Enhances Virus Entry Research Articles

IP-10 acts early in CV-A16 infection to induce BBB destruction and promote virus entry into the CNS by increasing TNF-α expression

The mechanisms underlying pathological changes in the central nervous system (CNS) following Coxsackievirus A16 (CV-A16) infection have not yet been elucidated. IFN-γ-inducible protein-10 (IP-10) is often used as a predictive factor to monitor early virus infection. It has also been reported that IP-10 plays a pivotal role in neuroinflammation. In this study, we aimed to explore the role of IP-10 in the neuropathogenesis of CV-A16 infection. We observed that the level of IP-10, as well as the TLR3-TRIF-TRAF3-TBK1-NF-κB and RIG-I/MDA5-MAVS-TRAFS-TBK1-NF-κB pathways, which are the upstream of IP-10, were significantly elevated during the course of CV-A16 infection. This increase was accompanied by an increase in a series of inflammatory cytokines at different time-points during CV-A16 infection. To determine whether IP-10 influences BBB integrity, we examined junctional complexes. Our results revealed that the expression levels of Claudin5, Occludin, ZO-1 and VE-Cadherin were notably decreased in CV-A16-infected HUVECs, but these indicators were restored in CV-A16-infected HUVECs with Eldelumab treatment. Nevertheless, IP-10 is only a chemokine that primarily traffics CXCR3-positive immune cells to inflammatory sites or promotes the production of inflammatory cytokines. Therefore, the interactions between IP-10 and inflammatory cytokines were evaluated. Our data revealed that IP-10 mediated the production of TNF-α, which was also observed to change the junctional complexes. Moreover, in a suckling mouse model, IP-10 and TNF-α treatments exacerbated clinical symptoms, mortality and pathological changes in the brain of CV-A16-infected mice, but Anti-IP-10 and Anti-TNF-α treatments alleviated these changes. Our data also revealed that IP-10 may be detected early in CV-A16 infection, whereas TNF-α was detected late in CV-A16 infection, and the production of TNF-α was also found to be positively correlated with IP-10. In addition, IP-10 and TNF-α were observed to reduce junctional complexes and enhance virus entry into the CNS. Taken together, this study provides the first evidence that CV-A16 activates the IP-10/TNF-α regulatory axis to cause BBB damage and accelerate the formation of neuroinflammation in infected hosts, which not only provides a new understanding of the neuropathogenesis caused by CV-A16, but also offers a promising target for the development of CV-A16 antiviral drugs.

Lineage-specific pathogenicity, immune evasion, and virological features of SARS-CoV-2 BA.2.86/JN.1 and EG.5.1/HK.3.

SARS-CoV-2 JN.1 with an additional L455S mutation on spike when compared with its parental variant BA.2.86 has outcompeted all earlier variants to become the dominant circulating variant. Recent studies investigated the immune resistance of SARS-CoV-2 JN.1 but additional factors are speculated to contribute to its global dominance, which remain elusive until today. Here, we find that SARS-CoV-2 JN.1 has a higher infectivity than BA.2.86 in differentiated primary human nasal epithelial cells (hNECs). Mechanistically, we demonstrate that the gained infectivity of SARS-CoV-2 JN.1 over BA.2.86 associates with increased entry efficiency conferred by L455S and better spike cleavage in hNECs. Structurally, S455 altered the mode of binding of JN.1 spike protein to ACE2 when compared to BA.2.86 spike at ACE2H34, and modified the internal structure of JN.1 spike protein by increasing the number of hydrogen bonds with neighboring residues. These findings indicate that a single mutation (L455S) enhances virus entry in hNECs and increases immune evasiveness, which contribute to the robust transmissibility of SARS-CoV-2 JN.1. We further evaluate the in vitro and in vivo virological characteristics between SARS-CoV-2 BA.2.86/JN.1 and EG.5.1/HK.3, and identify key lineage-specific features of the two Omicron sublineages that contribute to our understanding on Omicron antigenicity, transmissibility, and pathogenicity.

Severe COVID-19-A Review of Suggested Mechanisms Based on the Role of Extracellular Matrix Stiffness.

