Poxvirus entry and membrane fusion

The study of poxvirus entry and membrane fusion has been invigorated by new biochemical and microscopic findings that lead to the following conclusions: (1) the surface of the mature virion (MV), whether isolated from an infected cell or by disruption of the membrane wrapper of an extracellular virion, is comprised of a single lipid membrane embedded with non-glycosylated viral proteins; (2) the MV membrane fuses with the cell membrane, allowing the core to enter the cytoplasm and initiate gene expression; (3) fusion is mediated by a newly recognized group of viral protein components of the MV membrane, which are conserved in all members of the poxvirus family; (4) the latter MV entry/fusion proteins are required for cell to cell spread necessitating the disruption of the membrane wrapper of extracellular virions prior to fusion; and furthermore (5) the same group of MV entry/fusion proteins are required for virus-induced cell-cell fusion. Future research priorities include delineation of the roles of individual entry/fusion proteins and identification of cell receptors.     

Extracellular vesicles in autoimmune vasculitis - Little dirts light the fire in blood vessels

Systemic vasculitis is diverse group of autoimmune disorders which are characterized by inflammation of blood vessel walls with deep aching and burning pain. Their underlying etiology and pathophysiology still remain poorly understood. Extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, are membrane vesicular structures that are released either during cell activation, or when cells undergo programmed cell death, including apoptosis, necroptosis, and pyroptosis. Although EVs were thought as cell dusts, but now they have been found to be potently active since they harbor bioactive molecules, such as proteins, lipids, nucleic acids, or multi-molecular complexes. EVs can serve as novel mediators for cell-to-cell communications by delivery bioactive molecules from their parental cells to the recipient cells. Earlier studies mainly focused on MVs budding from membrane surface. Recent studies demonstrated that EVs may also carry molecules from cytoplasm or even from nucleus of their parental cells, and these EVs may carry autoantigens and are important in vasculitis. EVs may play important roles in vasculitis through their potential pathogenic involvements in inflammation, autoimmune responses, procoagulation, endothelial dysfunction/damage, angiogenesis, and intimal hyperplasia. EVs have also been used as specific biomarkers for diagnostic use or disease severity monitoring. In this review, we have focused on the aspects of EV biology most relevant to the pathogenesis of vasculitis, discussed their perspective insights, and summarized the exist literature on EV relevant studies in vasculitis, therefore provides an integration of current knowledge regarding the novel role of EVs in systemic vasculitis.     

Impact of lysosome status on extracellular vesicle content and release

Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells.     

Extracellular vesicles,news about their role in immune cells: physiology,pathology and diseases

Two types of extracellular vesicles (EVs), exosomes and ectosomes, are generated and released by all cells, including immune cells. The two EVs appear different in many properties: size, mechanism and site of assembly, composition of their membranes and luminal cargoes, sites and processes of release. In functional terms, however, these differences are minor. Moreover, their binding to and effects on target cells appear similar, thus the two types are considered distinct only in a few cases, otherwise they are presented together as EVs. The EV physiology of the various immune cells differs as expected from their differential properties. Some properties, however, are common: EV release, taking place already at rest, is greatly increased upon cell stimulation; extracellular navigation occurs adjacent and at distance from the releasing cells; binding to and uptake by target cells are specific. EVs received from other immune or distinct cells govern many functions in target cells. Immune diseases in which EVs play multiple, often opposite (aggression and protection) effects, are numerous; inflammatory diseases; pathologies of various tissues; and brain diseases, such as multiple sclerosis. EVs also have effects on interactive immune and cancer cells. These effects are often distinct, promoting cytotoxicity or proliferation, the latter together with metastasis and angiogenesis. Diagnoses depend on the identification of EV biomarkers; therapies on various mechanisms such as (1) removal of aggression-inducing EVs; (2) EV manipulations specific for single targets, with insertion of surface peptides or luminal miRNAs; and (3) removal or re-expression of molecules from target cells.     

