Molecular Determinants of Hepatitis B and D Virus Entry Restriction in Mouse Sodium Taurocholate Cotransporting Polypeptide

Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections.     

Granzymes in cardiovascular injury and disease

Chronic inflammation and impaired wound healing play important roles in the pathophysiology of cardiovascular diseases. Moreover, the aberrant secretion of proteases plays a critical role in pathological tissue remodeling in chronic inflammatory conditions. Human Granzymes (Granule secreted enzymes – Gzms) comprise a family of five (GzmA, B, H, K, M) cell-secreted serine proteases. Although each unique in function and substrate specificities, Gzms were originally thought to share redundant, intracellular roles in cytotoxic lymphocyte-induced cell death. However, an abundance of evidence has challenged this dogma. It is now recognized, that individual Gzms exhibit unique substrate repertoires and functions both intracellularly and extracellularly. In the extracellular milieu, Gzms contribute to inflammation, vascular dysfunction and permeability, reduced cell adhesion, release of matrix-sequestered growth factors, receptor activation, and extracellular matrix cleavage. Despite these recent findings, the non-cytotoxic functions of Gzms in the context of cardiovascular disease pathogenesis remain poorly understood. Minimally detected in tissues and bodily fluids of normal individuals, GzmB is elevated in patients with acute coronary syndromes, coronary artery disease, and myocardial infarction. Pre-clinical animal models have exemplified the importance of GzmB in atherosclerosis, aortic aneurysm, and cardiac fibrosis as animals deficient in GzmB exhibit reduced tissue remodeling, improved disease phenotypes and increased survival. Although a role for GzmB in cardiovascular disease is described, further work to elucidate the mechanisms that underpin the remaining human Gzms activity in cardiovascular disease is necessary. The present review provides a summary of the pre-clinical and clinical evidence, as well as emerging areas of research pertaining to Gzms in tissue remodeling and cardiovascular disease.     

Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 μM for DENV-2 and 1.3 μM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.     

Permanent inhibition of viral entry by covalent entrapment of HIV gp41 on the virus surface

HIV entry occurs by concerted conformational changes in the envelope protein complex on the surface of the virus. This complex is made up of a trimer of heterodimers of two subunits: surface subunit, gp120, and transmembrane subunit, gp41. Conformational changes in the envelope complex allow gp41 to mediate membrane fusion leading to exposure of two gp41 regions: N-heptad repeat (NHR) and C-heptad repeat (CHR). Peptides from the NHR or the CHR have been found to inhibit HIV entry. Herein we show that we can covalently inhibit HIV viral entry by permanently trapping the gp41 intermediate on the virus surface using a covalently reactive group on inhibitory peptides. This is evidence showing that vulnerable conformational intermediates exist transiently during HIV viral entry, and the details presented herein will facilitate development of envelope as a target for therapeutics and potential chemopreventive agents that could disable the virus before contact with the host cell.     

Role of lipids in virus replication

Viruses intricately interact with and modulate cellular membranes at several stages of their replication, but much less is known about the role of viral lipids compared to proteins and nucleic acids. All animal viruses have to cross membranes for cell entry and exit, which occurs by membrane fusion (in enveloped viruses), by transient local disruption of membrane integrity, or by cell lysis. Furthermore, many viruses interact with cellular membrane compartments during their replication and often induce cytoplasmic membrane structures, in which genome replication and assembly occurs. Recent studies revealed details of membrane interaction, membrane bending, fission, and fusion for a number of viruses and unraveled the lipid composition of raft-dependent and -independent viruses. Alterations of membrane lipid composition can block viral release and entry, and certain lipids act as fusion inhibitors, suggesting a potential as antiviral drugs. Here, we review viral interactions with cellular membranes important for virus entry, cytoplasmic genome replication, and virus egress.     

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability,cleavage, and cell–cell fusion

The trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell–cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2–infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell–cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and transmembrane serine protease 2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell–cell fusion and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell–cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell–cell fusion. In addition, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell–cell fusion, all of which help give a more comprehensive understanding of this high-profile therapeutic target.     