The severity of COVID-19 commonly depends on age-related tissue stiffness. The aim was to review publications that explain the effect of microenvironmental extracellular matrix stiffness on cellular processes. Platelets and endothelial cells are mechanosensitive. Increased tissue stiffness can trigger cytokine storm with the upregulated expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin IL-6, and tissue integrity disruption, leading to enhanced virus entry and disease severity. Increased tissue stiffness in critically ill COVID-19 patients triggers platelet activation and initiates plague formation and thrombosis development. Cholesterol content in cell membrane increases with aging and further enhances tissue stiffness. Membrane cholesterol depletion decreases virus entry to host cells. Membrane cholesterol lowering drugs, such as statins or novel chitosan derivatives, have to be further developed for application in COVID-19 treatment. Statins are also known to decrease arterial stiffness mitigating cardiovascular diseases. Sulfated chitosan derivatives can be further developed for potential use in future as anticoagulants in prevention of severe COVID-19. Anti-TNF-α therapies as well as destiffening therapies have been suggested to combat severe COVID-19. The inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells pathway must be considered as a therapeutic target in the treatment of severe COVID-19 patients. The activation of mechanosensitive platelets by higher matrix stiffness increases their adhesion and the risk of thrombus formation, thus enhancing the severity of COVID-19.

An entropic safety catch controls hepatitis C virus entry and antibody resistance.

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.

Dual control of tick-borne encephalitis virus replication by autophagy in mouse macrophages

Autophagy is a lysosomal degradative pathway responsible for recycling cytosolic proteins and organelles and also functions as an innate defense mechanism that host cells use against viral infection. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. For flaviviruses, both the positive and negative role of autophagy in virus replication has been reported. The interplay between autophagy and tick-borne encephalitis virus (TBEV) in innate immune cells is largely unknown. Here we report the relationship between an autophagy and TBEV replication in mouse macrophage cell line PMJ2-R using Hypr strain of TBEV. First, we examined the effect of Hypr infection on the autophagy pathway. We detected a mild and a temporary increase of autophagy marker LC3-II in Hypr-infected cells. The role of autophagy in TBEV replication was evaluated in autophagy related gene 5 (Atg5) knockdown cells (shAtg5). Our results showed that during an early stage of Hypr infection the viral titers were increased, while later on, at 72 hpi, the titers have declined in shAtg5 cells compared to control. Moreover, the higher number of virus-positive cells was observed in shAtg5 cells in early stage of infection and correlated with enhanced virus entry. Finally, we found an increased production of IFN-β in Hypr-infected shAtg5 cells in comparison to control at 48 and 72 hpi implicating that autophagy restricts the amount of IFN produced by TBEV-infected macrophages. To conclude, in mouse macrophages TBEV replication is controlled by autophagy in time dependent manner, having temporally an antiviral and then a pro-viral role during infection. Our study points out to a delicate and complex involvement of autophagy machinery at level of virus entry and IFN-β production when controlling TBEV infection.

The D614G Virus Mutation Enhances Anosmia in COVID-19 Patients: Evidence from a Systematic Review and Meta-analysis of Studies from South Asia.

The prevalence of chemosensory dysfunction in patients with COVID-19 varies greatly between populations. It is unclear whether such differences are due to factors at the level of the human host, or at the level of the coronavirus, or both. At the host level, the entry proteins which allow virus binding and entry have variants with distinct properties, and the frequency of such variants differs between ethnicities. At the level of the virus, the D614G mutation enhances virus entry to the host cell. Since the two virus strains (D614 and G614) coexisted in the first six months of the pandemic in most populations, it has been difficult to distinguish between contributions of the virus and contributions of the host for anosmia. To answer this question, we conducted a systematic review and meta-analysis of studies in South Asian populations when either the D614 or the G614 virus was dominant. We show that populations infected predominantly with the G614 virus had a much higher prevalence of anosmia (pooled prevalence of 31.8%) compared with the same ethnic populations infected mostly with the D614 virus strain (pooled anosmia prevalence of 5.3%). We conclude that the D614G mutation is a major contributing factor that increases the prevalence of anosmia in COVID-19, and that this enhanced effect on olfaction constitutes a previously unrecognized phenotype of the D614G mutation. The new virus strains that have additional mutations on the background of the D614G mutation can be expected to cause a similarly increased prevalence of chemosensory dysfunctions.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Antibody dependent enhancement: Unavoidable problems in vaccine development.