Extracellular Vesicles and Cell–Cell Communication in the Cornea

One question that has intrigued cell biologists for many years is, “How do cells interact to influence one another's activity?” The discovery of extracellular vesicles (EVs) and the fact that they carry cargo, which directs cells to undergo changes in morphology and gene expression, has revolutionized this field of research. Little is known regarding the role of EVs in the cornea; however, we have demonstrated that EVs isolated from corneal epithelial cells direct corneal keratocytes to initiate fibrosis. Intriguingly, our data suggest that EVs do not penetrate epithelial basement membrane (BM), perhaps providing a mechanism explaining the importance of BM in the lack of scarring in scrape wounds. Since over 100-million people worldwide suffer from visual impairment as a result of corneal scarring, the role of EVs may be vital to understanding the mechanisms of wound repair. Therefore, we investigated EVs in ex vivo and in vivo-like three-dimensional cultures of human corneal cells using transmission electron microscopy. Some of the major findings were all three major cell types (epithelial, fibroblast, and endothelial cells) appear to release EVs, EVs can be identified using TEM, and EVs appeared to be involved in cell–cell communication. Interestingly, while our previous publication suggests that EVs do not penetrate the epithelial BM, it appears that EVs penetrate the much thicker endothelial BM (Descemet's membrane). These findings indicate the huge potential of EV research in the cornea and wound healing, and suggest that during homeostasis the endothelium and stromal cells are in communication. Anat Rec, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Extracellular vesicles and reproduction-promotion of successful pregnancy

Extracellular vesicles （EVs） are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell-cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.     

The anti-HIV drug nelfinavir mesylate (Viracept) is a potent inhibitor of cell fusion caused by the SARSCoV-2 spike (S) glycoprotein warranting further evaluation as an antiviral against COVID-19 infections

Severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) is the causative agent of the coronavirus disease-2019 (COVID-19) pandemic. Coronaviruses enter cells via fusion of the viral envelope with the plasma membrane and/or via fusion of the viral envelope with endosomal membranes after virion endocytosis. The spike (S) glycoprotein is a major determinant of virus infectivity. Herein, we show that the transient expression of the SARS CoV-2 S glycoprotein in Vero cells caused extensive cell fusion (formation of syncytia) in comparison to limited cell fusion caused by the SARS S glycoprotein. Both S glycoproteins were detected intracellularly and on transfected Vero cell surfaces. These results are in agreement with published pathology observations of extensive syncytia formation in lung tissues of patients with COVID-19. These results suggest that SARS CoV-2 is able to spread from cell-to-cell much more efficiently than SARS effectively avoiding extracellular neutralizing antibodies. A systematic screening of several drugs including cardiac glycosides and kinase inhibitors and inhibitors of human immunodeficiency virus (HIV) entry revealed that only the FDA-approved HIV protease inhibitor, nelfinavir mesylate (Viracept) drastically inhibited S-n- and S-o-mediated cell fusion with complete inhibition at a 10-μM concentration. In-silico docking experiments suggested the possibility that nelfinavir may bind inside the S trimer structure, proximal to the S2 amino terminus directly inhibiting S-n- and S-o-mediated membrane fusion. Also, it is possible that nelfinavir may act to inhibit S proteolytic processing within cells. These results warrant further investigations of the potential of nelfinavir mesylate to inhibit virus spread at early times after SARS CoV-2 symptoms appear.     

Significance of basolateral domain of polarized MDCK cells for Sendai virus-induced cell fusion

Summary Fusion (fusion from within) of polarized MDCK monolayer cells grown on porous membranes was examined after infection with Sendai viruses. Wild-type virus, that buds at the apical membrane domain, did not induce cell fusion even when the F glycoprotein expressed at the apical domain was activated with trypsin. On the other hand, a protease activation mutant, F 1-R, with F protein in the activated form and that buds bipolarly at the apical and basolateral domains, caused syncytia formation in the absence of exogenous protease. Anti-Sendai virus antibodies added to the basolateral side, but not at the apical side, inhibited cell fusion induced by F 1-R. In addition, T-9, a mutant with bipolar budding phenotype of F 1-R but with an uncleavable F protein phenotype like wild-type virus, induced cell fusion exclusively when trypsin was added to the basolateral medium. By electron microscopy, cell-to-cell fusion was shown to occur at the lateral domain of the plasma membrane. These results indicate that in addition to proteolytic activation of the F protein, basolateral expression of Sendai virus envelope glycoproteins is required to induce cell fusion.     

The function of the neuraminidase in membrane fusion induced by myxoviruses

Interaction of cell membranes with liposomes containing the glycoproteins of myxoviruses (ortho- and paramyxoviruses) was investigated. Liposomes containing the individual or mixed viral glycoproteins were reacted with chicken embryo cells and observed for fusion by electron microscopy. It was found that the presence of neuraminidase is essential for fusion between the liposomes and cells. A hypothesis explaining the cooperative role of neuraminidase for myxovirus-induced membrane fusion is presented.     

