Anti-SARS-CoV-2 and anticoagulant properties of Pentacta pygmaea fucosylated chondroitin sulfate depend on high molecular weight structures.

Fucosylated chondroitin sulfate (FucCS) is a unique marine glycosaminoglycan that exhibits diverse biological functions, including antiviral and anticoagulant activity. In previous work, the FucCS derived from Pentacta pygmaea (PpFucCS) showed moderate anticoagulant effect but high inhibitory activity against the Wuhan strain of severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this study, we perform free-radical depolymerization of PpFucCS by the copper-based Fenton method to generate low molecular weight (MW) oligosaccharides. PpFucCS oligosaccharides were structurally analyzed by 1H nuclear magnetic resonance spectroscopy and were used to conduct structure-activity relationship studies regarding their effects against SARS-CoV-2 and clotting. Anticoagulant properties were measured by activated partial thromboplastin time, protease (factors Xa and IIa) inhibition by serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]), and competitive surface plasmon resonance (SPR) assay using AT, HCII, and IIa. Anti-SARS-CoV-2 properties were measured by the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 Delta variant spike (S)-protein and competitive SPR assays using multiple S-proteins (Wuhan, N501Y [Alpha], K417T/E484K/N501Y [Gamma], L542R [Delta], and Omicron [BA.2 subvariant]). Cytotoxicity of native PpFucCS and oligosaccharides was also assessed. The PpFucCS-derived oligosaccharide fraction of the highest MW showed great anti-SARS-CoV-2 Delta activity and reduced anticoagulant properties. Results have indicated no cytotoxicity and MW dependency on both anti-SARS-CoV-2 and anticoagulant effects of PpFucCS, as both actions were reduced accordingly to the MW decrease of PpFucCS. Our results demonstrate that the high-MW structures of PpFucCS is a key structural element to achieve the maximal anti-SARS-CoV-2 and anticoagulant effects.

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells.

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.

The Sea Cucumber Thyonella gemmata Contains a Low Anticoagulant Sulfated Fucan with High Anti-SARS-CoV-2 Actions against Wild-Type and Delta Variants.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-ß-N-acetylgalactosamine-(1→4)-ß-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection.

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10-60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-ß-GalNAcX-(1→4)-ß-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3-. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.

Inhibition of SARS-CoV-2 wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) strains by marine sulfated glycans.

The Coronavirus disease pandemic has steered the global therapeutic research efforts toward the discovery of potential anti-severe acute respiratory syndrome coronavirus (SARS-CoV-2) molecules. The role of the viral spike glycoprotein (S-protein) has been clearly established in SARS-CoV-2 infection through its capacity to bind to the host cell surface heparan sulfate proteoglycan (HSPG) and angiotensin-converting enzyme-2. The antiviral strategies targeting these 2 virus receptors are currently under intense investigation. However, the rapid evolution of the SARS-CoV-2 genome has resulted in numerous mutations in the S-protein posing a significant challenge for the design of S-protein-targeted inhibitors. As an example, the 2 key mutations in the S-protein receptor-binding domain (RBD), L452R, and T478K in the SARS-CoV-2 Delta variant (B.1.617.2) confer tighter binding to the host epithelial cells. Marine sulfated glycans (MSGs) demonstrate excellent inhibitory activity against SARS-CoV-2 via competitive disruption of the S-protein RBD-HSPG interactions and thus have the potential to be developed into effective prophylactic and therapeutic molecules. In this study, 7 different MSGs were evaluated for their anti-SARS-CoV-2 activity in a virus entry assay utilizing a SARS-CoV-2 pseudovirus coated with S-protein of the wild-type (Wuhan-Hu-1) or the Delta (B.1.617.2) strain. Although all tested MSGs showed strong inhibitory activity against both strains, no correlations between MSG structural features and virus inhibition could be drawn. Nevertheless, the current study provides evidence for the maintenance of inhibitory activity of MSGs against evolving SARS-CoV-2 strains.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Safety and Pharmacokinetics of Intranasally Administered Heparin.

PURPOSE: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. METHODS: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 µg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. RESULTS: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 h, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 h. CONCLUSIONS: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 h after dosing.

Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Diabetes and COVID-19 risk: an miRNA perspective.

