scholarly journals Phosphatidylserine within the Viral Membrane Enhances Chikungunya Virus Infectivity in a Cell-type Dependent Manner

2022 ◽  
Kerri L Miazgowicz ◽  
Judith Mary Reyes Ballista ◽  
Marissa D Acciani ◽  
Ariana R Jimenez ◽  
Ryan S Belloli ◽  

Chikungunya virus (CHIKV), an alphavirus of the Togaviridae family, is the causative agent of the human disease chikungunya fever (CHIKF), which is characterized by debilitating acute and chronic arthralgia. No licensed vaccines or antivirals exist for CHIKV. Preventing the attachment of viral particles to host cells is an attractive intervention strategy. Viral entry of enveloped viruses from diverse families including Filoviridae and Flaviviridae is mediated or enhanced by phosphatidylserine receptors (PSRs). PSRs facilitate the attachment of enveloped viruses to cells by binding to exposed phosphatidylserine (PS) in the viral lipid membrane - a process termed viral apoptotic mimicry. To investigate the role of viral apoptotic mimicry during CHIKV infection, we produced viral particles with discrete amounts of exposed PS on the virion envelope by exploiting the cellular distribution of phospholipids at the plasma membrane. We found that CHIKV particles containing high outer leaflet PS (produced in cells lacking flippase activity) were more infectious in Vero cells than particles containing low levels of outer leaflet PS (produced in cells lacking scramblase activity). However, the same viral particles were similarly infectious in NIH3T3 and HAP1 cells, suggesting PS levels can influence infectivity only in cells with high levels of PSRs. Interestingly, PS-dependent CHIKV entry was observed in mosquito Aag2 cells, but not C6/36 cells. These data demonstrate that CHIKV entry via viral apoptotic mimicry is cell-type dependent. Furthermore, viral apoptotic mimicry has a mechanistic basis to influence viral dynamics in vivo in both the human and mosquito host.

2021 ◽  
Vol 17 (8) ◽  
pp. e1009803
Dipanwita Mitra ◽  
Mohammad H. Hasan ◽  
John T. Bates ◽  
Michael A. Bierdeman ◽  
Dallas R. Ederer ◽  

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.

2020 ◽  
Vol 94 (17) ◽  
Kevin Furlong ◽  
Scott B. Biering ◽  
Jayoung Choi ◽  
Craig B. Wilen ◽  
Robert C. Orchard ◽  

ABSTRACT Human norovirus is the leading cause of gastroenteritis worldwide, yet basic questions about its life cycle remain unanswered due to an historical lack of robust experimental systems. Recent studies on the closely related murine norovirus (MNV) have identified CD300LF as an indispensable entry factor for MNV. We compared the MNV susceptibilities of cells from different mouse strains and identified polymorphisms in murine CD300LF which are critical for its function as an MNV receptor. Bone marrow-derived macrophages (BMDMs) from I/LnJ mice were resistant to infection from multiple MNV strains which readily infect BMDMs from C57BL/6J mice. The resistance of I/LnJ BMDMs was specific to MNV, since the cells supported infection of other viruses comparably to C57BL/6J BMDMs. Transduction of I/LnJ BMDMs with C57BL/6J CD300LF made the cells permissible to MNV infection, suggesting that the cause of resistance lies in the entry step of MNV infection. In fact, we mapped this phenotype to a 4-amino-acid difference at the CC′ loop of CD300LF; swapping of these amino acids between C57BL/6J and I/LnJ CD300LF proteins made the mutant C57BL/6J CD300LF functionally impaired and the corresponding mutant of I/LnJ CD300LF functional as an MNV entry factor. Surprisingly, expression of the I/LnJ CD300LF in other cell types made the cells infectible by MNV, even though the I/LnJ allele did not function as an MNV receptor in macrophage-like cells. Correspondingly, I/LnJ CD300LF bound MNV virions in permissive cells but not in nonpermissive cells. Collectively, our data suggest the existence of a cell type-specific modifier of MNV entry. IMPORTANCE MNV is a prevalent model system for studying human norovirus, which is the leading cause of gastroenteritis worldwide and thus a sizeable public health burden. Elucidating mechanisms underlying susceptibility of host cells to MNV infection can lead to insights on the roles that specific cell types play during norovirus pathogenesis. Here, we show that different alleles of the proteinaceous receptor for MNV, CD300LF, function in a cell type-dependent manner. In contrast to the C57BL/6J allele, which functions as an MNV entry factor in all tested cell types, including human cells, I/LnJ CD300LF does not function as an MNV entry factor in macrophage-like cells but does allow MNV entry in other cell types. Together, these observations indicate the existence of cell type-specific modifiers of CD300LF-dependent MNV entry.

