A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor α (PILRα) Abrogates PILRα-Dependent Viral Entry and Reduces Pathogenesis

Paired immunoglobulin-like type 2 receptor α (PILRα) is a herpes simplex virus 1 (HSV-1) entry receptor that associates with O-glycans on HSV-1 envelope glycoprotein B (gB). Two threonine residues (Thr-53 and Thr-480) in gB, which are required for the addition of the principal gB O-glycans, are essential for binding to soluble PILRα. However, the role of the two threonines in PILRα-dependent viral entry remains to be elucidated. Therefore, we constructed a recombinant HSV-1 carrying an alanine replacement of gB Thr-53 alone (gB-T53A) or of both gB Thr-53 and Thr-480 (gB-T53/480A) and demonstrated that these mutations abrogated viral entry in CHO cells expressing PILRα. In contrast, the mutations had no effect on viral entry in CHO cells expressing known host cell receptors for HSV-1 gD, viral entry in HL60 cells expressing myelin-associated glycoprotein (MAG) (another HSV-1 gB receptor), viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. These results support the hypothesis that gB Thr-53 and Thr-480 as well as gB O-glycosylation, probably at these sites, are critical for PILRα-dependent viral entry. Interestingly, following corneal inoculation in mice, the gB-T53A and gB-T53/480A mutations significantly reduced viral replication in the cornea, the development of herpes stroma keratitis, and neuroinvasiveness. The abilities of HSV-1 to enter cells in a PILRα-dependent manner and to acquire specific carbohydrates on gB are therefore linked to an increase in viral replication and virulence in the experimental murine model.Herpes simplex virus 1 (HSV-1) entry into host cells depends on interactions between cell surface receptors and HSV-1 virion envelope glycoproteins (39). Five of the 12 HSV-1 envelope glycoproteins that have been identified thus far (i.e., glycoprotein B [gB], gC, gD, gH, and gL) have roles in viral entry (39). Both gB and gC mediate virion attachment by interacting with cell surface glycosaminoglycan, primarily heparan sulfate (16, 17). Although not essential for entry, this step provides stable interactions between the virion and the cell that favor the next steps (39). These steps include gD binding to one of its identified receptors, i.e., herpesvirus entry mediator (HVEM), nectin-1, and specific sites on heparan sulfate 3-O-sulfated heparan sulfate (3-O-S-HS) generated by certain 3-O-sulfotransferases (3-O-STs) (14, 28, 38, 51). Subsequent fusion between the virion envelope and host cell membrane, which requires the cooperative function of gB, heterodimer gH/gL, gD, and a gD receptor, then produces nucleocapsid penetration into the cell (31, 46).In addition to the interaction of gD with a gD receptor, gB binding to a cellular receptor other than heparan sulfate has been suggested to mediate viral entry, based on the observation that a soluble form of gB binds to heparan sulfate-deficient cells and blocks HSV-1 infection of some cell lines (3). Consistent with this observation, we have reported that paired immunoglobulin-like type 2 receptor α (PILRα) associates with gB and functions as an HSV-1 entry receptor (36). Viral entry via PILRα appears to be conserved among alphaherpesviruses, but there is a PILRα preference based on the observation that PILRα is able to mediate the entry of pseudorabies virus, a porcine alphaherpesvirus, but not of HSV-2 (1). Importantly, HSV-1 infection of human primary monocytes expressing both HVEM and PILRα was blocked by either an anti-PILRα or anti-HVEM antibody, suggesting that cellular receptors for both gD and gB are required for HSV-1 infection (36). However, CHO-K1 cells, which are resistant to HSV-1 infection, can become susceptible to HSV-1 entry and HSV-1-induced cell fusion after the overexpression of either a gD receptor, such as nectin-1, or PILRα (14, 36). It was thought that CHO-K1 cells express endogenously low levels of gB and gD receptors that allow the single overexpression of either a gB or gD receptor to support detectable levels of HSV-1 entry and HSV-1-induced cell fusion (36). More recently, myelin-associated glycoprotein (MAG), which has homology to PILRα, was also reported to serve as the gB receptor for HSV-1 and varicella-zoster virus (40). However, the importance of PILRα- or MAG-dependent viral entry in HSV-1 infection and pathogenesis in vivo remains to be elucidated.PILRα is one of the paired receptor families, in which one receptor has inhibitory functions and the other mediates activation functions, and is expressed mainly in immune system cells (13, 29). In addition, PILRα was previously reported to be expressed in certain types of cells in neural tissues (36). We previously identified one of the PILRα ligands as CD99 (37). Interestingly, PILRα recognition of CD99 is dependent on the addition of sialylated O-linked sugar chains at particular CD99 threonines (50). Similarly, we recently demonstrated that a specific sialylated O-glycan(s) on gB is critical for PILRα binding, based on observations that neuraminidase, which removes sialic acid, and benzyl-α-GalNAc treatment, which blocks O-glycan synthesis, inhibited gB binding to a soluble PILRα (49). More importantly, one (Thr-53) or both (Thr-53 and Thr-480) putative O-glycosylation sites identified by bioinformatics analysis are required for the binding of gB to soluble PILRα, and the replacement of both Thr-53 and Thr-480 with alanine significantly inhibited the addition of O-glycans to gB (49). These observations suggest that Thr-53 and Thr-480 in gB are O-glycosylated, and these sites, and probably the addition of specific carbohydrates to them, are required for the interaction of gB with PILRα. However, it remains uncertain whether gB Thr-53 and Thr-480, and probably the gB O-glycosylation of these sites, are required for PILRα-dependent viral entry in natural infections.In the present study, we have shown that the alanine replacement of gB Thr-53 (gB-T53A) alone or of both gB Thr-53 and Thr-480 (gB-T53/480A) significantly inhibited cell-cell fusion in CHO cells expressing PILRα, gB, gD, gH, and gL, whereas the mutations had no effect on cell-cell fusion in CHO cells expressing nectin-1, gB, gD, gH, and gL. Furthermore, we constructed recombinant HSV-1 carrying the gB-T53A and gB-T53/480A mutations and found that these mutations abrogated PILRα-dependent viral entry but had no effect on viral entry via known receptors for HSV-1 gD and MAG, viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. We also tested these recombinant viruses in mice and present data showing that the mutations in gB significantly reduced viral replication, the development of herpes stromal keratitis (HSK), and neuroinvasiveness.     

