Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Investigating the Effects of HMGB1 Overexpression on Colorectal Cancer Cell Migration via Oncolytic Herpes simplex Virus Type 1 (oHSV-1).

Background: Colorectal Cancer (CRC) represents a significant global health challenge, and its progression, resistance to therapy, and metastasis are strongly influenced by the tumor microenvironment, including factors like hypoxia. This study explores the impact of High Mobility Group Box 1 (HMGB1) overexpression on CRC cell migration, while identifying potential genes associated with this process. Methods: To explore this, we developed oncolytic virotherapy, resulting in HSVHMGB1, an oncolytic Herpes simplex virus that expresses HMGB1. HMGB1 is known its role in cancer progression, particularly in the context of cancer cell migration. Results: Contrary to expectations, our scratch assays indicated that HSV-HMGB1 did not significantly induce migration in CRC cells, suggesting that HMGB1 might not directly contribute to this process. Employing microarray analysis, we investigated gene expression changes linked to CRC cell migration, leading to construction of a Protein-Protein Interaction (PPI) network. This network revealed the presence of hub proteins, including as NDRG1, LGALS1, and ANGPTL4, which are recognized for their roles in cancer cell migration. The differential expression of these genes under hypoxic conditions was further validated using quantitative RT-PCR, aligning with the findings from our microarray data. Conclusion: Our findings emphasize the complex regulation of CRC cell migration, and provides valuable insights into potential molecular mechanisms and pathways. These findings have implications for further research into cancer progression and the development of therapeutic strategies.

Structure-based evaluation of the envelope domain III-nonstructural protein 1 (EDIII-NS1) fusion as a dengue virus vaccine candidate.

The lack of effective medicines or vaccines, combined with climate change and other environmental factors, annually subjects a significant proportion of the world's inhabitants to the risk of dengue virus (DENV) infection. These conditions increase the likelihood of exposure to mosquito-borne diseases such as dengue fever. Hence, many research approaches tend to develop efficient vaccine candidates against the dengue virus. Therefore, we used immunoinformatics and bioinformatics to design a construction for developing a candidate vaccine against dengue virus serotypes. In this study, the in silico structure, containing the non-structural protein 1 region (NS1) (consensus and epitope), the envelope domain III protein (EDIII) as the structural part of the virus construction, and the bc-loop of envelope domain II (EDII) as the neutralizing and protected epitope, were employed. We utilized in silico tools to enhance the immunogenicity and effectiveness of dengue virus vaccine candidates. Evaluations included refining and validating physicochemical characteristics, B and T-cell epitopes, homology modeling, and the three-dimensional structure to assess the designed vaccine's quality. In silico results for tertiary structure prediction and validation revealed high-quality modeling for all vaccine constructs. Additionally, the instructed model demonstrated stability throughout molecular dynamics simulation. The results of the immune simulation suggested that the titers of IgG and IgM could be raised to desirable values following injection into in vivo models. It can be concluded that the designed construct effectively induce humoral and cellular immunity and can be proposed as effective vaccine candidate against four dengue serotypes.Communicated by Ramaswamy H. Sarma.

The chicken chorioallantoic membrane model for isolation of CRISPR/cas9-based HSV-1 mutant expressing tumor suppressor p53.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

Looking for biomarkers in interferon response pathway to predict response to oncolytic HSV-1 in breast cancer: An ex vivo study.

Breast cancer is the most common malignancy in women worldwide. Administration of oncolytic viruses is one of the novel promising cancer therapy approaches. Replication of these viruses is usually limited to cancer cells that have interferon (IFN) signaling defects. However, Interferon signaling is not completely impaired in all cancer cells which may limit the benefits of virotherapy.    Identification of realistic IFN-mediated biomarkers to identify patients who most likely respond to virotherapy would be helpful. In this study, eight patients-derived primary tumor cultures were infected with an ICP34.5 deleted oHSV, then the rate of infectivity, cell survival, and expression of the gene involved in IFN pathway were analyzed.Data showed that mRNA expressions of Myeloid differentiation primary response protein (Myd88) is significantly higher in tumors whose primary cultures showed less cell death and resistance to oHSV infectivity (P-value < 0.05). The differentiating cut off of Myd88 expression, inferred from the receiver operating characteristic (ROC) curve, predicted that only 13 out of 16 other patients could be sensitive to this oHSV. Identifying such biomarker improves our ability to select the patients who do not exhibit resistance to virotherapy.