2003 ◽  
Vol 77 (23) ◽  
pp. 12543-12551 ◽  
Xiangjie Sun ◽  
Gary R. Whittaker

ABSTRACT Enveloped viruses are highly dependent on their lipid envelopes for entry into and infection of host cells. Here, we have examined the role of cholesterol in the virus envelope, using methyl-β-cyclodextrin depletion. Pretreatment of virions with methyl-β-cyclodextrin efficiently depleted envelope cholesterol from influenza virus and significantly reduced virus infectivity in a dose-dependent manner. A nonenveloped virus, simian virus 40, was not affected by methyl-β-cyclodextrin treatment. In the case of influenza virus, infectivity could be partially rescued by the addition of exogenous cholesterol. Influenza virus morphology, binding, and internalization were not affected by methyl-β-cyclodextrin depletion, whereas envelope cholesterol depletion markedly affected influenza virus fusion, as measured by a specific reduction in the infectivity of viruses induced to fuse at the cell surface and by fluorescence-dequenching assays. These data suggest that envelope cholesterol is a critical factor in the fusion process of influenza virus.

2020 ◽  
Vol 11 ◽  
Débora Moraes de Oliveira ◽  
Igor de Andrade Santos ◽  
Daniel Oliveira Silva Martins ◽  
Yasmim Garcia Gonçalves ◽  
Léia Cardoso-Sousa ◽  

Chikungunya fever is a disease caused by the Chikungunya virus (CHIKV) that is transmitted by the bite of the female of Aedes sp. mosquito. The symptoms include fever, muscle aches, skin rash, and severe joint pains. The disease may develop into a chronic condition and joint pain for months or years. Currently, there is no effective antiviral treatment against CHIKV infection. Treatments based on natural compounds have been widely studied, as many drugs were produced by using natural molecules and their derivatives. Alpha-phellandrene (α-Phe) is a naturally occurring organic compound that is a ligand for ruthenium, forming the organometallic complex [Ru2Cl4(p-cymene)2] (RcP). Organometallic complexes have shown promising as candidate molecules to a new generation of compounds that presented relevant biological properties, however, there is a lack of knowledge concerning the anti-CHIKV activity of these complexes. The present work evaluated the effects of the RcP and its precursors, the hydrate ruthenium(III) chloride salt (RuCl3⋅xH2O) (Ru) and α-Phe, on CHIKV infection in vitro. To this, BHK-21 cells were infected with CHIKV-nanoluciferase (CHIKV-nanoluc), a viral construct harboring the nanoluciferase reporter gene, at the presence or absence of the compounds for 16 h. Cytotoxicity and impact on infectivity were analyzed. The results demonstrated that RcP exhibited a strong therapeutic potential judged by the selective index > 40. Antiviral effects of RcP on different stages of the CHIKV replicative cycle were investigated; the results showed that it affected early stages of virus infection reducing virus replication by 77% at non-cytotoxic concentrations. Further assays demonstrated the virucidal activity of the compound that completely blocked virus infectivity. In silico molecular docking calculations suggested different binding interactions between aromatic rings of RcP and the loop of amino acids of the E2 envelope CHIKV glycoprotein mainly through hydrophobic interactions. Additionally, infrared spectroscopy spectral analysis indicated interactions of RcP with CHIKV glycoproteins. These data suggest that RcP may act on CHIKV particles, disrupting virus entry to the host cells. Therefore, RcP may represent a strong candidate for the development of anti-CHIKV drugs.

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 206 ◽  
Corina Stewart ◽  
Stephanie Dorion ◽  
Marie Ottenbrite ◽  
Nicholas LeBlond ◽  
Tyler Smith ◽  

Filoviruses, such as Ebola virus (EBOV) and Marburg virus, are causative agents of unpredictable outbreaks of severe hemorrhagic fevers in humans and non-human primates. For infection, filoviral particles need to be internalized and delivered to intracellular vesicles containing cathepsin proteases and the viral receptor Niemann-Pick C1. Previous studies have shown that EBOV triggers macropinocytosis of the viral particles in a glycoprotein (GP)-dependent manner, but the molecular events required for filovirus internalization remain mostly unknown. Here we report that the diacylglycerol kinase inhibitor, R-59-022, blocks EBOV GP-mediated entry into Vero cells and bone marrow-derived macrophages. Investigation of the mode of action of the inhibitor revealed that it blocked an early step in entry, more specifically, the internalization of the viral particles via macropinocytosis. Finally, R-59-022 blocked viral entry mediated by a panel of pathogenic filovirus GPs and inhibited growth of replicative Ebola virus. Taken together, our studies suggest that R-59-022 could be used as a tool to investigate macropinocytic uptake of filoviruses and could be a starting point for the development of pan-filoviral therapeutics.

