scholarly journals Inhibition of Human Cytomegalovirus Entry into Host Cells through A Pleiotropic Small Molecule

2020 ◽  
Vol 21 (5) ◽  
pp. 1676 ◽  
Author(s):  
James Elste ◽  
Dominik Kaltenbach ◽  
Vraj R. Patel ◽  
Max T. Nguyen ◽  
Harsh Sharthiya ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Small Molecule ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Host Cells ◽  
Early Gene ◽  
Surface Receptors ◽  
Viral Spread ◽  
Multiple Receptors ◽  
Life Threatening

Human cytomegalovirus (HCMV) infections are wide-spread among the general population with manifestations ranging from asymptomatic to severe developmental disabilities in newborns and life-threatening illnesses in individuals with a compromised immune system. Nearly all current drugs suffer from one or more limitations, which emphasizes the critical need to develop new approaches and new molecules. We reasoned that a ‘poly-pharmacy’ approach relying on simultaneous binding to multiple receptors involved in HCMV entry into host cells could pave the way to a more effective therapeutic outcome. This work presents the study of a synthetic, small molecule displaying pleiotropicity of interactions as a competitive antagonist of viral or cell surface receptors including heparan sulfate proteoglycans and heparan sulfate-binding proteins, which play important roles in HCMV entry and spread. Sulfated pentagalloylglucoside (SPGG), a functional mimetic of heparan sulfate, inhibits HCMV entry into human foreskin fibroblasts and neuroepithelioma cells with high potency. At the same time, SPGG exhibits no toxicity at levels as high as 50-fold more than its inhibition potency. Interestingly, cell-ELISA assays showed downregulation in HCMV immediate-early gene 1 and 2 (IE 1&2) expression in presence of SPGG further supporting inhibition of viral entry. Finally, HCMV foci were observed to decrease significantly in the presence of SPGG suggesting impact on viral spread too. Overall, this work offers the first evidence that pleiotropicity, such as demonstrated by SPGG, may offer a new poly-therapeutic approach toward effective inhibition of HCMV.

scholarly journals Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA.

1996 ◽  
Vol 40 (9) ◽  
pp. 2004-2011 ◽  
Author(s):  
K P Anderson ◽  
M C Fox ◽  
V Brown-Driver ◽  
M J Martin ◽  
R F Azad
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Antiviral Activity ◽  
Protein Expression ◽  
Human Cytomegalovirus ◽  
Host Cells ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Virus Adsorption

ISIS 2922 is a phosphorothioate oligonucleotide that is complementary to human cytomegalovirus (CMV) immediate-early (IE) RNA and that exhibits potent and specific antiviral activity against CMV in cell culture assays. Specific assay systems were developed to separately characterize the antisense and nonantisense components of the antiviral activity mediated by ISIS 2922. In U373 cells transformed with cDNA encoding the CMV IE 55-kDa (IE55) protein, expression was inhibited at nanomolar concentrations comparable to effective concentrations in antiviral assays. The specificity of inhibition was demonstrated by using control oligonucleotides incorporating progressive base changes to destabilize oligonucleotide-RNA base pairing and by showing a lack of inhibition of the CMV IE72 product expressed from the same promoter. Inhibition of IE55 protein expression correlated with a reduction in mRNA levels consistent with an RNase H-mediated termination event. Studies with virus-infected cells demonstrated that antisense and nonantisense mechanisms contribute to the antiviral activity of ISIS 2922. Base complementarity to target RNA was important for optimal activity in antiviral assays, but base changes affecting parameters other than hybridization affinity also influenced antiviral activity. Sequence-independent inhibition of virus adsorption to host cells by phosphorothioate oligonucleotides was also observed at high concentrations. Therefore, at least three different mechanisms may contribute to the antiviral activity of ISIS 2922 in cell culture: antisense-mediated inhibition of target gene expression; nonantisense, sequence-dependent inhibition of virus replication; and sequence-independent inhibition of virus adsorption to host cells.

scholarly journals Viral Hepatitis and Iron Dysregulation: Molecular Pathways and the Role of Lactoferrin

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1997 ◽  
Author(s):  
Romina Mancinelli ◽  
Luigi Rosa ◽  
Antimo Cutone ◽  
Maria Stefania Lepanto ◽  
Antonio Franchitto ◽  
...  
Keyword(s):  
Viral Hepatitis ◽  
Viral Entry ◽  
Iron Homeostasis ◽  
Iron Chelation ◽  
Host Cells ◽  
Surface Receptors ◽  
Inflammatory Processes ◽  
Related Proteins ◽  
Binding Glycoprotein

