Human Herpesvirus 6 and Measles Virus Employ Distinct CD46 Domains for Receptor Function

We employed a quantitative cell fusion assay to identify structural domains of CD46 required for its function as a receptor for human herpesvirus 6 (HHV-6). We examined the activities of recombinant variants of CD46, including different isoforms as well as engineered truncations and molecular chimeras with decay-accelerating factor, a related protein in the family of regulators of complement activation (RCA). We observed strong receptor activity for all four CD46 isoforms, which differ in the membrane-proximal extracellular and cytoplasmic domains, indicating that the critical determinants for HHV-6 receptor activity reside outside the C-terminal portion of CD46. Analysis of the short consensus repeat (SCR) regions that comprise most of the extracellular portion of CD46 indicated a strong dependence on SCRs 2 and 3 and no requirement for SCRs 1 or 4. Fusion-inhibition studies with SCR-specific monoclonal antibodies supported the essential role of SCRs 2 and 3 in HHV-6 receptor activity. These findings contrast markedly with fusion mediated by measles virus glycoproteins for which we observed a strict dependence on SCRs 1 and 2, consistent with previous reports. These results expand the emerging notion that CD46 and other members of the RCA family are co-opted in distinct manners by different infectious pathogens.     

Determinants of Human CD134 Essential for Entry of Human Herpesvirus 6B

We identified two key amino acid residues within human CD134 (hCD134) that are required for its interaction with human herpesvirus 6B (HHV-6B) and for HHV-6B entry into cells. One of the residues (K79) allows access of the HHV-6B ligand to hCD134. Murine CD134 (mCD134) functioned as an HHV-6B receptor when these two amino acid residues were replaced with homologous human residues. This study identifies both the HHV-6B receptor-ligand interaction and the species-specific determinants of hCD134 essential for HHV-6B entry.     

Inhibition of p53-Dependent,but Not p53-Independent,Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.     

Development of Virus-Specific CD4+ and CD8+ Regulatory T Cells Induced by Human Herpesvirus 6 Infection

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4⁺ and CD8⁺ HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4⁺ and CD8⁺ Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4⁺ effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.     

Prevalence of Human Herpesvirus 6 Variants A and B in Patients with Chronic Fatigue Syndrome

Peripheral blood mononuclear cells collected from 13 patients with chronic fatigue syndrome and 13 healthy controls were analyzed for the presence of human herpesvirus 6 (HHV-6) DNA by variant-specific polymerase chain reaction and dot blot hybridization. HHV-6 DNA was detected in 7 of 13 (53%) patients, and of those 7 patients, 4 were positive for HHV-6 variant A DNA and 3 were for variant B. No HHV-6 DNA was detected in the controls. Serum antibody titers to the late antigen and antibody prevalence to the early antigen of HHV-6 were significantly higher in the patient group. These results suggest active replication of HHV-6 in patients with chronic fatigue syndrome.     

Tetraspanin CD9 Regulates Cell Contraction and Actin Arrangement via RhoA in Human Vascular Smooth Muscle Cells

The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC) morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active) RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology.     

Comparison of the Complete DNA Sequences of Human Herpesvirus 6 Variants A and B

Human herpesvirus 6 (HHV-6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. Two variants of HHV-6 have been distinguished on the basis of differences in several properties. We have determined the complete DNA sequence of HHV-6 variant B (HHV-6B) strain HST, the causative agent of exanthem subitum, and compared the sequence with that of variant A strain U1102. A total of 115 potential open reading frames (ORFs) were identified within the 161,573-bp contiguous sequence of the entire HHV-6 genome, including some genes with remarkable differences in amino acid identity. All genes with <70% identity between the two variants were found to contain deleted regions when ORFs that could not be expressed were excluded from the comparison. Except in the case of U47, these differences were found in immediate-early/regulatory genes, DR2, DR7, U86/90, U89/90, and U95, which may represent characteristic differences of variants A and B. Also, we have successfully typed 14 different strains belonging to variant A or B by PCR using variant-specific primers; the results suggest that the remarkable differences observed were conserved evolutionarily as variant-specific divergence.     

Human herpesvirus 6 variant A glycoprotein H-glycoprotein L-glycoprotein Q complex associates with human CD46

Human CD46 is a cellular receptor for human herpesvirus 6 (HHV-6). Virus entry into host cells requires a glycoprotein H (gH)-glycoprotein L (gL) complex. We show that the CD46 ectodomain blocked HHV-6 infection and bound a complex of gH-gL and the 80-kDa U100 gene product, designated glycoprotein Q, indicating that the complex is a viral ligand for CD46.     

Tetraspanin 6 (TSPAN6) Negatively Regulates Retinoic Acid-inducible Gene I-like Receptor-mediated Immune Signaling in a Ubiquitination-dependent Manner

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.     

TSG-6 promotes Cancer Cell aggressiveness in a CD44-Dependent Manner and Reprograms Normal Fibroblasts to create a Pro-metastatic Microenvironment in Colorectal Cancer

Tumor necrosis factor α stimulated gene 6 (TSG-6), a 30-KD secretory protein, plays an essential role in modulating inflammatory responses and extracellular matrix remodeling. However, little is known regarding the role of TSG-6 in human cancers. Here, we investigated the mechanism of action and the role of TSG-6 in colorectal cancer (CRC) metastasis. We found that TSG-6 was highly expressed in tumor tissues and was associated with poor prognosis and metastasis in CRC. Mechanistically, TSG-6 overexpression in CRC cells resulted in ERK activation and epithelial-mesenchymal transition by means of stabilizing CD44 and facilitating the CD44-EGFR complex formation on the cell membrane. Consequently, this resulted in the promotion of tumor migration and invasion both in vitro and in vivo. Notably, our data showed that CRC cells secreted TSG-6 could trigger a paracrine activation of JAK2-STAT3 signaling and reprogram normal fibroblasts into cancer-associated fibroblasts, which exhibited upregulation of pro-metastatic cytokines (CCL5 and MMP3) and higher movement ability. In animal models, the co-injection of cancer cells and TSG6-reprogrammed fibroblasts led to a significant increase in tumor metastasis. Our findings indicated that TSG-6 overexpression in CRC cells could promote cancer metastasis in both an autocrine and paracrine manner. Therefore, targeting TSG-6 might be a potential therapeutic strategy for the treatment of metastatic CRC.