activity of Nef or HIV-1 infectivity or replication.