Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 ( approximately 75 kDa), encoded by the UL56 and UL89 genes. In a recent investigation, we demonstrated that the main ATPase activity is associated with the large terminase subunit pUL56. The protein has two putative ATP-binding sites, which were suggested to be composed of the sequence (amino acids 463-470) for ATP-binding site 1 and YNETFGKQ (amino acids 709-716) for the second site. We now demonstrate using a 1.5 kb fragment encoding the C-terminal half of pUL56 that ATP-binding site 1 is not critical for the function, whereas ATP-binding site 2 is required for the enzymatic activity. Mutation G714A in this protein reduced the ATPase activity to approximately 65% and the double mutation G714A/K715N showed a reduction up to 75%. However, the substitution of E711A revoked the effect of the substitutions. The functional character of the ATP-binding site was demonstrated by transfer of YNETFGKQLSIACLR (709-723) to glutathione-S-transferase (GST). Interestingly, vanadate, an ATPase inhibitor, has the ability to block the ATPase activity of pUL56 as well as of Apyrase, while the antitumor ATP-mimetic agent geldanamycin, did not affect the ATP-binding of pUL56. Furthermore, in contrast to an inactive control compound, the specific HCMV terminase inhibitor BDCRB showed a partial inhibition of the pUL56-specific ATPase activity. Our results clearly demonstrated that (i) the enzymatic activity of the terminase subunit pUL56 could be inhibited by vanadate, (ii) only the ATP-binding site 2 is critical for the pUL56 function and (iii) glycine G714 is an invariant amino acid.

Cytomegalovirus (CMV) infection in patients receiving hematopoietic stem cell transplants (HSCT)is associated with morbidity and mortality. Adoptive T cell immunotherapy has been used to treat viral reactivation but is hardly feasible in high-risk constellations of CMV-positive HSCT patients and CMV-negative stem cell donors. We endowed human effector T cells with a chimeric immunoreceptor (cIR) directed against CMV glycoprotein B. These cIR-engineered primary T cells mediated antiviral effector functions such as cytokine production and cytolysis. This first description of cIR-redirected CMV-specific T cells opens up a new perspective for HLA-independent immunotherapy of CMV infection in high-risk patients.Primary infection by human cytomegalovirus (CMV) and reactivation of latent virus are major problems after hematopoietic stem cell transplantation (HSCT), resulting in inflammation of a wide range of organs, systemic disease, and an increased rate of graft-versus-host disease (GvHD) (3,5,21). Antiviral chemotherapy with nucleoside analogs is used prophylactically and preemptively in the early phase after transplantation, but long-term treatment is often associated with toxicity, selection of resistant virus variants, and the inability to prevent all CMV-associated complications (4,7,28). Sustained control of latent CMV infection depends on the restoration of a functional antiviral immune response (15,25).Adoptive T cell transfer has been used successfully to bridge the critical phase of delayed or insufficient antiviral response in patients with immune suppression. In CMV and Epstein-Barr virus (EBV) infection, the adoptive transfer of ex vivo-expanded, donor-derived, virus-specific T cells reduced virus titers in the recipient to levels similar to those in immunocompetent, healthy, seropositive controls (10,23,29,31). Ex vivo expansion of these cells can be carried out by different procedures (9). In naïve seronegative persons, however, virus-specific T cells occur at very low frequencies, generally insufficient for expansion.As an alternative, T cells can be grafted with defined specificities using recombinant immunoreceptors (11). The receptor specificity is determined by extracellular single-chain fragments of the variable region (scFv) that recognize predefined antigens and can easily be altered by selecting an appropriate scFv (16). Recombinant immunoreceptors have been successfully developed against a number of tumor antigens (16) but against only a few viral proteins from HIV and hepatitis B virus (6,20,22) and not against CMV.In human CMV infection, the analysis of the physiological cytotoxic T lymphocyte (CTL) response has been focused on a limited set of proteins, namely, the proteins pp65, IE1, and IE2; recent and more extensive studies have shown that glycoprotein B (gB) as well as other CMV glycoproteins is also able to evoke adaptive T cell responses (32, 34). Notably, the CMV gB is expressed at the cell surface during the early or delayed early phase of CMV replication, even in the presence of ...

Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFk-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFk-B induction was found, which indicates that internalization of bacteria is crucial for NFk-B induction.Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFk-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFk-B induction.