scholarly journals Polyamines mediate enterovirus attachment directly and indirectly through cellular heparan sulfate synthesis.

2021 ◽  
Author(s):  
Bridget M Hulsebosch ◽  
Oreoluwa S Omoba ◽  
Natalie J LoMascolo ◽  
Bryan C. Mounce
Keyword(s):  
Heparan Sulfate ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Initiation Factor ◽  
Direct Role ◽  
Viral Attachment ◽  
Cellular Expression ◽  
Cellular Translation ◽  
Heparan Sulfates

Productive viral infection begins with attachment to a susceptible cell, and viruses have evolved complex mechanisms to attach to and subsequently enter cells. Prior to engagement with a cellular receptor, viruses frequently interact with nonspecific attachment factors that can facilitate virus-receptor interactions and viral entry. Polyamines, small positively-charged molecules abundant in mammalian cells, mediate viral attachment, though the mechanism was not fully understood. Using the Coxsackievirus B3 (CVB3) enterovirus model system, we show that polyamines mediate viral attachment both directly and indirectly. The polyamine putrescine specifically enhances viral attachment to cells depleted of polyamines. Putrescine's positive charge mediates its ability to enhance viral attachment, and polyamine analogs are less efficient at mediating viral attachment. In addition to this direct role of polyamines in attachment, polyamines facilitate the cellular expression of heparan sulfates, negatively-charged molecules found on the cell surface. In polyamine-depleted cells, heparan sulfates are depleted from the surface of cells, resulting in reduced viral attachment. We find that this is due to polyamines' role in the process of hypusination of eukaryotic initiation factor 5A, which facilitates cellular translation. These data highlight the important role of polyamines in mediating cellular attachment, as well as their function in facilitating cellular heparan sulfate synthesis.

scholarly journals Melatonin reduces oxidative damage in mouse granulosa cells via restraining JNK-dependent autophagy

Reproduction ◽  
2018 ◽  
Vol 155 (3) ◽  
pp. 307-319 ◽  
Author(s):  
Yan Cao ◽  
Ming Shen ◽  
Yi Jiang ◽  
Shao-chen Sun ◽  
Honglin Liu
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Mammalian Cells ◽  
Therapeutic Strategies ◽  
Oxygen Species ◽  
Protective Effects ◽  
Follicular Atresia ◽  
Direct Role ◽  
Jnk Pathway

Oxidative stress-induced granulosa cell (GCs) injury is believed to be a common trigger for follicular atresia. Emerging evidence indicates that excessive autophagy occurs in mammalian cells with oxidative damage. N-acetyl-5-methoxytrypamine (melatonin) has been shown to prevent GCs from oxidative injury, although the exact mechanism remains to be elucidated. Here, we first demonstrated that the suppression of autophagy through the JNK/BCL-2/BECN1 signaling is engaged in melatonin-mediated GCs protection against oxidative damage. Melatonin inhibited the loss of GCs viability, formation of GFP-MAP1LC3B puncta, accumulation of MAP1LC3B-II blots, degradation of SQSTM1 and the expression of BECN1, which was correlated with impaired activation of JNK during oxidative stress. On the other hand, blocking of autophagy and/or JNK also reduced the level of H2O2-induced GCs death, but failed to further restore GCs viability in the presence of melatonin. Particularly, the suppression of autophagy provided no additional protective effects when GCs were pretreated with JNK inhibitor and/or melatonin. Importantly, we found that the enhanced interaction between BCL-2 and BECN1 might be a responsive mechanism for autophagy suppression via the melatonin/JNK pathway. Moreover, blocking the downstream antioxidant system of melatonin using specific inhibitors further confirmed a direct role of melatonin/JNK/autophagy axis in preserving GCs survival without scavenging reactive oxygen species (ROS). Taken together, our findings uncover a novel function of melatonin in preventing GCs from oxidative damage by targeting JNK-mediated autophagy, which might contribute to develop therapeutic strategies for patients with ovulation failure-related disorders.

scholarly journals The Golgi-associated Protein GRASP65 Regulates Spindle Dynamics and Is Essential for Cell Division

2005 ◽  
Vol 16 (7) ◽  
pp. 3211-3222 ◽  
Author(s):  
Christine Sütterlin ◽  
Roman Polishchuk ◽  
Matt Pecot ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Direct Role ◽  
Golgi Stack ◽  
Spindle Poles ◽  
C Terminus ◽  
Spindle Dynamics ◽  
Negative Role ◽  
Golgi Fragmentation