2005 ◽  
Vol 79 (17) ◽  
pp. 10852-10863 ◽  
Reyes R. Novoa ◽  
Gloria Calderita ◽  
Pilar Cabezas ◽  
Richard M. Elliott ◽  
Cristina Risco

ABSTRACT Several complex enveloped viruses assemble in the membranes of the secretory pathway, such as the Golgi apparatus. Among them, bunyaviruses form immature viral particles that change their structure in a trans-Golgi-dependent manner. To identify key Golgi factors for viral structural maturation, we have purified and characterized the three viral forms assembled in infected cells, two intracellular intermediates and the extracellular mature virion. The first viral form is a pleomorphic structure with fully endo-β-N-acetylglucosaminidase H (Endo-H)-sensitive, nonsialylated glycoproteins. The second viral intermediate is a structure with hexagonal and pentagonal contours and partially Endo-H-resistant glycoproteins. Sialic acid is incorporated into the small glycoprotein of this second viral form. Growing the virus in glycosylation-deficient cells confirmed that acquisition of Endo-H resistance but not sialylation is critical for the trans-Golgi-dependent structural maturation and release of mature viruses. Conformational changes in viral glycoproteins triggered by changes in sugar composition would then induce the assembly of a compact viral particle of angular contours. These structures would be competent for the second maturation step, taking place during exit from cells, that originates fully infectious virions.

H.M. Mazzone ◽  
W.F. Engler ◽  
G. Wray ◽  
A. Szirmae ◽  
J. Conroy ◽  

Viral inclusion bodies isolated from infected pest insects are being evaluated by the U.S. Dept. of Agriculture as biological insecticides against their hosts. Our research on these inclusion bodies constitutes part of an effort to support their approval by the Environmental Protection Agency as insect control agents. The inclusion bodies in this study are polyhedral in shape and contain rod-shaped viral particles. When ingested by pest insects, the inclusion bodies are broken down in the insect gut and release the viral particles which infect and multiply in the nuclei of host cells. These viruses are termed nucleopolyhedrosis viruses (NPV) and are representatives of the baculoviruses (Wildy, P. 1971 IN J.L. Melnick, ed., Monographs in Virology, vol. 5, S.Karger, New York).

2021 ◽  
Vol 22 (14) ◽  
pp. 7669
Cassio Luiz Coutinho Almeida-da-Silva ◽  
Harmony Matshik Dakafay ◽  
Kaitlyn Liu ◽  
David M. Ojcius

A large body of evidence shows the harmful effects of cigarette smoke to oral and systemic health. More recently, a link between smoking and susceptibility to coronavirus disease 2019 (COVID-19) was proposed. COVID-19 is due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which uses the receptor ACE2 and the protease TMPRSS2 for entry into host cells, thereby infecting cells of the respiratory tract and the oral cavity. Here, we examined the effects of cigarette smoke on the expression of SARS-CoV-2 receptors and infection in human gingival epithelial cells (GECs). We found that cigarette smoke condensates (CSC) upregulated ACE2 and TMPRSS2 expression in GECs, and that CSC activated aryl hydrocarbon receptor (AhR) signaling in the oral cells. ACE2 was known to mediate SARS-CoV-2 internalization, and we demonstrate that CSC treatment potentiated the internalization of SARS-CoV-2 pseudovirus in GECs in an AhR-dependent manner. AhR depletion using small interference RNA decreased SARS-CoV-2 pseudovirus internalization in CSC-treated GECs compared with control GECs. Our study reveals that cigarette smoke upregulates SARS-CoV-2 receptor expression and infection in oral cells. Understanding the mechanisms involved in SARS-CoV-2 infection in cells of the oral cavity may suggest therapeutic interventions for preventing viral infection and transmission.

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1320
Yogesh B Narkhede ◽  
Karen J Gonzalez ◽  
Eva-Maria Strauch

The emergence of novel viral infections of zoonotic origin and mutations of existing human pathogenic viruses represent a serious concern for public health. It warrants the establishment of better interventions and protective therapies to combat the virus and prevent its spread. Surface glycoproteins catalyzing the fusion of viral particles and host cells have proven to be an excellent target for antivirals as well as vaccines. This review focuses on recent advances for computational structure-based design of antivirals and vaccines targeting viral fusion machinery to control seasonal and emerging respiratory viruses.

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

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