Virus-infected Host Cells Research Articles

Diesel exhaust particles modify natural killer cell function and cytokine release

BackgroundNatural killer (NK) cells are an important lymphocyte population in the nasal mucosa and play important roles in linking the innate and the adaptive immune response. Their two main functions are direct cell-mediated cytotoxicity and the release of cytokines. They are important during viral infections and cancer. Due to their location in the nasal mucosa, NK cells are likely exposed to inhaled pollutants, such as diesel exhaust. Whether and how exposure to diesel exhaust particles (DEP) affects NK cell function in the context of viral infections has not been investigated.MethodsNK cells were isolated from peripheral blood obtained from normal healthy volunteers and subsequently stimulated with the viral mimetic polyinosinic:polycytidylic acid (pI:C), DEP, or pI:C+DEP for 18 hours. NK cells were subsequently analyzed for changes in surface marker expression, cytokine production, gene expression changes, and cytotoxic function using flow cytometry, ELISA, qRT-PCR, and cell-mediated cytotoxicity assay, respectively.ResultsStimulation of NK cells with pI:C and pI:C+DEP, but not DEP alone, increased the release of IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12p70, IFN-γ and TNF-α. As compared to pI:C alone or pI:C+DEP, the release of IL-1β, IL-8 and TNF-α was significantly lower after DEP stimulation alone. Stimulation with pI:C alone increased the gene and protein expression of granzyme B and perforin, which was completely blunted by adding DEP. Addition of DEP further reduced CD16 expression in pI:C stimulated cells. Similarly, cell-mediated cytotoxicity was significantly reduced by the addition of DEP.ConclusionsIn the context of viral infection, DEP potentially reduces NK cells' ability to kill virus-infected host cells, in spite of normal cytokine levels, and this may increase susceptibility to viral infections . This reduction in the potential ability of NK cells to kill virus-infected host cells may increase the susceptibility to viral infections after DEP exposure.

Surface proteins of C6/36 cells involved in dengue virus 4 binding and entry

Dengue virus (DENV) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. It is transmitted to humans by Aedes mosquitoes. The first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. There are several molecules that participate in DENV infection of mosquitoes, but only a few have been identified. In this work, we co-purified 4 proteins from C6/36 cells using a recombinant DENV 4 E protein and identified them as 70 kDa Heat Shock and 70 kDa Heat Shock cognate proteins (HSP70/HSc70), Binding immunoglobulin protein (BiP), Thioredoxin/protein disulphide isomerase (PDI), and 44 kDa Endoplasmic reticulum resident protein (ERp44) via matrix-assisted laser desorption/ionisation time of flight (Maldi-ToF) analysis. Using immunofluorescence and flow cytometry assays, we observed re-localisation of HSP70/HSc70 and, to a lesser extent, BiP to the plasma membrane under stress conditions, such as during DENV infection. By performing binding and infection assays independently, we found that all 4 proteins participate in both processes, but to differing extents: HSP70/HSc70 is the most critical component, while ERp44 is less important. Viral infection was not inhibited when the cells were incubated with antibodies against all of the surface proteins after virus binding, which suggests that DENV entry to C6/36 cells is mediated by these proteins at the same step and not sequentially.

Quantitative omics and its application to study virus-host interactions—a new frontier

Quantitative omics and its application to study virus-host interactions—a new frontier

Small Interfering RNA Targeting the Nonstructural Gene 1 Transcript Inhibits Influenza A Virus Replication in Experimental Mice

Nonstructural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the double-stranded RNA-activated protein kinase-mediated inhibition of viral RNA translation. Reduction of NS1 gene product in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We used small interfering RNAs (siRNAs) synthesized against the viral mRNA to down regulate the NS1 gene and observed its effect on inhibition of virus replication. When NS1 gene-specific siRNA were transfected in Madin Darby canine kidney (MDCK) cells followed by influenza A virus infection, approximately 60% inhibition in intracellular levels of NS1 RNA was observed. When siRNA was administered in BALB/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and cytokine levels was also detected in the presence of siRNAs as compared with the untreated control. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus-infected host cells and may be used as potential candidates for nucleic acid-based antiviral therapy for prevention of influenza A virus infection.

The Packaging of Different Cargo into Enveloped Viral Nanoparticles

Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting. Alphavirus virus-like particles, or viral nanoparticles, can be generated from in vitro self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research.