Coronavirus disease 2019 (COVID-19) and diabetes outcomes (CORONADO) trial revealed that 10.6% of patients with diabetes mellitus hospitalized for COVID-19 (COVID-19) die within 7 days. Several studies from New York, Italy, and China confirm that patients with diabetes are at a much higher risk for mortality due to COVID-19. Besides respiratory illness, COVID-19 increases cardiac injury and diabetic ketoacidosis. In the absence of specific guidelines for the prevention and treatment of COVID-19 for patients with diabetes, they remain at higher risk and are more susceptible to COVID-19. Furthermore, there is a scarcity of basic knowledge on how diabetes affects pathogenesis of severe acute respiratory coronavirus (SARS-CoV-2) infection. In patients with diabetes, impaired glucose use alters metabolic and consequently biological processes instigating pathological remodeling, which has detrimental effects on cardiovascular systems. A majority of biological processes are regulated by noncoding microRNAs (miRNAs), which have emerged as a promising therapeutic candidate for several diseases. In consideration of the higher risk of mortality in patients with diabetes and COVID-19, novel diagnostic test and treatment strategy are urgently warranted in post-COVID-19 era. Here, we describe potential roles of miRNA as a biomarker and therapeutic candidate, especially for heart failure, in patients with diabetes and COVID-19.

Inhibition of Human Cytomegalovirus Entry into Host Cells Through a Pleiotropic Small Molecule.

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.

Dynamin Is Required for Efficient Cytomegalovirus Maturation and Envelopment.

Cytomegalovirus secondary envelopment occurs in a virus-induced cytoplasmic assembly compartment (vAC) generated via a drastic reorganization of the membranes of the secretory and endocytic systems. Dynamin is a eukaryotic GTPase that is implicated in membrane remodeling and endocytic membrane fission events; however, the role of dynamin in cellular trafficking of viruses beyond virus entry is only partially understood. Mouse embryonic fibroblasts (MEF) engineered to excise all three isoforms of dynamin were infected with mouse cytomegalovirus (MCMV-K181). Immediate-early (IE1; m123) viral protein was detected in these triple dynamin knockout (TKO) cells, as well as in mock-induced parental MEF, at early times postinfection, although levels were reduced in TKO cells, indicating that virus entry was affected but not eliminated. Levels of IE1 protein and another viral early protein (m04) were normalized by 48 h postinfection; however, late protein (m55; gB) expression was reduced in infected TKO cells compared to parental MEF. Ultrastructural analysis revealed intact stages of nuclear virus maturation in both cases with equivalent numbers of nucleocapsids containing packaged viral DNA (C-capsids), indicating successful viral DNA replication, capsid assembly, and genome packaging. Most importantly, severe defects in virus envelopment were visualized in TKO cells but not in parental cells. Dynamin inhibitor (dynasore)-treated MEF showed a phenotype similar to TKO cells upon mouse cytomegalovirus infection, confirming the role of dynamin in late maturation processes. In summary, dynamin-mediated endocytic pathways are critical for the completion of cytoplasmic stages of cytomegalovirus maturation.IMPORTANCE Viruses are known to exploit specific cellular functions at different stages of their life cycle in order to replicate, avoid immune recognition by the host and to establish a successful infection. Cytomegalovirus (CMV)-infected cells are characterized by a prominent cytoplasmic inclusion (virus assembly compartment [vAC]) that is the site of virus maturation and envelopment. While endocytic membranes are known to be the functional components of vAC, knowledge of specific endocytic pathways implicated in CMV maturation and envelopment is lacking. We show here that dynamin, which is an integral part of host endocytic machinery, is largely dispensable for early stages of CMV infection but is required at a late stage of CMV maturation. Studies on dynamin function in CMV infection will help us understand the host-virus interaction pathways amenable to targeting by conventional small molecules, as well as by newer generation nucleotide-based therapeutics (e.g., small interfering RNA, CRISPR/CAS gRNA, etc.).

Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline-peroxides against Human Cytomegalovirus and Glioblastoma ∥.