The Uptake of Extracellular Vesicles is Affected by the Differentiation Status of Myeloid Cells

Intercellular communication includes the exchange of various membrane vesicles including exosomes. Exosomes are intraluminal nanovesicles generated from multivesicular bodies, a late endosomal compartment. Cancer cells release exosomes that influence their proximate and distant environment to facilitate angiogenesis, metastatic dissemination and immune escape. Cancer‐derived vesicles may also trigger an anti‐tumour response by transferring tumour antigens to immune cells. We wanted to investigate whether differentiation and maturation of myeloid cells changes their capacity to take up cancer‐derived extracellular vesicles (EV). We compared the efficiency of vesicle uptake by monocytes, macrophages and dendritic cells. To visualize and quantify the cellular uptake, EV were labelled with two different dyes, carboxyfluoresceine diacetate succinimidyl‐ester (CFSE), or DSSN+, a water soluble distyrylstilbene oligoelectrolyte which preferentially intercalates into the cell membrane. With the help of cytokines, THP‐1 monocytes were differentiated into immature or mature dendritic cells, or macrophages. EV uptake was monitored by flow cytometry and immunofluorescence microscopy. The results show that macrophages and mature dendritic cells acquired stronger fluorescence transferred by EV than monocytes or immature dendritic cells indicating that the differentiation status influences the efficiency of EV uptake.     

Domains of herpes simplex virus I glycoprotein B that function in virus penetration, cell-to-cell spread, and cell fusion.

Herpes simplex virus 1 glycoprotein B (gB) is one of 10 glycoproteins in the virion envelope and in the membranes of infected cells. It is required for infection of cells in culture and functions in penetration of the cell by fusing the virion envelope with the plasma membrane. In studies to map the functional domains on HSV-1 gB, we reported that epitopes of potent neutralizing antibodies cluster in three major antigenic domains, D1, D2, and D5a. D1 contains continuous epitopes in the very amino terminus of gB. D2 comprises discontinuous epitopes that are assembled on gB derivatives 457 amino acids in length. D5a contains discontinuous epitopes that map between amino acids 600 and 690. We have now analyzed the function of these domains in virion infectivity by a detailed examination of the effects of 16 neutralizing antibodies on virion adsorption, penetration, plaque development, and cell fusion. Our results are as follows. (i) Ten antibodies with complement-independent neutralizing activity blocked penetration of virions into cells but not their adsorption to the cell surface. Treating cell-bound, neutralized virus with the fusogenic agent polyethylene glycol promoted their entry into cells. (ii) Ten antibodies with complement-dependent and -independent neutralizing activity interfered with plaque development by preventing spread of virus from infected to neighboring uninfected cells. (iii) Nine neutralizing antibodies, all complement-independent, prevented cell fusion induced by strain HFEM syn. We conclude that domains mapping in three regions of gB function in penetration of virions into cells, and that most neutralizing antibodies to these domains also block cell-to-cell spread of virus and cell fusion. The findings that three complement-independent neutralizing antibodies that blocked penetration did not inhibit plaque development, and that only one of these blocked cell fusion, indicate that the cell-to-cell spread of virus and cell fusion are related processes, but not identical to the penetration function.     

Vaccinia virus antigens on the plasma membrane of infected cells. I. Viral antigens transferred from infecting virus particles and synthesized after infection

SDS-polyacrylamide gel electrophoretic analysis of plasma membranes prepared from L cells infected with radioiodinated vaccinia virus particles showed that at 2.0 hr postinfection, 125I-labeled virion polypeptides with molecular weights of 58K-60K, 32K-34K, 17K, and 12K-14K were associated with infected cell plasma membranes. By 4 hr postinfection, only the 32- to 34-kDa polypeptide, derived from infecting virus particles, could be detected on infected cell surfaces. A variety of techniques were applied to analyzing purified plasma cell membranes to define the viral antigens expressed on cell surfaces after infection, including (a) surface radioiodination of infected cells; (b) immune or Western blotting; (c) specific immunoprecipitation of viral proteins present in nonionic detergent extracts of membranes purified from [35S]methionine-labeled, virus-infected cells. It was determined that vaccinia virus-specified polypeptides with molecular weights of 78K-82K, 65K, 50K, 42K-45K, 35K-37K, 32K-34K, 30K, 20K, and 17K-18K were expressed by 3 hr postadsorption, on the plasma membranes of infected cells and were accessible to binding by exogenous antiviral antibodies. Viral antigens with molecular weights similar to those expressed on cell surfaces were secreted or shed from infected cells and could be detected in the medium harvested from virus-infected mouse L-cell cultures.     