Ultrasensitive analysis of binding affinity of HIV receptor and neutralizing antibodies using solution-phase electrochemiluminescence assay

Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein–protein (ligand–receptor, antigen–antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution-phase ECL assay with a wide dynamic range (0–2nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein–protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions.

3D Reconstruction of Varicella-Zoster Virus Infected Host Cell Nuclei and PML Cages by Serial Section Array-SEM and TEM Tomography.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Xenografting from Pigs, a Solved Problem Needing Urgent Application

The histocompatibility system is responsible for the rejection of allograft. The system exists to counter the explosive speed of viral replication. It does this by directing the defensive immune attack by cytotoxic T cells on to histocompatibility antigens on the infected cell’s surface. This enables destruction of the virus factories that the infected cells become, before the cytotoxic T cells are swamped by the myriad numbers of new virions, a thousand coming from each infected cell every 10 hours. The immunity system mistakes alloantigens for virus infected host cells that need swift destruction. For transplantation, Sykes has improved Kaplan’s technique by adding recipient bone marrow cells to the donor ones injected for reconstitution of the recipient after immune ablation. This protocol should enable xenografting from untreated pigs, offering instant and unlimited supply of grafts for man.

Why the histocompatibility system exists and how transplant surgeons can xenograft without rejection

The histocompatibility system is responsible for the rejection of allografts. The system exists to counter the explosive speed of viral replication by directing the defensive immune attack by cytotoxic T cells on to histocompatibility antigens on the infected cell's surface. This enables destruction of the virus factories before the cytotoxic T cells are swamped by the myriad numbers of new virions, a thousand coming from each infected cell every 10 h. The immunity system mistakes alloantigens for virus-infected host cells that need swift destruction. For transplantation, Sykes has improved Kaplan's technique by adding recipient bone marrow cells to the donor ones injected for reconstitution of the recipient after immune ablation. This technique should enable the use of xenografts from pigs.

Inhibition of Germinal Centre Apoptotic Programmes by Epstein-Barr Virus

To establish a persistent latent infection, Epstein-Barr virus (EBV) faces a challenge in that the virus-infected host cell must transit through the germinal centre reaction. This is a site of B cell differentiation where antibody responses are optimised, and the selection criteria for B cells are stringent. The germinal centre environment is harsh, and the vast majority of B cells here die by apoptosis. Only cells receiving adequate survival signals will differentiate fully to be released into the periphery as long-term memory B cells (the site of persistence). In this review, we detail the apoptotic pathways potentially encountered by EBV-infected B cells during the process of infection, and we describe the functions of those EBV-regulated cellular and viral genes that help promote survival of the host B cell.

The Role of Apoptosis in Influenza Virus Pathogenesis and the Mechanisms Involved in Anti-Influenza Therapies

Because pandemic influenza virus infection can result in large-scale human death and have a major economic impact, influenza virus is one of the most important microbial pathogens. To control influenza virus, both clarification of the mechanisms by which the virus causes disease and the development of effective drugs and other therapies based on these mechanisms are needed. Virus infection often induces cell death, which can play an important role in disease pathogenesis, and influenza virus, in particular, has been shown to induce significant levels of apoptosis in vivo and in vitro. Over the past decade, the mechanisms by which viral and cellular factors modulate apoptosis induction in influenza virus-infected cells have been revealed. Viral proteins such as hemagglutinin, neuraminidase, M1, NS1, and PB1-F2 have been shown to be involved in apoptosis induction through extrinsic or intrinsic apoptotic pathways. On the other hand, several drugs with different mechanisms of action and a neutralizing monoclonal antibody (mAb) are currently used to control influenza virus. A better understanding of both how influenza virus infects host cells and the intracellular signaling pathways that are activated by infection is essential for the development of antiviral drugs and other effective therapies. Keywords: Apoptosis, drugs, haemagglutinin, H5N1, influenza virus, therapies

