TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-β (TRIF), TRIF-related adapter molecule (TRAM), and sterile α motifs and β-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-κB activation. The domain that was required for induction of apoptosis could activate NF-κB but not IFN regulatory factor-3, yet the activation of NF-κB could be blocked by superrepressor IκBα without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-κB but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.
Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mM) are used, whereas reduction of butyrate concentration to 0n3 mM decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20 % of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIP I, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0n3 mM delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate concentration to 0n3 mM, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.
To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the lacZ gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the lacZ gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.Attachment and entry represent the first essential steps of viral replication. Enveloped viruses have evolved two main pathways to mediate their entry into the cells after attachment to cell surface moieties (reviewed in reference 25). The first one, low-pH-dependent pathway, involves endocytosis of viral particles followed by viral-cell membrane fusion in endosomes or lysosomes. This fusion is triggered by an acidic-pH-dependent conformational change of viral glycoprotein(s) and allows release of capsid into the cytoplasm. Entry of vesicular stomatitis virus (VSV), the prototype rhabdovirus, exemplifies this pathway. In contrast, in the second one, pH-independent pathway, viral-cell membrane fusion takes place on the plasma membrane at neutral pH. Most retroviruses and paramyxoviruses use this pathway. The pH-independent entry was also demonstrated for herpesviruses, herpes simplex virus (55), and cytomegalovirus (CMV) (16). However, Epstein-Barr virus (EBV) uses membrane fusion both in endosomes and on the plasma membrane differentially (40).Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is a member of the gammaherpesvirus subfamily and is etiologically associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (reviewed in references 1 and 11). It interacts with target cells by binding of glycoprotein B (gB) and K8.1 with glycosaminoglycans, such as heparan sulfate, on the cell surface (5, 10, 54). Recent studies found that gB also bound to ␣31 integrin through its RGD motif and induced ERK signaling pathway, implicating that ␣31 integrin functions as a cellular receptor for HHV-8 entry (6, 42). One of the major constraints to study the entry mechanisms of HHV-8 is a lack of fully permissive cell lines to conduct traditional virological assays b...
Fatal cases of filoviral infection are accompanied by a marked immunosuppression. Endothelial cells play a vital role in the host immune response through the expression of several immunomodulatory genes in addition to the expression of the antiviral genes, 2',5'-oligoadenylate synthetase [2'-5'(A)N], and the double-stranded RNA (dsRNA)-activated protein kinase (PKR). dsRNA, an intermediate generated during viral replication and gene transcription of many viruses, leads to the induction of immunomodulatory genes in endothelial cells. In this report, we show that induction of the major histocompatibility complex class I family of genes, 2'-5'(A)N, interleukin-6 (IL-6), PKR, interferon (IFN)-regulatory factor-1, and intercellular adhesion molecule-1 (ICAM-1) by dsRNA in human umbilical vein endothelial cells is suppressed by infection with the filovirus Ebola-Zaire (EZ). In contrast, induction of IL-6 and ICAM-1 by IL-1 is intact in EZ-infected cells. Gel shift analysis demonstrates that dsRNA-induced protein binding to IFN-responsive elements is strongly suppressed by EZ-IFN, whereas NF-kappa B activation by dsRNA remains intact. We previously reported that IFN signaling is suppressed by EZ infection, and these data strongly suggest that elements shared between IFN and dsRNA signaling are being inhibited by EZ. Inhibition of IFN and dsRNA responsiveness could play a role in the immunosuppression seen in EZ infections and would play a role in the pathogenesis of disease caused by EZ.
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