In silico Identification of Hypoxic Signature followed by reverse transcription-quantitative PCR Validation in Cancer Cell Lines

Background: Hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. This study aimed to computationally identify hypoxia-related messenger RNA (mRNA) signatures in nine hypoxic-conditioned cancer cell lines and investigate their role during hypoxia. Methods: Nine RNA sequencing (RNA-Seq) expression data sets were retrieved from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in each cancer cell line. Then 23 common DEGs were selected by comparing the gene lists across the nine cancer cell lines. Reverse transcription-quantitative PCR (qRT-PCR) was performed to validate the identified DEGs. Results: By comparing the data sets, GAPDH, LRP1, ALDOA, EFEMP2, PLOD2, CA9, EGLN3, HK, PDK1, KDM3A, UBC, and P4HA1 were identified as hub genes. In addition, miR-335-5p, miR-122-5p, miR-6807-5p, miR-1915-3p, miR-6764-5p, miR-92-3p, miR-23b-3p, miR-615-3p, miR-124-3p, miR-484, and miR-455-3p were determined as common micro RNAs. Four DEGs were selected for mRNA expression validation in cancer cells under normoxic and hypoxic conditions with qRT-PCR. The results also showed that the expression levels determined by qRT-PCR were consistent with RNA-Seq data. Conclusion: The identified protein-protein interaction network of common DEGs could serve as potential hypoxia biomarkers and might be helpful for improving therapeutic strategies.

Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP.

BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

Fiber manipulation and post-assembly nanobody conjugation for adenoviral vector retargeting through SpyTag-SpyCatcher protein ligation.

For adenoviruses (Ads) to be optimally effective in cancer theranostics, they need to be retargeted toward target cells and lose their natural tropism. Typically, this is accomplished by either engineering fiber proteins and/or employing bispecific adapters, capable of bonding Ad fibers and tumor antigen receptors. This study aimed to present a simple and versatile method for generating Ad-based bionanoparticles specific to target cells, using the SpyTag-SpyCatcher system. The SpyTag peptide was inserted into the HI loop of fiber-knob protein, which could act as a covalent anchoring site for a targeting moiety fused to a truncated SpyCatcher (SpyCatcherΔ) pair. After confirming the presence and functionality of SpyTag on the Ad type-5 (Ad5) fiber knob, an adapter molecule, comprising of SpyCatcherΔ fused to an anti-vascular endothelial growth factor receptor 2 (VEGFR2) nanobody, was recombinantly expressed in Escherichia coli and purified before conjugation to fiber-modified Ad5 (fmAd5). After evaluating fmAd5 detargeting from its primary coxsackie and adenovirus receptor (CAR), the nanobody-decorated fmAd5 could be efficiently retargeted to VEGFR2-expressing 293/KDR and human umbilical vein endothelial (HUVEC) cell lines. In conclusion, a plug-and-play platform was described in this study for detargeting and retargeting Ad5 through the SpyTag-SpyCatcher system, which could be potentially applied to generate tailored bionanoparticles for a broad range of specific targets; therefore, it can be introduced as a promising approach in cancer nanotheranostics.

Modifying oncolytic virotherapy to overcome the barrier of the hypoxic tumor microenvironment. Where do we stand?

Viruses are completely dependent on host cell machinery for their reproduction. As a result, factors that influence the state of cells, such as signaling pathways and gene expression, could determine the outcome of viral pathogenicity. One of the important factors influencing cells or the outcome of viral infection is the level of oxygen. Recently, oncolytic virotherapy has attracted attention as a promising approach to improving cancer treatment. However, it was shown that tumor cells are mostly less oxygenated compared with their normal counterparts, which might affect the outcome of oncolytic virotherapy. Therefore, knowing how oncolytic viruses could cope with stressful environments, particularly hypoxic environments, might be essential for improving oncolytic virotherapy.

Adenovirus vector-based vaccines as forefront approaches in fighting the battle against flaviviruses.

Flaviviruses are arthropod-borne viruses (arboviruses) that have been recently considered among the significant public health problems in defined geographical regions. In this line, there have been vaccines approved for some flaviviruses including dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), although the efficiency of such vaccines thought to be questionable. Surprisingly, there are no effective vaccine for many other hazardous flaviviruses, including West Nile and Zika viruses. Furthermore, in spite of approved vaccines for some flaviviruses, for example DENV, alternative prophylactic vaccines seem to be still needed for the protection of a broader population, and it originates from the unsatisfying safety, and the efficacy of vaccines that have been introduced. Thus, adenovirus vector-based vaccine candidates are suggested to be effective, safe, and reliable. Interestingly, recent widespread use of adenovirus vector-based vaccines for the COVID-19 pandemic have highlighted the importance and feasibility of their widespread application. In this review, the applicability of adenovirus vector-based vaccines, as promising approaches to harness the diseases caused by Flaviviruses, is discussed.