The association between glioblastoma (GBM) and human cytomegalovirus (HCMV) infection has been the intensely debated topic over the decades for developing new therapeutic options. In this regard, the peroxides from natural and synthetic sources served as potential antiviral and anticancer agents in the past. Herein, a concise and efficient strategy has been demonstrated to access a novel class of peroxides containing a spiro-isoxazoline to primarily investigate the biological activities. The synthetic compounds were evaluated for in vitro antiviral and antiproliferative activity against HCMV and glioblastoma cell line (GBM6), respectively. While compound 13m showed moderate anti-CMV activity (IC50 = 19 µM), surprisingly, an independent biological assay for compound 13m revealed its antiproliferative activity against the human glioblastoma cell line (GBM6) with an IC50 of 10 µM. Hence, the unification of an isoxazoline and peroxide heterocycles could be a potential direction to initiate the HCMV-GBM drug discovery program.

Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress.

UNLABELLED: Human cytomegalovirus (HCMV) pUL93 and pUL77 are both essential for virus growth, but their functions in the virus life cycle remain mostly unresolved. Homologs of pUL93 and pUL77 in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) are known to interact to form a complex at capsid vertices known as the capsid vertex-specific component (CVSC), which likely stabilizes nucleocapsids during virus maturation and also aids in nuclear egress. In herpesviruses, nucleocapsids assemble and partially mature in nuclear replication compartments and then travel to the inner nuclear membrane (INM) for nuclear egress. The factors governing the recruitment of nucleocapsids to the INM are not known. Kinetic analysis of pUL93 demonstrates that this protein is expressed late during infection and localizes primarily to the nucleus of infected cells. pUL93 associates with both virions and capsids and interacts with the components of the nuclear egress complex (NEC), namely, pUL50, pUL53, and pUL97, during infection. Also, multiple regions in pUL93 can independently interact with pUL77, which has been shown to help retain viral DNA during capsid assembly. These studies, combined with our earlier report of an essential role of pUL93 in viral DNA packaging, indicate that pUL93 serves as an important link between nucleocapsid maturation and nuclear egress. IMPORTANCE: HCMV causes life-threatening disease and disability in immunocompromised patients and congenitally infected newborns. In this study, we investigated the functions of HCMV essential tegument protein pUL93 and determined that it interacts with the components of the nuclear egress complex, namely, pUL50, pUL53, and pUL97. We also found that pUL93 specifically interacts with pUL77, which helps retain viral DNA during capsid assembly. Together, our data point toward an important role of pUL93 in linking virus maturation to nuclear egress. In addition to expanding our knowledge of the process of HCMV maturation, information from these studies will also be utilized to develop new antiviral therapies.

Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging.

Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.

A role for 3-O-sulfated heparan sulfate in promoting human cytomegalovirus infection in human iris cells.

Human cytomegalovirus (HCMV) has emerged as a clinically opportunistic pathogen that targets multiple types of ocular cells and tissues, including the iris region of the uveal tract during anterior uveitis. In this report, we used primary cultures of human iris stroma (HIS) cells derived from human eye donors to investigate HCMV entry. The following lines of evidence suggested the role of 3-O-sulfated heparan sulfate (3-OS HS) during HCMV-mediated entry and cell-to-cell fusion in HIS cells. First, 3-O-sulfotransferase-3 (3-OST-3) expression in HIS cells promoted HCMV internalization, while pretreatment of HIS cells with heparinase enzyme or with anti-3-OS HS (G2) peptide significantly reduced the HCMV-mediated formation of plaques/foci. Second, coculture of the HCMV-infected HIS cells with CHO-K1 cells expressing 3-OS HS significantly enhanced cell fusion. Finally, a similar trend of enhanced fusion was observed with cells expressing HCMV glycoproteins (gB, gO, and gH-gL) cocultured with 3-OS HS cells. Taken together, these results highlight the role of 3-OS HS during HCMV plaque formation and cell-to-cell fusion and identify a novel target for future therapeutic interventions.

Highly acidic C-terminal region of cytomegalovirus pUL96 determines its functions during virus maturation independently of a direct pp150 interaction.