The liver is a frontline immune site specifically designed to check and detect potential pathogens from the bloodstream to maintain a general state of immune hyporesponsiveness. One of the main functions of the liver is the regulation of iron homeostasis. The liver detects changes in systemic iron requirements and can regulate its concentration. Pathological states lead to the dysregulation of iron homeostasis which, in turn, can promote infectious and inflammatory processes. In this context, hepatic viruses deviate hepatocytes’ iron metabolism in order to better replicate. Indeed, some viruses are able to alter the expression of iron-related proteins or exploit host receptors to enter inside host cells. Lactoferrin (Lf), a multifunctional iron-binding glycoprotein belonging to the innate immunity, is endowed with potent antiviral activity, mainly related to its ability to block viral entry into host cells by interacting with viral and/or cell surface receptors. Moreover, Lf can act as an iron scavenger by both direct iron-chelation or the modulation of the main iron-related proteins. In this review, the complex interplay between viral hepatitis, iron homeostasis, and inflammation as well as the role of Lf are outlined.

scholarly journals Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 707 ◽  
Author(s):  
Susan Realegeno ◽  
Lalita Priyamvada ◽  
Amrita Kumar ◽  
Jessica B. Blackburn ◽  
Claire Hartloge ◽  
...  
Keyword(s):  
Viral Entry ◽  
Membrane Trafficking ◽  
Gene Knockout ◽  
Host Cells ◽  
Viral Fusion ◽  
Genetic Screens ◽  
Viral Spread ◽  
Cog Complex ◽  
Conserved Oligomeric Golgi ◽  
The Individual

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1–COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection.

scholarly journals The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

2020 ◽  
Author(s):  
Mohammed N.A. Siddiquey ◽  
Eric P. Schultz ◽  
Qin Yu ◽  
Diego Amendola ◽  
Giacomo Vezzani ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Opportunistic Infections ◽  
Host Cells ◽  
Efficient Production ◽  
Viral Glycoprotein ◽  
New Host ◽  
Glycoprotein H ◽  
Covalently Linked ◽  
Viral Membrane Fusion

ABSTRACTHeterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), a glycoprotein encoded by UL116 noncovalently assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. However, physiological roles for UL116 or its complex with gH remain to be identified. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of the two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Furthermore, gH/UL116 complexes comprise a substantial constituent of virions since an abundant gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is readily detected from wild-type but not UL116-null virions.Interestingly, UL116 co-immunoprecipitates with UL148, a viral ER resident glycoprotein previously shown to attenuate ER-associated degradation (ERAD) of gO, and we observe elevated levels of UL116 in UL148-null virions.Collectively, our findings suggest that UL116 may serve as a chaperone for gH to support the assembly, maturation, and incorporation of gH/gL complexes into virions.IMPORTANCEHCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients, as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH) / glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes, and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding of HCMV cell tropism as well as for the development of vaccines against the virus.

scholarly journals Fluoxetine Is a Potent Inhibitor of Coxsackievirus Replication

2012 ◽  
Vol 56 (9) ◽  
pp. 4838-4844 ◽  
Author(s):  
Jun Zuo ◽  
Kevin K. Quinn ◽  
Steve Kye ◽  
Paige Cooper ◽  
Robert Damoiseaux ◽  
...  
Keyword(s):  
Small Molecule ◽  
Viral Entry ◽  
Safety Profile ◽  
Viral Genome ◽  
Serotonin Reuptake ◽  
Potent Inhibitor ◽  
Antiviral Agent ◽  
Molecular Screening ◽  
Small Molecule Libraries ◽  
Life Threatening

ABSTRACTNo antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine did not interfere with either viral entry or translation of the viral genome. Instead, fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and protein. In view of its favorable pharmacokinetics and safety profile, fluoxetine warrants additional study as a potential antiviral agent for enterovirus infections.

scholarly journals 1524 Design and Implementation Of ICE-COVID, A Double-Blind Randomised Placebo-Controlled Trial on The Efficacy of Iota-Carrageenan Nasal and Throat Spray for Covid-19 Prophylaxis

2021 ◽  
Vol 108 (Supplement_6) ◽  
Author(s):  
J Y Lim ◽  
Z M Jessop ◽  
J A G Gibson ◽  
T H Jovic ◽  
E Combellack ◽  
...  
Keyword(s):  
Viral Entry ◽  
Controlled Trial ◽  
Host Cells ◽  
Upper Respiratory Tract ◽  
Double Blind ◽  
Edible Seaweed ◽  
Surgical Department ◽  
Surface Receptors ◽  
Tract Infections

