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Purpose : To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods : Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results : Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion : Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells. Keywords : Herpes simplex virus type 1, UL43 protein, Membrane fusion, Mutant viruses, Virion, Mutagenesis

Purpose : To search for effective antagonist peptide of CC chemokine receptor 1 (CCR1), and evaluate the potential role and mechanism of peptide C18P derived from viral macrophage inflammatory protein II (vMIP-II). Methods: Alignment, simulated peptide-cut, bioinformatics and protease digestion were used to screen and prepare antagonist peptide. Interactions between C18P and CCR1 were determined by radioligand binding assays and [ 35 S]GTPγS binding experiment. Chemotaxis assay was utilized to assess the potency for inducing or inhibiting peripheral blood mononuclear cells (PBMCs) migration. Ligandinduced intracellular calcium mobilization was tested by flow cytometry. Results: A peptide containing 18 amino acids (C18P) was screened. C18P bound to CCR1 with a Kd of 5.7 ng/ml and displaced 125 I-labeled MIP-1α and 125 I-labeled RANTES on human CCR1-transfected HEK293 cells (IC 50 = 11.2 and 9.6 ng/ml, respectively) in radioligand binding studies. C18P lacked intrinsic agonist activity but strongly inhibited HCC-1 (100 nM) induced [ 35 S]GTPγS binding (IC 50 = 3.7 ug/ml), chemotaxis induced by HCC-1, MIP-1α and RANTES (IC 50 = 23, 25 and 13.1 ng/ml, respectively), and intracellular calcium mobilization. Conclusion: These results demonstrate that bioinformatics and protease digestion are feasible to screen and prepare C18P, and that C18P is a novel and specific small molecule peptide antagonist of CCR1 with therapeutic potential for preventing cell migration. Keywords: CC Chemokine receptor 1; Simulated peptide-cut; Antagonist peptide; Viral macrophage inflammatory protein II; Bioinformatics; Protease digestion; HEK293 cells; Radioligand binding

Purpose : To evaluate lymphatic system targeting and inhibitory ability of N15P nano-liposomal preparation (naLipo-N15P) of CXCR4 receptor antagonist in HIV infection. Methods: Chemotactic and chemotaxic inhibition activity assays were used to analyze the biological activity of naLipo-N15P. The anti-HIV potential of NaLipo-N15P in vitro was evaluated when NaLipo- N15P combined with the peripheral blood mononuclear cells of Macaca fascicularis which carry the Simian immunodeficiency virus. Furthermore, the anti-HIV potential in vivo of NaLipo-N15P was evaluated by the plasma concentration and tissue distribution (Ki) Results: The half-maximal inhibitory concentration of naLipo-N15P binding to CXCR4 as an antagonist in competition with SDF-1α was 1.89 pM while Ki was 2.4 pM. Viral load was 289 ± 45. NaLipo- N15P majorly accumulated in liver and spleen. Conclusion: When N15P is encapsulated into nano-liposomes, it not only retains specific binding to CXCR4 and facilitates cell-type-specific targeting of nano-liposomes to PBMCs with high CXCR4 expression, but also shows enhanced anti-HIV effect. Therefore, we propose that naLipo-N15P as a CXCR4 antagonist will play an important role in inflammation and blocking of HIV infection. Keywords : Antagonist, CXCR4, Liposomes, Receptor, Inflammation, HIV

Purpose : To evaluate the broad-spectrum antiviral activity of peptide H9 (H9) in vitro in order to gain insight into its underlying molecular mechanisms. Method: Antiviral activity against Herpes simplex virus type 1 (HSV-1) was determined using thiazolyl blue (MTT) assay. Polymerase Chain Reaction (PCR) was employed to assay H9 antiviral activity against human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The inhibitory effect of H9 on the replication of these viral genes including early genes was assayed by real time-Ppolymerase chain reaction (RT-PCR) and Western blot. Results: H9 possessed significant inhibitory effect on the four different herpesviruses with 50 % inhibitory concentration (IC 50 ) of 1.21 ng/mL (HSV-1). AD169 infection was strongly inhibited with an EC 50 value of 0.46 ng/ml. The anti-herpesviral activity of H9 was dose-dependent. The peptide acted primarily during the early stage of infection by detection of the early genes. Conclusion: The results demonstrate that H9 can inhibit the infection of HSV-1, EBV and HCMV. Furthermore, H9 has a broad-spectrum anti-herpesviral effect in vitro based on targeted killing of infected cells expressing genes. Keywords: Antagonist, Trapping receptor/ligand, Broad-spectrum, Anti-herpesvirus, H9 peptide, Gene expression