At the onset of mitosis, the pericentriolar Golgi apparatus of mammalian cells is converted into small fragments, which are dispersed throughout the cytosol. The Golgi-associated protein GRASP65 is involved in this process. To address the role of GRASP65 in mitotic Golgi fragmentation, we depleted the protein from HeLa cells by RNAi. In the absence of GRASP65, the number of cisternae per Golgi stack is reduced without affecting the overall organization of Golgi membranes and protein transport. GRASP65-depleted cells entered mitosis, but accumulated in metaphase with condensed chromatin and multiple aberrant spindles and eventually died. Although Centrin2 and g-tubulin were detected in two of the spindle poles, the other spindle poles contained g-tubulin, but not Centrin2. Furthermore, we provide evidence that the expression of the C-terminus of GRASP65 interferes with entry of cells into mitosis. Our results suggest the requirement for GRASP65 in the regulation of spindle dynamics rather than a direct role in the stacking of Golgi cisternae. This novel function is in addition to the previously established negative role of GRASP65 at the G2/M transition, which is mediated by its C-terminus.

scholarly journals Detection of Cell-Cell Fusion Mediated by Ebola Virus Glycoproteins

2006 ◽  
Vol 80 (6) ◽  
pp. 2815-2822 ◽  
Author(s):  
Séverine Bär ◽  
Ayato Takada ◽  
Yoshihiro Kawaoka ◽  
Marc Alizon
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Ebola Virus ◽  
Low Ph ◽  
Viral Envelope ◽  
Target Cells ◽  
Direct Role ◽  
Influenza Virus Hemagglutinin ◽  
Ph Dependent

ABSTRACT Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a β-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.

Aminopeptidase N is an entry co-factor triggering porcine deltacoronavirus entry via an endocytotic pathway

2021 ◽  
Author(s):  
Yue-Lin Yang ◽  
Jianbo Liu ◽  
Tong-Yun Wang ◽  
Meng Chen ◽  
Gang Wang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Viral Entry ◽  
Aminopeptidase N ◽  
Swine Industry ◽  
Potential Threat ◽  
Viral Attachment ◽  
Efficient Replication ◽  
Entry Assay ◽  
Functional Receptor

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the role of porcine APN (pAPN) during PDCoV infection of non-susceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter non-susceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly co-localized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters non-susceptible cells, but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate anti-viral drugs.

scholarly journals mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor

2001 ◽  
Vol 75 (21) ◽  
pp. 10272-10280 ◽  
Author(s):  
Pinghui Feng ◽  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrnas ◽  
Cellular Translation ◽  
Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

scholarly journals HEPARAN SULFATES EXPRESSION IN SURGERY MATERIAL OF OVARIAN ENDOMETRIAL CYSTS

2020 ◽  
Vol 76 (4) ◽  
pp. 96-99
Author(s):  
S.V. Aidagulova ◽  
◽  
Yu.S. Timofeeva ◽  
A.V. Volchek ◽  
V.M. Kuleshov ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycans ◽  
Simultaneous Increase ◽  
Ovarian Endometriosis ◽  
Core Proteins ◽  
Heparan Sulfates ◽  
Pathological Tissues

The expression of heparan sulfate proteoglycans in eutopic and heterotopic endometrium in patients with ovarian endometriosis was studied using the method of immunohistochemistry. The simultaneous increase in the level of expression of core proteins of all studied heparan sulfate proteoglycans and the content of heparan sulfate in pathological tissues indicates the important role of glycosylated molecules in the pathogenesis of ovarian endometriosis.

Metabolic engineering of mammalian cells to produce heparan sulfates

2018 ◽  
Vol 2 (3) ◽  
pp. 443-452 ◽  
Author(s):  
Bryan E. Thacker ◽  
Susan T. Sharfstein
Keyword(s):  
Metabolic Engineering ◽  
Heparan Sulfate ◽  
Genetic Modification ◽  
Mammalian Cells ◽  
Biosynthetic Pathway ◽  
Therapeutic Benefits ◽  
Challenges And Opportunities ◽  
Heparan Sulfates

Heparan sulfate (HS) is a glycosaminoglycan produced by all mammalian cells that plays important roles in physiology and various pathologies. Heparin is a highly sulfated form of HS that is used clinically as an anticoagulant. Heparin and HSs may also have therapeutic benefits for a wide variety of other indications. Cultured mammalian cells produce HS and, through genetic modification, have been used to elucidate the biosynthetic pathway. Recently, metabolic engineering has been used to produce HS from cultured mammalian cells for clinical purposes. This review describes the HS biosynthetic pathway and its manipulation through metabolic engineering to produce bioengineered HSs. We also discuss current challenges and opportunities to advance the field of HS metabolic engineering.