Designing vaccine candidates against dengue virus by in silico studies on structural and nonstructural domains.

One-third of the world's population is at risk of Dengue infection. Envelope domain 3 (EDIII) and nonstructural protein1 (NS1) proteins as the potent antigenicity regions for humoral immunity in addition to the bc loop region as a completely conserved region have been used for designing protective vaccines. We aimed to design vaccine candidates according to the bc loop, EDIII, and NS1 regions of Dengue serotype2 to be used as vaccine candidates for all serotypes of Dengue virus especially serotype 2. Firstly the bc loop region with EDII fragments at both ends as well as EDIII and NS1 regions were used which were linked with the GGGGS linker to the bc loop region. In two other strategies, the bc loop with EDII and NS1 fragments at both ends was used to increase its structural stability. Tertiary structure prediction and validation of vaccine constructs indicated that all vaccine constructs were modeled with high quality and stable structure during molecular dynamics simulation. B cell epitope mapping by Bepipred and ElliPro methods confirmed the existence of high potent epitopes in the bc loop, EDIII, and NS1 regions in both linear and conformational B cell epitopes. Furthermore, molecular docking for the bc loop region demonstrated that all designed vaccines have a higher affinity to interact with 1C19 monoclonal antibody than only the bc loop region or bc loop epitope in the protein EII. Our data of in silico studies indicated that the designed vaccines could effectively induce humoral immunity against four dengue serotypes.

Cell type-specific response of colon cancer tumor cell lines to oncolytic HSV-1 virotherapy in hypoxia.

BACKGROUND: Novel strategies are required since the hypoxic tumor microenvironment is one of the important impediments for conventional cancer therapy. High mobility group box 1 (HMGB1) protein can block aerobic respiration in cancer cells. We hypothesized that HMGB1could also kill the colorectal cancer cells during hypoxia. METHODS: In this study, we developed oncolytic herpes simplex virus type 1 expressing HMGB1 protein (HSV-HMGB1) and investigated the cytotoxic effect of HSV-HMGB1 and its parental virus (HSV-ble) on three colorectal cancer cells (HCT116, SW480, and HT29) under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. We further identified potential autophagy- related genes in HT29 cells by retrieving mRNA expression microarray datasets from the Gene Expression Omnibus database. These genes were then detected in HT29 cells infected with HSV-HMGB1 and HSV-ble during normoxia and hypoxia by Real-Time quantitative PCR (qRT-PCR). RESULTS: The cytotoxic effect of HSV-HMGB1 was significantly higher than that of HSV-ble during normoxia; however, during hypoxia, HSV-HMGB1 enhanced the viability of HT29 cells at MOI 0.1. Analyzing the cell death pathway revealed that HSV-HMGB1 induced autophagy in HT29 cells under hypoxic conditions. CONCLUSION: In conclusion, it appears that oncolytic virotherapy is cell context-dependent. Therefore, understanding the cancer cells' characteristics, microenvironment, and cell signaling are essential to improve the therapeutic strategies.

Screening of candidate genes associated with high titer production of oncolytic measles virus based on systems biology approach.

The number of viral particles required for oncolytic activity of measles virus (MV) can be more than a million times greater than the reported amount for vaccination. The aim of the current study is to find potential genes and signaling pathways that may be involved in the high-titer production of MV. In this study, a systems biology approach was considered including collection of gene expression profiles from the Gene Expression Omnibus (GEO) database, obtaining differentially expressed genes (DEGs), performing gene ontology, functional enrichment analyses, and topological analyses on the protein-protein interaction (PPI) network. Then, to validate the in-silico data, total RNA was isolated from five cell lines, and full-length cDNA from template RNA was synthesized. Subsequently, quantitative reverse transcription-PCR (RT-qPCR) was employed. We identified five hub genes, including RAC1, HSP90AA1, DNM1, LTBP1, and FSTL1 associated with the enhancement in MV titer. Pathway analysis indicated enrichment in PI3K-Akt signaling pathway, axon guidance, proteoglycans in cancer, regulation of actin cytoskeleton, focal adhesion, and calcium signaling pathways. Upon verification by RT-qPCR, the relative expression of candidate genes was generally consistent with our bioinformatics analysis. Hub genes and signaling pathways may be involved in understanding the pathological mechanisms by which measles virus manipulates host factors in order to facilitate its replication. RAC1, HSP90AA1, DNM1, LTBP1, and FSTL1 genes, in combination with genetic engineering techniques, will allow the direct design of high-throughput cell lines to answer the required amounts for the oncolytic activity of MV.