Purpose: The aim of the present study was to develop peptide H9 as an efficient antagonist of human cytomegalovirus (HCMV) chemokine receptor US28. Methods: US28 gene was amplified from HCMV, and a stable expression system was constructed using NIH/3T3 cells. Interaction between peptide H9 and receptor US28 was tested by enzyme-linked immunosorbent assay. Flow cytometry was used to determine intracellular concentrations of Ca 2+ , and the possible role of H9 as an antagonist was evaluated by anti-viral experiments. Results: H9 interacts with the US28 receptor and prevents an increase of Ca 2+ resulting from an interaction of chemokine with its receptor. Anti-viral assays showed that H9 could inhibit cytopathic effects of HCMV. AD169 infection (EC 50 = 0.46 ng/ml), and the production of pp65 antigen were strongly inhibited with an EC 50 value of 0.34 ng/ml. Conclusion: The results demonstrate that H9 is an antagonist of US28, suggesting a possible role as a treatment for HCMV. Keywords: Human cytomegalovirus, US28, Peptide H9, Trapping receptor/ligand, Antagonist

Purpose : To evaluate the effect of synthetic polypeptide (N15P) derived from viral macrophage inflammatory protein II (vMIP-II) on the secretion of vascular endothelial growth factor (VEGF) as well as investigate the signaling pathways involved in stromal cell-derived factor-1α(SDF-1α)/CXC Chemokin Receptor 4 (CXCR4) axis-induced VEGF in glioblastoma U87 cells. Methods : Glioblastoma U87 cells were exposed to SDF-1a, N15P with various concentrations. The expression of CXCR4, SDF-1α and VEGF mRNA were assessed by RT-PCR, while expression level of VEGF was tested by ELISA and protein kinase B (Akt) phosphorylation detected by Western blot. Results : The results showed that CXCR4, SDF-1α, VEGF are expressed in human glioblastoma U87 cell lines. SDF-1α caused a dose-dependent sensitivity of cell proliferation with a maximum effect at 15 µmole/ml, while N15P decreased cell viability in U87 cells in a dose-dependent manner. SDF-1α stimulated the activation of VEGF, and N15P inhibited the activation of VEGF with or without SDF-1α stimulation. VEGF production in U87 cells was associated with Akt pathway. These changes in intracellular processes were blocked by N15P in a dose-dependent manner. Conclusion : The results suggest that N15P suppress SDF-1α/CXCR4 Mediated VEGF production through Akt signaling pathway and this may be a potent therapeutic strategy in glioblastoma. Keywords : Viral macrophage, Inflammatory protein II, Glioblastoma, CXC chemokin receptor 4, Stromal cell-derived factor-1α, Protein kinase B

Purpose : To evaluate the potential role and cellular mechanism of the CXCR4 antagonist (N15P) derived from the N-terminal of viral macrophage inflammatory protein-II (vMIP-II) on the apoptosis induced by HIV-1 extracellular nef protein in vitro. Method: Peripheral blood mononuclear cells (PBMCs) and Jurkat cells were treated with HIV-1 nef protein alone or together with N15P at different doses and time points. The competitive binding effect of N15P against nef was assessed via radioligand binding assays. Apoptosis was evaluated via terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. The level of nuclear FOXO3a and phospho-FOXO3a was assessed by Western blotting. Results: The interaction of 125 I-nef with Jurkat cells was inhibited by N15P in a dose-dependent manner, with IC50 value of 0.3516 ng/ml. N15P protect against nef protein-induced apoptosis in a timeand concentration- dependent manner. The proapoptotic effect of extracellular nef protein in cells was associated with FOXO3a pathway and the changes in intracellular processes were blocked by N15P in a dose-dependent manner. Conclusion: N15P inhibits the apoptosis of uninfected CD4 + T lymphocytes induced by HIV-1 extracellular nef protein. Therefore, N15P is a potential effective CXCR4 antagonist in the course of HIV and could prevent or delay the onset of AIDS. Keywords : HIV-1, Nef, vMIP-II, Bystander lymphocytes, Apoptosis, FOXO3a, CXCR4 antagonist, Macrophage, Inflammation