Current and Future Anti-Influenza Virus Drugs

In 2009, we have been experiencing a new pandemic of novel influenza virus type A (H1N1) infection. The human beings still face the threat of highly pathogenic avian influenza A (H5N1) virus. Many patients with influenza virus infection have died due to severe complications even though receiving intensive care. This suggests the need for new treatment strategies of severe influenza-associated complications. In cases of severe influenza-associated complications, pathological manifestations are as a result of complex biological consequences, such as apoptosis induction, macrophage activation, oxidative damage and increased production of pro-inflammatory cytokines. Recent studies have revealed that the pathogenesis of severe influenza-associated complications involves not only the virus replication-mediated apoptotic cell death in the infected cells but also non-infected cell injury by toxicity of reactive oxygen species derived from macro- phages phagocytosing apoptotic cells, and that pro-inflammatory cytokines produced by the virus-infected host cells play a critical role in the activation of macrophages. These findings provide a possibility that an agent with antiviral and antioxidant activities can be a drug of choice for the treatment of patients with severe influenza-associated complications. Selected antioxidants, such as pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, glutathione, nordihydroguaiaretic acid, thujaplicin and certain types of flavonoids, possess both activities. The combination of antioxidants, such as superoxide dismutase and N-acetyl-L-cysteine, with antiviral drug ribavirin synergistically reduced the lethal effect of influenza virus infection. Accumulating a number of evidence highlights a potential of selected antioxidants for treatment of severe influenza-associated complications and a possibility that combination of antioxidants with current anti-influenza drugs can improve conventional influenza chemotherapy.

Human Natural Killer Cells Are Able to Kill Aspergillus Fumigatus but Not Via the Perforin - Granzyme Pathway.

Abstract 1640Poster Board I-666 IntroductionNatural killer (NK) cells are CD3- CD56+ lymphocytes demonstrating confirmed cytotoxicity against neoplastic and virus infected host cells. Increasing data provide evidence of a direct NK cell effect against extracellular pathogens, such as bacteria, parasites and yeasts, but there is a relative lack of data on their interaction with filamentous fungus and especially with Aspergillus fumigatus. Aspergillus is an omnipresent mold, living in close vicinity with humans, being constantly inhaled in the lungs and thereafter cleared by the innate immune system. Otherwise harmless for healthy people, it is at the origin of invasive Aspergillosis (IA), an extremely devastating disease for immunocompromised subjects. Host's innate immune system controls Aspergillus growth through a complex system of potent effector cells, mediating their antifungal activity mainly by phagocytosis. Our study aims to shed light for the first time on the direct interaction between human NK cells, mediators of extracellular cytotoxicity, and Aspergillus. MethodsNK cells were isolated after magnetic depletion of the peripheral blood of healthy volunteers and they were used after 24h priming with 500 U/ml recombinant interleukin – 2 rhIL-2. To determine gene expression and cytokine release of interferon gamma (IFNg) and Tumor Necrosis Factor- a (TNF-a), NK cells were stimulated for 0, 3, 6 and 12h with different morphologies of Aspergillus: conidia and germlings. To evaluate the lethal impact of NK cells on Aspergillus, plate killing assays were performed at 0, 3 and 6h time points. To illustrate the role of antibody dependent cellular cytotoxicity, ADCC a monoclonal IgG antibody, against germlings, was tested. Transwell permeable membranes, with pores of 0,4 μm, prohibiting the direct contact of cells placed on their opposite sides, but allowing the free circulation of molecules, were used to estimate the effect of cell-fungal contact. To investigate the cytotoxic mechanism involved, NK cells were depleted from perforin and granzymes by treatment with strontium chloride and they had their death ligands, TNF- related apoptosis- inducing ligand (TRAIL) and FasL, neutralised by means of blocking antibodies. The release of cytotoxic granules was estimated by the NK cell surface expression of the marker of degranulation CD107a/b. ResultsObserving the in vitro interaction of NK cells with Aspergillus, fungal germinated morphologies (germlings) showed to be highly immunogenic towards NK cells, compared to conidia, inducing the gene expression and cytokine release of Th1 immune mediators such as IFN-g (p <0,05) and TNF-a.(p <0,1). NK cells demonstrated also a strong lethal impact against germlings (p <0,05). Moreover, the presence of antifungal antibody further potentiated both immunoregulatory and cytotoxic activities. Investigating the means engaged by NK cells to perceive and kill Aspergillus, direct effector–pathogen cell to cell contact was revealed as prerequisite; when this condition was not present there was neither cytokine induction, nor fungal damage (p <0,05). This finding was confirmed by the lack of surface expression of CD107a/b, after NK cell- Aspergillus co-incubation. Investigating the killing pathway we compared the effectiveness of perforin – granzymes depleted NK cells to this of intact cells against germlings and it was found equivalent (p =NS). In a similar way, neutralisation of TRAIL and FasL ligands did not alter the cytotoxic ability of NK cells towards Aspergillus. ConclusionOur data show that human NK cells are stimulated in vitro by Aspergillus germlings, which triggers an immunoregulatory Th1 orientated response and causes important fungal killing. NK cells are not aware of conidia, they are not stimulated by them and par consequence they do not kill them. Finally, we showed that NK cells do not mediate their cytotoxic effect via perforin – granzymes pathway, neither through the engagement of TRAIL, FasL death receptors, suggesting that another pathway is involved in NK cell – Aspergillus fumigatus interplay. We suggest that further investigation of these striking findings might offer a potent immunotherapeutic tool against IA. DisclosuresNo relevant conflicts of interest to declare.