In some cases, antibodies can enhance virus entry and replication in cells. This phenomenon is called antibody-dependent infection enhancement (ADE). ADE not only promotes the virus to be recognized by the target cell and enters the target cell, but also affects the signal transmission in the target cell. Early formalin-inactivated virus vaccines such as aluminum adjuvants (RSV and measles) have been shown to induce ADE. Although there is no direct evidence that there is ADE in COVID-19, this potential risk is a huge challenge for prevention and vaccine development. This article focuses on the virus-induced ADE phenomenon and its molecular mechanism. It also summarizes various attempts in vaccine research and development to eliminate the ADE phenomenon, and proposes to avoid ADE in vaccine development from the perspective of antigens and adjuvants.

Intrinsic ADE: The Dark Side of Antibody Dependent Enhancement During Dengue Infection.

Dengue fever is an Aedes mosquito-borne illness caused by any one of the four different dengue virus (DENV) serotypes (1–4) and manifests in the form of symptoms ranging from mild or asymptomatic to severe disease with vascular leakage, leading to shock, and viral hemorrhagic syndrome. Increased risk of severe disease occurs during secondary infection with a virus serotype distinct from that of prior dengue infection. This occurs by antibody dependent enhancement (ADE) of infection, wherein sub-neutralizing antibodies against the virus particles opsonize dengue virus entry via formation of immune complexes that interact with fragment crystallizable gamma receptors (FcγR) on monocytes, dendritic cells, and macrophages. The ADE phenomenon has two components: Extrinsic and Intrinsic ADE. While extrinsic ADE contributes to enhanced virus entry, intrinsic ADE results in heightened virus production by inhibition of type1 interferon and activation of interleukin-10 biosynthesis, thereby favoring a Th2 type immune response. Intrinsic ADE has greater contribution in enhancing Dengue replication as compared to extrinsic ADE. Detailed elucidation of intrinsic ADE during secondary dengue infection can increase our understanding of DENV-pathogenesis and aid in the development of host-targeting antivirals. Here we review literature focusing on intrinsic factors contributing to severe dengue pathology and suggest possible avenues for further research.

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Zika virus (ZIKV) belongs to the Flaviviridae family and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurologic disorders and replicates efficiently in reproductive tissues1–3,. Here, we show that ZIKV envelope (E) protein is K63-linked polyubiquitinated by the E3-ubiquitin ligase TRIM7. Accordingly, ZIKV replicates less efficiently in brain and reproductive tissues of Trim7−/− mice. Ubiquitinated E is present on infectious Zika virions when released from specific cell types and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the Tim-1 (HAVCR1) receptor, enhancing virus replication in cells and in vivo in brain tissue. Recombinant ZIKV mutants lacking ubiquitination are attenuated in human cells and in a mouse model, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viremia in mice. Collectively, the results demonstrate that ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

On the unpredictability of outcomes of immunotherapy and preventive immunization against COVID-19

This article analyzes the possibility of employing immunotherapy and preventive immunization to fight COVID-19. The authors think that treatment and prevention of the infection with anti-SARS-CoV-2 antibodies can have unpredictable outcomes. Although these antibodies can neutralize virus antigens (S-proteins), they also have the ability to enhance virus entry into the host cell. The article emphasizes the importance of solid evidence of efficacy and safety for candidate anti-COVID-19 therapies and protective measures.