Adjuvant Effects of Pseudomonas aeruginosa Flagellin on the Immunological Patterns of the HIV-1 Vaccine Candidate: Vaccine Formulations Versus Different Routes of Immunization.

New strategies to increase the immune response to HIV-1 vaccine using immunological adjuvants such as Toll-like receptor agonists are needed. In this study, HIV-1 p24-Nef and conjugated form of the vaccine candidate to type-A flagellin (FLA) were injected in the BALB/c mice in different routes. Two weeks after the last immunization, lymphocyte proliferation was measured by the BrdU method. The IL-4 and IFN-γ levels, as well as the total IgG antibody and its isotypes titer, were evaluated by the enzyme-linked immunosorbent assay method. The IFN-γ ELISPOT was also performed. Our data showed that the HIV-1 p24-Nef alone and conjugated to type-A flagellin (FLA) significantly increased lymphocyte proliferation responses as well as higher levels of cytokines and IFN-γ producing lymphocytes and the level of humoral immune responses compared with the control groups. The cell-mediated immune responses through the subcutaneous route and humoral immune responses through the intramuscular route were significantly higher in the conjugated form than in the mere vaccine candidate. In conclusion, when the FLA as an adjuvant is constructed in the HIV-1 vaccine candidate, it could effectively improve both humoral and cellular immune responses. Furthermore, modification in the vaccine formulation could change the optimal route of vaccine inoculation.

Genomic and serological assessment of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in child labor.

Since working children have limited access to testing and monitoring for COVID-19, we decided to measure SARS-CoV-2 prevalence among them and compare it to non-working children. Our objective is to compare the frequency of SARS-CoV-2 genome and anti-SARS-CoV-2 antibody among working and non-working children. Volunteer child labor studying at Defense of Child Labor and Street Children and randomly selected 5-18-year-old (same range as child labor group) unemployed children participated in this study. The groups, respectively, had 65 and 137 members. This is an analytical cross-sectional study that surveys molecular prevalence of SARS-CoV-2 infection by RT-PCR, and seroprevalence of SARS-CoV-2 antibody by ELISA in working and non-working children. The IBM SPSS statistics software version 25 was used for data analysis. The χ2 or Fisher's exact test was used to analyze categorical dependent variables, for calculating odds ratios and 95% confidence intervals. Among the children enrolled in this study, molecular prevalence of SARS-CoV-2 turned out to be 18.5% in working children while it was 5.8% in unemployed children [aOR: 3.00 (CI95%: 1.00-7.00); P value: 0.003] and seroprevalence turned out to be 20% in working children vs 13.9% in non-working children [aOR: 1.000 (CI95%: 0.00-2.00); > P 0.001]. Equal SARS-CoV-2 viral load as adults and no symptoms or mild ones in children, coupled with working children's strong presence in crowded areas and their higher rate of COVID-19 prevalence, make them a probable source for spread of the virus.

The ins and outs of SARS-CoV-2 variants of concern (VOCs).

SARS-CoV-2, a newly emerging coronavirus that caused the COVID-19 epidemic, has been spreading quickly throughout the world. Despite immunization and some fairly effective therapeutic regimens, SARS-CoV-2 has been ravaging patients, health workers, and the economy. SARS-CoV-2 mutates and evolves to adapt to its host as a result of extreme selection pressure. As a consequence, new SARS-CoV-2 variants have emerged, some of which are classified as variants of concern (VOC) because they exhibit greater transmissibility, cause more-severe disease, are better able to escape immunity, or cause higher mortality than the original Wuhan strain. Here, we introduce these VOCs and review their characteristics, such as transmissibility, immune escape, mortality risk, and diagnostics.

In-silico identification of new inhibitors for Low-density lipoprotein receptor-related protein6 (LRP6).