Abstract Introduction The severity of Covid-19 infection is associated with viral load. For infection to occur, viruses including SARS-CoV-2 must first penetrate the respiratory mucus to attach to the host cell surface receptors. Iota-carrageenan (I-C), a sulphated polysaccharide extracted from red edible seaweed, has shown efficacy against a range of viruses in clinical trials, through prevention of viral entry into respiratory host cells and in-vitro activity against SARS-CoV-2. Our aim, as an academic surgical department, was to design and implement a clinical trial to investigate whether I-C nasal and throat sprays are effective in reducing the rate and severity of Covid-19 infection. Method The study is a single centre, double-blinded randomised controlled trial. Recruitment of 480 participants aged ≥18 years without previous Covid-19 infection and who have not yet been vaccinated, commenced in December 2020. Participants are randomised to either the treatment (0.12% I-C in 0.5% saline spray) or placebo (0.5% saline spray) arm and will prophylactically apply the spray to their nose and throat while completing a daily symptom tracker for a total of 10 weeks. The primary outcome is the acquisition of Covid-19 infection. Secondary outcomes include symptom type, severity and duration, subsequent familial/household Covid-19 infection and infection with non-Covid-19 upper respiratory tract infections. Discussion Our hypothesis is that I-C sprays will reduce SARS-CoV-2 attachment to the naso- and oropharyngeal mucosal epithelial cells thus reducing the effective viral infective dose. If proven effective, the self-administered prophylactic spray would have wider utility for key workers and the general population. Trial registration number NCT04590365

scholarly journals Protease, Growth Factor, and Heparanase-Mediated Syndecan-1 Shedding Leads to Enhanced HSV-1 Egress

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1748
Author(s):  
Ghadah A. Karasneh ◽  
Divya Kapoor ◽  
Navya Bellamkonda ◽  
Chandrashekhar D. Patil ◽  
Deepak Shukla
Keyword(s):  
Growth Factor ◽  
Heparan Sulfate ◽  
Cell Fusion ◽  
Viral Entry ◽  
Plaque Formation ◽  
Host Cells ◽  
Herpes Simplex Virus 1 ◽  
Extracellular Release ◽  
Infected Cells ◽  
Hsv 1

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells.

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 An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen

2020 ◽  
Author(s):  
Yao Yao ◽  
Rameshwar U. Kadam ◽  
Chang-Chun David Lee ◽  
Jordan L. Woehl ◽  
Nicholas C. Wu ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Viral Entry ◽  
Fluorescence Polarization ◽  
Influenza A ◽  
Cyclic Peptide ◽  
Host Cells ◽  
Influenza Hemagglutinin ◽  
Group 1

AbstractInfluenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs) and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low μM to nM affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 HA trimer against approximately 72,000 compounds. The crystal structure of H1/Puerto Rico/8/1934 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.SummaryInfluenza hemagglutinin (HA) glycoprotein enables viral entry into host cells and is the main target for antibodies in our immune system. While HA has now been established as a validated target for drug discovery, no FDA-approved small molecules are available that specifically prevent HA from binding host receptors or inhibit its membrane fusion activity and thus prevent infection. We therefore designed a fluorescence polarization probe to enable rapid identification of small molecules that bind to the stem fusion machinery of group 1 HAs. Application of our assay yielded a small molecule to the influenza A group 1 HA stem with antiviral efficacy.

scholarly journals The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

2021 ◽  
Author(s):  
Mohammed N. A. Siddiquey ◽  
Eric P. Schultz ◽  
Qin Yu ◽  
Diego Amendola ◽  
Giacomo Vezzani ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Opportunistic Infections ◽  
Host Cells ◽  
Efficient Production ◽  
Viral Glycoprotein ◽  
Term Infants ◽  
New Host ◽  
Glycoprotein H ◽  
Covalently Linked

Heterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), the glycoprotein UL116 assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Kifunensine, a mannosidase inhibitor that interferes with ER-associated degradation (ERAD) of terminally misfolded glycoproteins, restored levels of gH, gL and gO in UL116-null infected cells, indicating that constituents of HCMV gH complexes are unstable in the absence of UL116. Further, we find that gH/UL116 complexes are abundant in virions since a major gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is detected from WT but not UL116-null virions. Interestingly, UL116 co-immunoprecipitates with UL148, a viral ER resident glycoprotein that attenuates ERAD of gO, and we observe elevated levels of UL116 in UL148-null virions. Collectively, our findings argue that UL116 is chaperone for gH that supports the assembly, maturation, and incorporation of gH/gL complexes into virions. IMPORTANCE. HCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients, as well as in the immune-naive fetus and pre-term infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH)/glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes, and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding of HCMV cell tropism as well as for the development of vaccines against the virus.

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