Production of biologically active IgG hinge-tag soluble epidermal growth factor receptors (ErbB)

The extracellular domains (ECD) of epidermal growth factor receptors, ErbB1, 2, 3 and 4, were designed as soluble dimeric forms. Each ECD was fused to a short hinge region derived from IgG, such that the stable dimer could be formed with disulfide bridges. This hinge-tagged design minimized the molecular weight to approximately 50% of the conventional Fc-fusion design without an Fc domain of IgG. The refolded dimers could be easily analyzed and characterized by SDS-PAGE. Hinge-tagged soluble ErbBs demonstrated significant affinity for betacellulin and heregulin. The IgG hinge-tag should be a simple method to design soluble dimers that would be useful for high throughput screening of ligands, antagonists or derivatives.     

Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1^−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1^−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1^−/− mice. Osteoclastogenesis induced from cultured Ccr1^−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1^−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1^−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1^−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.     

The gefitinib-sensitizing mutant epidermal growth factor receptor enables transformation of a mouse fibroblast cell line

A specific inhibitor of the Epidermal Growth Factor Receptor (EGFR), Gefitinib, displays significant antitumor effects against non-small cell lung cancers (NSCLC) that express EGFR with mutations in their tyrosine kinase domain. Although previous reports have already demonstrated that oncogenic transformation can be induced by some mutant EGFR forms, the precise differences between mutant and wild-type EGFR in terms of mechanisms of transformation have not been fully elucidated. We show here that a murine fibroblast cell line, NR6 becomes transformed by an expression level of the mutant EGFR form lacking E746-A750 that is far less than that needed with transfected wild-type EGFR. However, the mutant EGFR was unable to transform NR6 in a ligand-independent manner, as was seen with the wild-type EGFR. The consequent biological features after transformation, including DNA synthesis or cell cycle progression and biochemical characteristics such as MAPK activation mediated by the mutant EGFR are comparable and equivalent to those mediated by wild-type EGFR. These data suggest that the mutant EGFR possesses greater ligand-dependent transformation when compared with wild-type EGFR, although the exact mechanisms to account for this characteristic remain to be defined.     

Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.     

Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr³⁰⁸ and Ser⁴⁷³) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ~75% in CHO cells and ~30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.     

Ischemia Induces a Reduction in the Content of Ulinastatin-Like Substance in the Murine Hippocampus

Effects of ischemia on the content of a ulinastatin (UT)-like substance in the murine cerebral cortex and hippocampus were studied. At 24 h post-ischemia, a significant (p < 0.05) decrease in the content of UT-like substance in the hippocampus but not the cerebral cortex and a concurrent increase in the activity of -calpain were observed. In in vitro experiments, a decrease was registered in the content of UT-like substance in the hippocampus in the presence of calcium. This decrease was inhibited by both EDTA and calpastatin treatments. These results implicate the destruction of UT-like substance by -calpain in the ischemic hippocampus.     

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.