Low-density lipoprotein receptor-related protein 6 (LRP6) is an important therapeutic target for diseases such as osteoporosis, Alzheimer, cancer, and neurodegenerative disease. Computational methods such as ligand-based and structure-based virtual screening have been introduced as an extremely efficient and accurate tool for finding new drug targets and candidates. In this study, we aimed to screen the National Cancer Institute (NCI) Diversity Set II and parts of the ZINC database by virtual screening to identify potential and safe compounds that can inhibit the LRP6 protein. By utilizing various screening methods such as rigid and flexible molecular docking and Lipinski's rule of five, we identified 10 potential compounds. Then, their validity was further tested by molecular dynamics simulation and MMPBSA binding free energy calculations. Eventually, it was concluded that ZINC03954520, ZINC01729523, ZINC03898665, ZINC13152226, ZINC26730911 and ZINC01069082 compounds can be potential drug compounds for inhibiting LRP6 protein. These compounds in complex with ß-propeller domains of LRP6 showed that they are capable of altering the backbone of these domains and interfere with their structural dynamics which may lead to the inhibition of the signal transmission. Communicated by Ramaswamy H. Sarma.

Computational design of antagonist peptides based on the structure of secreted frizzled-related protein-1 (SFRP1) aiming to inhibit Wnt signaling pathway.

Aberrant activation of Wnt/ß-catenin signaling pathway, due to the genetic or epigenetic changes, is responsible for tumorigenesis in epithelial cells of different types of cancer such as colorectal cancer. Secreted Frizzled-Related Protein-1 (SFRP1), as one of the antagonist proteins of this pathway, is hyper-methylated in colorectal cancer leading to the formation of Wnt-Fz-LRP and activation of Wnt/ß-catenin signaling pathway. We aimed to design antagonist peptides based on SFRP1 structure against wingless-type 2 (Wnt2), a highly expressed ligand in different cancers like colorectal cancer, to inhibit the formation of the initial triple complex of Wnt-Fz-LRP. After homology modeling of SFRP1, molecular docking showed that Wnt2 and SFRP1 interact in the same mode of xWnt8-mFz8 and hWnt3-mFz8 through the thumb and finger binding sites. These binding sites were selected for designing peptides using either substitution or deep learning-based approaches. The efficiency of each designed peptide in interacting with Wnt2 was evaluated by molecular docking. Stability assessment of Wnt2-peptide complexes via molecular dynamic (MD) revealed that the designed peptides could effectively interact with Wnt2 binding sites during the simulation. However, the designed peptides against the thumb site had higher binding affinity and hydrogen bonds compared to the initial sequence. The secondary structure of the designed peptides indicated an alpha-helix structure which is a favorable structure for peptide drugs. Computing the physicochemical properties of peptides predicted a fairly acceptable structure which made them promising candidates in the treatment of cancers like CRC.

Lentiviral Expression of Rabies Virus Glycoprotein in the Rat Hippocampus Strengthens Synaptic Plasticity.

Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two µl (108 T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat's brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.

Activating Natural Killer Cell Receptors, Selectins, and Inhibitory Siglecs Recognize Ebolavirus Glycoprotein.

Expression of the extensively glycosylated Ebolavirus glycoprotein (EBOV-GP) induces physical alterations of surface molecules and plays a crucial role in viral pathogenicity. Here we investigate the interactions of EBOV-GP with host surface molecules using purified EBOV-GP, EBOV-GP-transfected cell lines, and EBOV-GP-pseudotyped lentiviral particles. Subsequently, we wanted to examine which receptors are involved in this recognition by binding studies to cells transfected with the EBOV-GP as well as to recombinant soluble EBOV-GP. As the viral components can also bind to inhibitory receptors of immune cells (e.g., Siglecs, TIM-1), they can even suppress the activity of immune effector cells. Our data show that natural killer (NK) cell receptors NKp44 and NKp46, selectins (CD62E/P/L), the host factors DC-SIGNR/DC-SIGN, and inhibitory Siglecs function as receptors for EBOV-GP. Our results show also moderate to strong avidity of homing receptors (P-, L-, and E-selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, to cells transfected with EBOV-GP, as well as to the envelope of a pseudotyped lentiviral vector carrying the EBOV-GP. The concomitant activation and inhibition of the immune system exemplifies the evolutionary antagonism between the immune system and pathogens. Altogether these interactions with activating and inhibitory receptors result in a reduced NK cell-mediated lysis of EBOV-GP-expressing cells. Modulation of these interactions may provide new strategies for treating infections caused by this virus.