scholarly journals Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2

2020 ◽  
Author(s):  
Asif Shajahan ◽  
Stephanie Archer-Hartmann ◽  
Nitin T. Supekar ◽  
Anne S. Gleinich ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Viral Entry ◽  
Glycosylation Site ◽  
Converting Enzyme ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Viral Binding ◽  
Glycosylation Sites

AbstractThe emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 human cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.

scholarly journals Protamine Sulfate Is a Potent Inhibitor of Human Papillomavirus InfectionIn Vitro and In Vivo

2021 ◽  
Author(s):  
Jesse M. Young ◽  
Amira Zine El Abidine ◽  
Ricardo A. Gómez-Martinez ◽  
Virginie Bondu ◽  
Rosa T. Sterk ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Close Contact ◽  
Hpv Infection ◽  
Protamine Sulfate ◽  
Vaccine Uptake ◽  
Host Cells ◽  
Viral Attachment

Human papillomavirus (HPV) infections are transmitted through sexual or other close contact and are etiologically associated with epithelial warts, papillomas, and intraepithelial lesions that may progress to cancer. Indeed, 4.8% of the global cancer burden is linked to HPV infection. Highly effective vaccines protect against two to nine of the most medically important HPV genotypes; yet vaccine uptake is inadequate and/or cost prohibitive in many settings. With HPV-related cancer incidence expected to rise over the coming decades, there is a need for effective HPV microbicides. Herein we demonstrate the strong inhibitory activity of the heparin-neutralizing drug protamine sulfate (PS) against HPV infection. Pretreatment of cells with PS greatly reduced infection regardless of HPV genotype or virus source. Vaginal application of PS prevented infection of the murine genital tract by HPV pseudovirions. Time-of-addition assays where PS was added to cells before infection, during infection, or after viral attachment demonstrated strong inhibitory activities on early infection steps. No effect on virus infection was found for cell lines deficient in heparan sulfate expression, suggesting that PS binds to heparan sulfate on the cell surface. Consistent with this, prophylactic PS exposure prevented viral attachment, including under low pH conditions akin to the human vaginal tract. Our findings suggest PS acts dually to prevent HPV infection: prophylactic treatment prevents HPV attachment to host cells and post-attachment administration alters viral entry. Clinical trials are warranted to determine whether protamine-based products are effective as topical microbicides against genital HPVs.

scholarly journals Sulfonated and Carboxymethylated β-Glucan Derivatives with Inhibitory Activity against Herpes and Dengue Viruses

2021 ◽  
Vol 22 (20) ◽  
pp. 11013
Author(s):  
José Louzinho Lopes ◽  
Vinicius Seiki Takemura Quinteiro ◽  
Jéssica Wouk ◽  
Maria Laura Darido ◽  
Robert F. H. Dekker ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Heparan Sulfate ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Detrimental Effect ◽  
Viral Envelope ◽  
Anionic Polysaccharides ◽  
Intermediate Degree ◽  
Simplex Virus ◽  
Hsv 1

The infection of mammalian cells by enveloped viruses is triggered by the interaction of viral envelope glycoproteins with the glycosaminoglycan, heparan sulfate. By mimicking this carbohydrate, some anionic polysaccharides can block this interaction and inhibit viral entry and infection. As heparan sulfate carries both carboxyl and sulfate groups, this work focused on the derivatization of a (1→3)(1→6)-β-D-glucan, botryosphaeran, with these negatively-charged groups in an attempt to improve its antiviral activity. Carboxyl and sulfonate groups were introduced by carboxymethylation and sulfonylation reactions, respectively. Three derivatives with the same degree of carboxymethylation (0.9) and different degrees of sulfonation (0.1; 0.2; 0.4) were obtained. All derivatives were chemically characterized and evaluated for their antiviral activity against herpes (HSV-1, strains KOS and AR) and dengue (DENV-2) viruses. Carboxymethylated botryosphaeran did not inhibit the viruses, while all sulfonated-carboxymethylated derivatives were able to inhibit HSV-1. DENV-2 was inhibited only by one of these derivatives with an intermediate degree of sulfonation (0.2), demonstrating that the dengue virus is more resistant to anionic β-D-glucans than the Herpes simplex virus. By comparison with a previous study on the antiviral activity of sulfonated botryosphaerans, we conclude that the presence of carboxymethyl groups might have a detrimental effect on antiviral activity.

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