Difference in susceptibility to MxA protein between a coxsackievirus B1 isolate and prototype,impact of serial cell culture passage

Human enteroviruses (EVs) cause a broad spectrum of acute and chronic diseases including meningitis and myocarditis. The type I interferon‐induced MxA protein has been shown to inhibit the replication of an EV, coxsackievirus B4 (CVB4), but not cardioviruses such as encephalomyocarditis virus and mengo virus, members of the Picornaviridae family. EVs consist of more than 60 distinct serotypes against which the antiviral activity of MxA was not investigated yet. The main aim of this study was to explore the antiviral activity of MxA protein against a clinical CVB1 isolate and other EV prototypes. Vero cells expressing constituvely MxA protein were infected with EVs, and the percentage of inhibiton of expression of enteroviral RNA and capsid VP1 protein was determined. Following infection of MxA‐transfected Vero cells with EVs, the expression of enteroviral RNA was inhibited by up to 99%, and that of VP1 protein by up to 85%. However, there was a difference in the percentage of MxA inhibition of EV replication between the different EV prototypes. This difference in MxA sensitivity was not due to a difference in the viral replication rates. The MxA protein was inactive against the clinical CVB1 isolate, and the replication rate of CVB1 isolate in MxA‐transfected Vero cells was higher than that in mock‐transfected Vero cells. A serial passage of the clinical CVB1 isolate and other EV prototypes resulted in an increase in their susceptibility to MxA protein. These results suggest the presence of MxA‐resistant EV variants that may escape innate immunity and cause disease. J. Med. Virol. 82:424–432, 2010. © 2010 Wiley‐Liss, Inc.     

Neutralization of animal virus infectivity by antibody

Summary Neutralization is the ability of antibody to bind to and inactivate virus infectivity under defined conditions in vitro. Most neutralizing antibodies also protect animals in vivo, but protection is more complex as it also involves interaction of antibody with cells and molecules of the innate immune system. Neutralization by antibody can be mediated by a number of different mechanisms: by aggregation of virions, destabilization of the virion structure, inhibition of virion attachment to target cells, inhibition of the fusion of the virion lipid membrane with the membrane of the host cell, inhibition of the entry of the genome of non-enveloped viruses into the cell cytoplasm, inhibition of a function of the virion core through a signal transduced by an antibody, transcytosing IgA, and binding to nascent virions to block their budding or release from the cell surface. The mechanism of neutralization is determined by the properties of both a virion epitope and the antibody that reacts with it. Further, since a virus has at least several unique epitopes sited in different locations on the virion, and since the paratope and other properties of the reacting antibody can vary, this means that a virus can be neutralized by several different mechanisms. Understanding the processes of neutralization informs the creation of modern vaccines, and gives valuable insights into virus-cell interactions.     

Experimental Adenovirus Infection of the Mouse Adrenal Gland: II. Electron Microscopic Observations

A strain of mouse adenovirus, found to have a striking tropism for the weanling mouse adrenal gland, enabled electron microscopic examination of adrenals in various stages of infection. Nucleolar hypertrophy and the successive formation of three types of inclusion bodies in association with nucleoli preceded virion production. Angular crystals of virions formed in the affected nuclei. Virus was released by lysis of nuclear membranes; rapid degeneration of cytoplasmic organelles followed. Rupture of external cell membranes released virus into the extracellular spaces where virions crossed vascular basement membranes to enter endothelial cells. Virions were also phagocytized by inflammatory cells which reentered vascular sinusoids, and by adrenal parenchymal cells. Disruption of virus-laden phagocytic vacuoles in parenchymal cells released virions into the cytoplasm. Typical viral inclusion bodies also formed in vascular endothelial cells and in inflammatory cells, but virion replication was not detected. The possibility that virus directly entered parenchymal cells through the external cell membrane without phagocytosis is discussed.     

Exosomes or microvesicles? Two kinds of extracellular vesicles with different routes to modify protozoan-host cell interaction

Parasite-host cell interaction can be modulated by a dynamic communication between extracellular vesicles (EVs). They should play key roles in cell-cell communications transferring biomolecules (miRNA, proteins, soluble factors) from one cell to another cell. While many names have been used to denominate EVs, a better comprehension to understand these vesicles is raised when we classify it according to biogenesis: originated from multivesicular bodies, named exosomes, and from plasmatic membranes, denominated microvesicles. Here, we have reviewed EV participation during the protozoan-host cell interaction and reinforced the differences and similarities between exosomes and microvesicles, suggesting different intracellular routes and functions. We also discussed perspectives to study EVs and the role of EVs in diagnosis and chemotherapies of infectious diseases.     