Bovine ephemeral fever virus triggers autophagy enhancing virus replication via upregulation of the Src/JNK/AP1 and PI3K/Akt/NF-\u03baB pathways and suppression of the PI3K/Akt/mTOR pathway

Autophagy plays an important role in cellular response to pathogens. However, the impact of the autophagy machinery on bovine ephemeral fever virus (BEFV) infection is not yet determined. A recent study in our laboratory demonstrated that BEFV triggers simultaneously the PI3K/Akt/NF-κB and Src/JNK-AP1 pathways in the stage of virus binding to enhance virus entry. In this work, we report that BEFV induces autophagy via upregulation of the PI3K/Akt/NF-κB and Src/JNK/AP1 pathways in the early to middle stages of infection and suppresses the PI3K/Akt/mTOR pathway at the late stage of infection. To activate NF-κB, BEFV promotes degradation of IκBα and activates Akt to stimulate NF-κB translocation into the nucleus. Immunoprecipitation assays revealed that BEFV disrupts Beclin 1 and Bcl-2 interaction by JNK-mediated Bcl-2 phosphorylation, thereby activating autophagy. Overexpression of Bcl-2 reversed the BEFV-induced increase in the LC3 II levels. Suppression of autophagy either by knockdown of autophagy-related genes with shRNAs or treatment with a pharmacological inhibitor 3-MA reduced BEFV replication, suggesting that BEFV-induced autophagy benefits virus replication. Our results revealed that the BEFV M protein is one of the viral proteins involved in inducing autophagy via suppression of the PI3K/Akt/mTORC1 pathway. Furthermore, degradation of p62 was observed by immunoblotting, suggesting that BEFV infection triggers a complete autophagic response. Disruption of autophagosome-lysosome fusion by depleting LAMP2 resulted in reduction of virus yield, suggesting that formation of autolysosome benefits virus production.

Cytokine Effects on the Entry of Filovirus Envelope Pseudotyped Virus-Like Particles into Primary Human Macrophages.

Macrophages are one of the first and also a major site of filovirus replication and, in addition, are a source of multiple cytokines, presumed to play a critical role in the pathogenesis of the viral infection. Some of these cytokines are known to induce macrophage phenotypic changes in vitro, but how macrophage polarization may affect the cell susceptibility to filovirus entry remains largely unstudied. We generated different macrophage subsets using cytokine pre-treatment and subsequently tested their ability to fuse with beta-lactamase containing virus-like particles (VLP), pseudotyped with the surface glycoprotein of Ebola virus (EBOV) or the glycoproteins of other clinically relevant filovirus species. We found that pre-incubation of primary human monocyte-derived macrophages (MDM) with interleukin-10 (IL-10) significantly enhanced filovirus entry into cells obtained from multiple healthy donors, and the IL-10 effect was preserved in the presence of pro-inflammatory cytokines found to be elevated during EBOV disease. In contrast, fusion of IL-10-treated macrophages with influenza hemagglutinin/neuraminidase pseudotyped VLPs was unchanged or slightly reduced. Importantly, our in vitro data showing enhanced virus entry are consistent with the correlation established between elevated serum IL-10 and increased mortality in filovirus infected patients and also reveal a novel mechanism that may account for the IL-10-mediated increase in filovirus pathogenicity.

IFITM1 expression is crucial to gammaherpesvirus infection, in vivo

The oncogenic gammaherpesviruses, Epstein–Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV), are etiologically associated with a variety of human cancers, including Burkitt’s lymphoma (BL), Hodgkin lymphoma (HL), Kaposi’s sarcoma (KS), and primary effusion lymphoma (PEL). Recently, we demonstrated KSHV infection of B- and endothelial cells to significantly upregulate the expression of interferon induced transmembrane protein 1 (IFITM1) which in turn enhances virus entry. This is an extension of the above study. In here, we determined EBV infection of cells to trigger IFITM1 expression, in vitro. Silencing IFITM1 expression using siRNA specifically lowered gammaherpesvirus infection of cells at a post binding stage of entry. A natural model system to explore the effect of IFITM1 on gammaherpesvirus infection in vivo is infection of BALB/c mice with murine gammaherpesvirus 68 (MHV-68). Priming mice with siRNA specific to IFITM1 significantly lowered MHV-68 titers in the lung specimens compared to priming with (NS)siRNA or PBS. MHV-68 titers were monitored by plaque assay and qPCR. Taken together, for the first time, this study provides insight into the critical role of IFITM1 to promoting in vivo gammaherpesvirus infections.