UNLABELLED: Tegument proteins pp150 and pUL96 function at a late step in cytomegalovirus (CMV) maturation. Here, we show that pp150 interacts directly with pUL96; however, the N-terminal region of pp150 and the C-terminal region of pUL96, which are critical for these proteins to function, are not required for this interaction. Moreover, the largely dispensable C-terminal region of pp150 is critical for pp150-pUL96 interaction. To further study the role of pUL96, several point and clustered mutations were engineered into the CMV Towne bacterial artificial chromosome (Towne-BAC) genome, replacing the conserved negatively charged C-terminal residues of pUL96. Although individual point mutations (E122A, D124A, and D125A) reduced virus growth slightly, the clustered mutations of 122EVDDAV127 significantly reduced virus growth, produced small syncytial plaque phenotypes, and impacted a late stage of virus maturation. When the UL96 C-terminal alanine conversion mutant (B6-BAC) virus was serially passaged in cell culture, it gained a plaque size comparable to that of Towne-BAC, displayed an altered restriction fragment length pattern, and replicated with increased growth kinetics. Whole-genome sequencing of this passaged virus (UL96P10) and the similarly passaged Towne-BAC virus revealed major differences only in the RNA4.9 and UL96 regions. When one of the mutations in the UL96 coding region was engineered into the B6-BAC virus, it significantly increased the plaque size and rescued the virus growth rate. Thus, accumulation of compensatory mutations only in UL96 in this revertant and the specific involvement of functionally dispensable regions of pp150 in the pUL96-pp150 interaction point toward a role for pUL96 in virus maturation that does not depend upon pp150. IMPORTANCE: Human cytomegalovirus causes significant medical problems in newborns, as well as in people with low immunity. In this study, we investigated the functions of two essential virus proteins, pp150 and pUL96, and determined the impact of their mutual interaction on virus replication. These studies provide valuable information that is critical for the development of targeted antiviral therapies.

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive demyelinating disease of the spinal cord due to chronic inflammation. Hallmarks of disease pathology include dysfunctional anti-viral responses and the infiltration of HTLV-1-infected CD4+ T cells and HTLV-1-specific CD8+ T cells in the central nervous system. HAM/TSP individuals exhibit CD4+ and CD8+ T cells with elevated co-expression of multiple inhibitory immune checkpoint proteins (ICPs), but ICP blockade strategies can only partially restore CD8+ T-cell effector function. Exosomes, small extracellular vesicles, can enhance the spread of viral infections and blunt anti-viral responses. Here, we evaluated the impact of exosomes isolated from HTLV-1-infected cells and HAM/TSP patient sera on dendritic cell (DC) and T-cell phenotypes and function. We observed that exosomes derived from HTLV-infected cell lines (OSP2) elicit proinflammatory cytokine responses in DCs, promote helper CD4+ T-cell polarization, and suppress CD8+ T-cell effector function. Furthermore, exosomes from individuals with HAM/TSP stimulate CD4+ T-cell polarization, marked by increased Th1 and regulatory T-cell differentiation. We conclude that exosomes in the setting of HAM/TSP are detrimental to DC and T-cell function and may contribute to the progression of pathology with HTLV-1 infection.

Inhibition of Cytomegalovirus by Pentacta pygmaea Fucosylated Chondroitin Sulfate Depends on Its Molecular Weight.

Many viruses attach to host cells by first interacting with cell surface proteoglycans containing heparan sulfate (HS) glycosaminoglycan chains and then by engaging with specific receptor, resulting in virus entry. In this project, HS-virus interactions were targeted by a new fucosylated chondroitin sulfate from the sea cucumber Pentacta pygmaea (PpFucCS) in order to block human cytomegalovirus (HCMV) entry into cells. Human foreskin fibroblasts were infected with HCMV in the presence of PpFucCS and its low molecular weight (LMW) fractions and the virus yield at five days post-infection was assessed. The virus attachment and entry into the cells were visualized by labeling the purified virus particles with a self-quenching fluorophore octadecyl rhodamine B (R18). The native PpFucCS exhibited potent inhibitory activity against HCMV specifically blocking virus entry into the cell and the inhibitory activities of the LMW PpFucCS derivatives were proportional to their chain lengths. PpFucCS and the derived oligosaccharides did not exhibit any significant cytotoxicity; moreover, they protected the infected cells from virus-induced lytic cell death. In conclusion, PpFucCS inhibits the entry of HCMV into cells and the high MW of this carbohydrate is a key structural element to achieve the maximal anti-viral effect. This new marine sulfated glycan can be developed into a potential prophylactic and therapeutic antiviral agent against HCMV infection.

Structural requirements of Holothuria floridana fucosylated chondroitin sulfate oligosaccharides in anti-SARS-CoV-2 and anticoagulant activities.

Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.