Extracellular multivesicular bodies in tissues affected by inflammation/repair and tumors

Extracellular vesicles (EVs) are a heterogeneous population involved in intercellular communication. Little attention has been paid to a peculiar EV type with the appearance of a multivesicular body: extracellular multivesicular body (EMVB), also termed matrix vesicle cluster/multivesicular cargo. The aim of this work is to assess the ultrastructural characteristics, participation, and tissue location of EMVBs in inflammation/repair and tumors (with physiopathological processes involving intense intercellular communication), for which representative specimens were used. The results showed several forms of EMVBs: a) mature EMVBs, made up of clusters of vesicles surrounded by a plasma membrane, b) pre-EMVBs, with protruding grouped vesicles under the cell membrane, and c) post-EMVBs, releasing their vesicles. In tissues with inflammation/repair, EMVBs were observed in vessel lumens, interstitial spaces of vessel walls (between endothelial cells, pericytes, and smooth muscle cells) and between inflammatory and stromal cells. In tumors, such as basal cell carcinoma, craniopharyngioma, syringocystoadenoma, fibrous histiocytoma, alveolar rhabdomyosarcoma, lymphomas, neuroblastoma, astrocytomas, meningiomas, and hydatiform mole, EMVBs were present in tumor gland lumens and between tumor cells. In conclusion, in numerous physiopathological processes, we contribute EMVB ultrastructural characteristics (including different forms of mature, pre- and post-EMVBs, suggesting a more efficient EV transport), location and relationship with different types of cells. Further studies are required to assess the role of EMVBs in these physiopathological conditions.     

Diagnosis of hand,foot, and mouth disease caused by EV71 and other enteroviruses by a one‐step,single tube,duplex RT‐PCR

Hand, foot, and mouth disease (HFMD) is caused mainly by enterovirus 71 (EV71) and other enteroviruses (EVs) such as Coxsackie A16 in China. EV71 infection can lead to severe clinical manifestations and even death. Other EVs, however, generally cause mild symptoms. Thus, early and accurate distinction of EV71 from other EVs for HFMD will offer significant benefits. A one‐step, single tube, duplex RT‐PCR assay is described in the present study to detect simultaneously EV71 and other EVs. The primers used for the duplex RT‐PCR underwent screening and optimization. The detection threshold was 0.001 TCID₅₀/ml for EV71 and 0.01 TCID₅₀/ml for other EVs. The positive rate of enterovirus detection in 165 clinical samples reached 68.5%, including 46.1% for EV71 and 22.4% for other EVs. Of all the severe HFMD cases, EV71 was responsible for 85.3% cases. The positive rate of EV71 fell markedly by day 8 after onset. In addition, sequencing of EV71 specific amplicons from duplex RT‐PCR revealed that C4a was the predominant subgenotype of EV71 circulating in Nanjing, China. The accuracy and reliability of the assay suggest strongly that the one‐step, single tube, duplex RT‐PCR will be useful for early diagnosis and monitoring of EV71 and other EV infections. J. Med. Virol. 84:1803–1808, 2012. © 2012 Wiley Periodicals, Inc.     

Measles virus inhibits mitogen-induced T cell proliferation but does not directly perturb the T cell activation process inside the cell.

Measles virus (MV) inhibits lymphocyte function in patients, as well as in cells infected in vitro. The proliferation of phytohemagglutinin-stimulated T lymphocytes is suppressed by in vitro MV infection, as shown by the diminished incorporation of [3H]thymidine into DNA and the reduced frequency of cells in the S phase of the cell cycle, as compared with mock-infected cells. MV infection itself, however, does not completely block DNA synthesis in infected cells, because infected T cells expressing MV antigens on the cell surface, isolated by fluorescence-activated cell sorter, could still proliferate. Northern blot analysis indicated that the expression of genes induced during T cell activation, such as those encoding interleukin 2 (IL-2), c-myc, IL-2 receptor, IL-6, c-myb, and cdc-2, was not significantly suppressed in MV-infected cells, suggesting that MV does not interfere with the T cell activation process. When anti-MV serum or carbobenzoxy-D-Phe-L-Phe-Gly, a synthetic oligopeptide known to inhibit MV-induced fusion, was added 24 hr after infection, the inhibition of T cell proliferation was reversed in a dose-dependent manner. From these results we propose a model for the inhibition of T cell proliferation by MV; MV glycoproteins expressed on the cell surface of infected cells interact with the MV receptor or other molecules on the cell membrane of adjacent T cells, which in turn affects the proliferation of those T cells.