Adoptive Immunotherapy for CMV Infection Complicating Haploidentical Transplants: Challenges Posed by the HLA Restriction of Immuno- Dominant Donor T Cells

T-cell depleted hematopoietic stem cell transplants (HSCT) from HLA haplotypedisparate related donors are being increasingly used for the treatment of patients lacking an HLA-compatible related or unrelated donor. Acute and chronic graft versus host disease can be prevented without added prophylaxis if techniques achieving extensive T-cell depletion are employed. Improved conditioning regimens and transplants providing high doses of CD34+ cells have also insured consistent engraftment. However, these patients have a higher and more prolonged risk of serious viral infections than recipients of matched unmodified or T-cell depleted grafts.Adoptive immunotherapy with donor-derived pathogen-specific T-cells can restore effective resistance in these patients. However, T-cells sensitized and propagated in vitro usually respond to only 1–2 immunodominant epitopes from any antigeneic protein presented by different donor HLA alleles. We questioned how often T-cells generated from an HLA-haplotype disparate donor would be restricted by donor-unique, rather than shared HLA alleles since such T-cells would be inactive against virus-infected cells of a haplotype disparate host. Accordingly, we tested 8 cytomegalovirus (CMV) seropositive HLA haplotype disparate parent or sibling donors, as well as 4 patients, each transplanted from one of these donors 1 or more years previously. In each case, T-cells were sensitized with autologous DCs loaded with a pool of overlapping 15-mers spanning the sequence of CMVpp65, an immunodominant protein of cytomegalovirus, for 21–28 days. Using a matrix of peptide subpools, we then mapped the epitopes eliciting IFNγ+ T-cell responses and then determined their HLA-restriction by assaying their specific cytotoxicity against a panel of EBV B-cell lines loaded with the immunogenic epitope each sharing a single HLA allele with the donor. Of the 8 donors tested, 6 responded to CMV pp65 epitopes presented by HLA alleles shared with the haplotype matched recipient, while 2 donors responded exclusively to epitopes presented by donor unique HLA alleles. Recipients of transplants from the latter two donors experienced prolonged CMV viremia, one with CMV chorioretinitis, requiring months of antiviral treatment. CMV-specific T-cells isolated from each of the four transplanted patients over 1 year post transplant were specific for epitopes presented by HLA alleles shared by their donors. In the patients transplanted from donors whose CMV-pp65 specific T-cells were restricted by donor-unique HLA alleles, the epitope targeted and HLA restriction of the engrafted T-cell differed from that of the transplant donors own T-cells, suggesting that these T-cells may have developed from donor precursors differentiating in the host thymus.Thus, in a significant proportion of cases (2/8 in this study), virus-specific T-cells generated in vitro from HLA haplotype disparate donors may be restricted by donor-unique HLA alleles and, as a consequence, inactive against virus-infected host cells. For such patients, techniques insuring selective generation of T-cells specific for epitopes presented by shared alleles should be employed. Our findings may also in part explain the prolonged susceptibility of these patients to viral infection. Since immunodominant T-cells reactive against the same epitopes are also those detected at highest frequency in the donor's blood, they would likely be the only virus-reactive T-cells transferred in a T-cell depleted graft. As a consequence, effective CMV-specific responses might be delayed until new T cells are generated from precursors developing in the host thymus.