An antiviral peptide targets a coiled-coil domain of the human T-cell leukemia virus envelope glycoprotein

Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.     

Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells

Although granzymes (Gzms) A- and B-induced cell death pathways have been defined, little is known about how other orphan Gzms function in CTL-mediated cytotoxicity. GzmK and A are tryptases among all the Gzms of humans and they are closely linked on the same chromosome. In this study, we showed that GzmK can be efficiently delivered into target cells with a cationic lipid protein transfection reagent Pro-Ject. We found human GzmK triggers rapid cell death independently of caspase activation. The features of death are characterized by rapid externalization of phosphatidylserine, nuclear morphological changes and single-stranded DNA nicks. GzmK hydrolyzes the nucleosome assembly protein SET in its recombinant and native forms or in intact cells. Cleavage of SET by GzmK abrogates its nucleosome assembly activity. After GzmK loading, SET and DNase NM23H1 rapidly translocate into the nucleus and SET is cleaved, where the nuclease activity of NM23H1 is activated to nick chromosomal DNA.     

Heparanase as a potential player in SARS-CoV-2 infection and induced coagulopathy

During the current formidable COVID-19 pandemic, it is appealing to address ideas that may invoke therapeutic interventions. Clotting disorders are well recognized in patients infected with severe acute respiratory syndrome (SARS) caused by a novel coronavirus (SARS-CoV-2), which lead to severe complications that worsen the prognosis in these subjects.Increasing evidence implicate Heparan sulfate proteoglycans (HSPGs) and Heparanase in various diseases and pathologies, including hypercoagulability states. Moreover, HSPGs and Heparanase are involved in several viral infections, in which they enhance cell entry and release of the viruses.Herein we discuss the molecular involvement of HSPGs and heparanase in SARS-CoV-2 infection, namely cell entry and release, and the accompanied coagulopathy complications, which assumedly could be blocked by heparanase inhibitors such as Heparin and Pixatimod.     

Emerging roles for ubiquitin in adenovirus cell entry

Adenovirus relies on numerous interactions between viral and host cell proteins to efficiently enter cells. Undoubtedly, post-translational modifications of host and cellular proteins can impact the efficiency of this cell entry process. Ubiquitylation, once simply thought of as a modification targeting proteins for proteasomal degradation, is now known to regulate protein trafficking within cells, protein-protein interactions and cell signalling pathways. Accumulating evidence suggests that protein ubiquitylation can influence all stages of the life cycle of other viruses such as cell entry, replication and egress. Until recently, the influence of ubiquitylation has only been documented during adenovirus replication. This review highlights the most recent evidence demonstrating direct engagement of host ubiquitylation and SUMOylation machinery by adenovirus during cell entry. Additionally, potential roles for host protein ubiquitylation and the potential for adenovirus regulation of host ubiquitylation machinery during cell entry are discussed.     

A role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral infection

RANTES (CCL5) is a chemokine expressed by many hematopoietic and non-hematopoietic cell types that plays an important role in homing and migration of effector and memory T cells during acute infections. The RANTES receptor, CCR5, is a major target of anti-HIV drugs based on blocking viral entry. However, defects in RANTES or RANTES receptors including CCR5 can compromise immunity to acute infections in animal models and lead to more severe disease in humans infected with west Nile virus (WNV). In contrast, the role of the RANTES pathway in regulating T cell responses and immunity during chronic infection remains unclear. In this study, we demonstrate a crucial role for RANTES in the control of systemic chronic LCMV infection. In RANTES^−/− mice, virus-specific CD8 T cells had poor cytokine production. These RANTES^−/− CD8 T cells also expressed higher amounts of inhibitory receptors consistent with more severe exhaustion. Moreover, the cytotoxic ability of CD8 T cells from RANTES^−/− mice was reduced. Consequently, viral load was higher in the absence of RANTES. The dysfunction of T cells in the absence of RANTES was as severe as CD8 T cell responses generated in the absence of CD4 T cell help. Our results demonstrate an important role for RANTES in sustaining CD8 T cell responses during a systemic chronic viral infection.     