Spontaneous Mutation at Amino Acid 544 of the Ebola Virus Glycoprotein Potentiates Virus Entry and Selection in Tissue Culture.

Ebolaviruses have a surface glycoprotein (GP1,2) that is required for virus attachment and entry into cells. Mutations affecting GP1,2 functions can alter virus growth properties. We generated a recombinant vesicular stomatitis virus encoding Ebola virus Makona variant GP1,2 (rVSV-MAK-GP) and observed emergence of a T544I mutation in the Makona GP1,2 gene during tissue culture passage in certain cell lines. The T544I mutation emerged within two passages when VSV-MAK-GP was grown on Vero E6, Vero, and BS-C-1 cells but not when it was passaged on Huh7 and HepG2 cells. The mutation led to a marked increase in virus growth kinetics and conferred a robust growth advantage over wild-type rVSV-MAK-GP on Vero E6 cells. Analysis of complete viral genomes collected from patients in western Africa indicated that this mutation was not found in Ebola virus clinical samples. However, we observed the emergence of T544I during serial passage of various Ebola Makona isolates on Vero E6 cells. Three independent isolates showed emergence of T544I from undetectable levels in nonpassaged virus or virus passaged once to frequencies of greater than 60% within a single passage, consistent with it being a tissue culture adaptation. Intriguingly, T544I is not found in any Sudan, Bundibugyo, or Tai Forest ebolavirus sequences. Furthermore, T544I did not emerge when we serially passaged recombinant VSV encoding GP1,2 from these ebolaviruses. This report provides experimental evidence that the spontaneous mutation T544I is a tissue culture adaptation in certain cell lines and that it may be unique for the species Zaire ebolavirusIMPORTANCE The Ebola virus (Zaire) species is the most lethal species of all ebolaviruses in terms of mortality rate and number of deaths. Understanding how the Ebola virus surface glycoprotein functions to facilitate entry in cells is an area of intense research. Recently, three groups independently identified a polymorphism in the Ebola glycoprotein (I544) that enhanced virus entry, but they did not agree in their conclusions regarding its impact on pathogenesis. Our findings here address the origins of this polymorphism and provide experimental evidence showing that it is the result of a spontaneous mutation (T544I) specific to tissue culture conditions, suggesting that it has no role in pathogenesis. We further show that this mutation may be unique to the species Zaire ebolavirus, as it does not occur in Sudan, Bundibugyo, and Tai Forest ebolaviruses. Understanding the mechanism behind this mutation can provide insight into functional differences that exist in culture conditions and among ebolavirus glycoproteins.

The TAM receptor Mertk protects against neuroinvasive viral infection by maintaining blood-brain barrier integrity.

The TAM receptors Tyro3, Axl, and Mertk are receptor tyrosine kinases that dampen host innate immune responses following engagement with their ligands, Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells. In a form of apoptotic mimicry, many enveloped viruses display phosphatidylserine on the outer leaflet of their membranes, enabling TAM receptor activation and down-regulation of antiviral responses. Accordingly, we hypothesized that a deficiency of TAM receptors would enhance antiviral responses and protect against viral infection. Unexpectedly, mice lacking Mertk and/or Axl but not Tyro3 exhibited greater vulnerability to infection with neuroinvasive West Nile and La Crosse viruses. This phenotype was associated with increased blood-brain barrier permeability, which enhanced virus entry into and infection of the brain. Activation of Mertk synergized with IFN-β to tighten cell junctions and prevent virus transit across brain microvascular endothelial cells. Because TAM receptors restrict pathogenesis of neuroinvasive viruses, these findings have implications for TAM antagonists that are currently in clinical development.

Fc receptors in antibody-dependent enhancement of viral infections.