Hepatitis C Virus Infection Sensitizes Human Hepatocytes to TRAIL-Induced Apoptosis in a Caspase 9-Dependent Manner

Apoptosis of infected cells represents a key host defense mechanism against viral infections. The impact of apoptosis on the elimination of hepatitis C virus (HCV)-infected cells is poorly understood. The TRAIL has been implicated in the death of liver cells in hepatitis-infected but not in normal liver cells. To determine the impact of TRAIL on apoptosis of virus-infected host cells, we studied TRAIL-induced apoptosis in a tissue culture model system for HCV infection. We demonstrated that HCV infection sensitizes primary human hepatocytes and Huh7.5 hepatoma cells to TRAIL induced apoptosis in a dose- and time-dependent manner. Mapping studies identified the HCV nonstructural proteins as key mediators of sensitization to TRAIL. Using a panel of inhibitors targeting different apoptosis pathways, we demonstrate that sensitization to TRAIL is caspase-9 dependent and mediated in part via the mitochondrial pathway. Sensitization of hepatocytes to TRAIL-induced apoptosis by HCV infection represents a novel antiviral host defense mechanism that may have important implications for the pathogenesis of HCV infection and may contribute to the elimination of virus-infected hepatocytes.

Multivalent Presentation of Antihantavirus Peptides on Nanoparticles Enhances Infection Blockade

Viral entry into susceptible host cells typically results from multivalent interactions between viral surface proteins and host entry receptors. In the case of Sin Nombre virus (SNV), a New World hantavirus that causes hantavirus cardiopulmonary syndrome, infection involves the interaction between viral membrane surface glycoproteins and the human integrin alpha(v)beta(3). Currently, there are no therapeutic agents available which specifically target SNV. To address this problem, we used phage display selection of cyclic nonapeptides to identify peptides that bound SNV and specifically prevented SNV infection in vitro. We synthesized cyclic nonapeptides based on peptide sequences of phage demonstrating the strongest inhibition of infection, and in all cases, the isolated peptides were less effective at blocking infection (9.0% to 27.6% inhibition) than were the same peptides presented by phage (74.0% to 82.6% inhibition). Since peptides presented by the phage were pentavalent, we determined whether the identified peptides would show greater inhibition if presented in a multivalent format. We used carboxyl linkages to conjugate selected cyclic peptides to multivalent nanoparticles and tested infection inhibition. Two of the peptides, CLVRNLAWC and CQATTARNC, showed inhibition that was improved over that of the free format when presented on nanoparticles at a 4:1 nanoparticle-to-virus ratio (9.0% to 32.5% and 27.6% to 37.6%, respectively), with CQATTARNC inhibition surpassing 50% when nanoparticles were used at a 20:1 ratio versus virus. These data illustrate that multivalent inhibitors may disrupt polyvalent protein-protein interactions, such as those utilized for viral infection of host cells, and may represent a useful therapeutic approach.

Lipids as modulators of membrane fusion mediated by viral fusion proteins

Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called “rafts”, or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 (LMP1) Induces the Expression of the Cellular MicroRNA miR-146a

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g. the “vault” RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is up-regulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NF-κB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NF-κB-response elements. Conversely, a LMP1-mutant deficient in NF-κB-activation failed to activate the promoter. The “CAO”-LMP1 variant which has an increased potential to induce NF-κB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt’s lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt’s lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells up-regulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

Expression of mRNA and proteins for toll-like receptors, associated molecules, defensins and LL-37 by SRIK-NKL, a CD8 + NK/T cell line

Natural killer (NK) cells, innate lymphocyte effectors, kill virus-infected host and tumor cells in non-MHC, non-TCR restricted fashion, unlike T cells. The role of NK cells in recognition and killing of pathogens remains unknown. Expression of the ten human pathogen associated molecular pattern or toll-like receptors (TLR's), associated molecules, and antimicrobial peptides α, β defensins, and LL-37 by NK cells has not been investigated previously. We report our CD8+ NK/T cell line SRIK-NKL, derived from leukemic phase of acute lymphoblastic lymphoma, expresses mRNA and protein for 8/10 TLR's, associated proteins for signaling, defensins, cathelicidin/LL-37, and responds to live bacteria by cell proliferation and increased IFN-γ, TNF-α, TNF-β, and MIP-1α production.