The role of epithelial tight junctions involved in pathogen infections

Tight junctions (TJs) are sealing complexes between adjacent epithelial cells, functioning by controlling paracellular passage and maintaining cell polarity. These functions of TJs are primarily based on structural integrity as well as dynamic regulatory balance, indicating plasticity of TJ in response to external stimuli. An indispensable role of TJs involved in pathogen infection has been widely demonstrated since disruption of TJs leads to a distinct increase in paracellular permeability and polarity defects which facilitate viral or bacterial entry and spread. In addition to pathological changes in TJ integrity, TJ proteins such as occludin and claudins can either function as receptors for pathogen entry or interact with viral/bacterial effector molecules as an essential step for characterizing an infective stage. This suggests a more complicated role for TJ itself and especially specific TJ components. Thus, this review surveys the role of the epithelial TJs involved in various pathogen infections, and extends TJ targeted therapeutic and pharmacological application prospects.     

IFITM3‐containing exosome as a novel mediator for anti‐viral response in dengue virus infection

Interferon‐inducible transmembrane proteins 1, 2 and 3 (IFITM1, IFITM2 and IFITM3) have recently been identified as potent antiviral effectors that function to suppress the entry of a broad range of enveloped viruses and modulate cellular tropism independent of viral receptor expression. However, the antiviral effect and mechanisms of IFITMs in response to viral infections remain incompletely understood and characterized. In this work, we focused our investigation on the function of the extracellular IFITM3 protein. In cell models of DENV‐2 infection, we found that IFITM3 contributed to both the baseline and interferon‐induced inhibition of DENV entry. Most importantly, our study for the first time demonstrated the presence of IFITM‐containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver IFITM3 and thus transmit its antiviral effect from infected to non‐infected cells. Thus, our findings provide new insights in the basic mechanisms underlying the actions of IFITM3, which might lead to future development of exosome‐mediated anti‐viral strategies using IFITM3 as a therapeutic agent. Conceivably, variations in the basal and inducible levels of IFITMs, as well as in intracellular and extracellular levels of IFITMs, might predict the severity of dengue virus infections among individuals or across species.     

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.     

Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry.     

Antiviral cyclic D,L-alpha-peptides: targeting a general biochemical pathway in virus infections

Diverse virus families have evolved to exploit the acidification of endosomal compartments to gain entry into cells. We describe a supramolecular approach for selectively targeting and inhibiting viral infections through this central biochemical pathway. Using adenovirus as a model non-enveloped virus, we have determined that an eight-residue cyclic D,L-alpha-peptide, selected from a directed combinatorial library, can specifically prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection without an apparent adverse effect on cell viability. The likely generality of this approach against other pH-dependent viral infections is supported by the inhibition of type-A influenza virus escape from endosomes in the presence of the same peptide. Our studies suggest that self-assembling cyclic D,L-alpha-peptides hold considerable potential as a new rational supramolecular approach toward the design and discovery of broad-spectrum antiviral agents.     

Utilization of human DC-SIGN and L-SIGN for entry and infection of host cells by the New World arenavirus,Junín virus

The target cell tropism of enveloped viruses is regulated by interactions between viral proteins and cellular receptors determining susceptibility at a host cell, tissue or species level. However, a number of additional cell-surface moieties can also bind viral envelope glycoproteins and could act as capture receptors, serving as attachment factors to concentrate virus particles on the cell surface, or to disseminate the virus infection to target organs or susceptible cells within the host. Here, we used Junín virus (JUNV) or JUNV glycoprotein complex (GPC)-pseudotyped particles to study their ability to be internalized by the human C-type lectins hDC- or hL-SIGN. Our results provide evidence that hDC- and hL-SIGN can mediate the entry of Junín virus into cells, and may play an important role in virus infection and dissemination in the host.     

Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs)

Background

Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway.

Methodology/Principal Findings

Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16.

Conclusions/Significance

Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.     

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase γ1 (PLC-γ1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-γ1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-γ1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-γ1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-γ1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-γ1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-γ1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-γ1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.