SummarySensitization of the humoral immune response to invading viruses and production of antiviral antibodies forms part of the host antiviral repertoire. Paradoxically, for a number of viral pathogens, under certain conditions, antibodies provide an attractive means of enhanced virus entry and replication in a number of cell types. Known as antibody‐dependent enhancement (ADE) of infection, the phenomenon occurs when virus‐antibody immunocomplexes interact with cells bearing complement or Fc receptors, promoting internalization of the virus and increasing infection. Frequently associated with exacerbation of viral disease, ADE of infection presents a major obstacle to the prevention of viral disease by vaccination and is thought to be partly responsible for the adverse effects of novel antiviral therapeutics such as intravenous immunoglobulins. There is a growing body of work examining the intracellular signaling pathways and epitopes responsible for mediating ADE, with a view to aiding rational design of antiviral strategies. With in vitro studies also confirming ADE as a feature of infection for a growing number of viruses, challenges remain in understanding the multilayered molecular mechanisms of ADE and its effect on viral pathogenesis.

Cell entry of bovine ephemeral fever virus requires activation of Src-JNK-AP1 and PI3K-Akt-NF-κB pathways as well as Cox-2-mediated PGE2 /EP receptor signalling to enhance clathrin-mediated virus endocytosis.

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin-mediated and dynamin 2-dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src-JNK-AP1 and PI3K-Akt-NF-κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src-JNK signalling to enhance its entry. Activation of these pathways by ultraviolet-inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV-induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3'-dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox-2-catalysed prostaglandin E2 (PGE2) synthesis and induces expressions of G-protein-coupled E-prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src-JNK-AP1 and PI3K-Akt-NF-κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV-induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox-2-mediated PGE2/EP receptor signalling pathways, further enhancing Src-JNK-AP1 in a cAMP-dependent manner and PI3K-Akt-NF-κB in a cAMP-independent manner. Accordingly, BEFV stimulates PGE2/EP receptor signalling amplifying Src-JNK-AP1 and PI3K-Akt-NF-κB pathways in an autocrine or paracrine fashion to enhance virus entry.

Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral membrane. While it is known that the PtdSer binding is essential for the PVEER function of TIM-1, TIM-3 shares this binding activity but does not enhance virus entry. No comprehensive studies have been done to characterize the other domains of TIM-1. In this study, using a variety of chimeric proteins and deletion mutants, we define the features necessary for a functional PVEER. With these features in mind, we generated a TIM-1 mimic using functionally similar domains from other proteins. This mimic, like TIM-1, effectively enhanced transduction. These studies provide insight into the key features necessary for PVEERs and will allow for more effective identification of unknown PVEERs.

Amino acid differences in glycoproteins B (gB), C (gC), H (gH) and L(gL) are associated with enhanced herpes simplex virus type-1 (McKrae) entry via the paired immunoglobulin-like type-2 receptor α

BackgroundHerpes simplex virus type-1 (HSV-1) enters into cells via membrane fusion of the viral envelope with plasma or endosomal membranes mediated by viral glycoproteins. HSV-1 virions attach to cell surfaces by binding of viral glycoproteins gC, gD and gB to specific cellular receptors. Here we show that the human ocular and highly neurovirulent HSV-1 strain McKrae enters substantially more efficiently into cells via the gB-specific human paired immunoglobulin-like type-2 receptor-α (hPILR-α). Comparison of the predicted amino acid sequences between HSV-1(F) and McKrae strains indicates that amino acid changes within gB, gC, gH and gL may cause increased entry via the hPILR- α receptor.ResultsHSV-1 (McKrae) entered substantially more efficiently than viral strain F in Chinese hamster ovary (CHO) cells expressing hPIRL-α but not within CHO-human nectin-1, -(CHO-hNectin-1), CHO-human HVEM (CHO-hHVEM) or Vero cells. The McKrae genes encoding viral glycoproteins gB, gC, gD, gH, gL, gK and the membrane protein UL20 were sequenced and their predicted amino acid (aa) sequences were compared with virulent strains F, H129, and the attenuated laboratory strain KOS. Most aa differences between McKrae and F were located at their gB amino termini known to bind with the PILRα receptor. These aa changes included a C10R change, also seen in the neurovirulent strain ANG, as well as redistribution and increase of proline residues. Comparison of gC aa sequences revealed multiple aa changes including an L132P change within the 129-247 aa region known to bind to heparan sulfate (HS) receptors. Two aa changes were located within the H1 domain of gH that binds gL. Multiple aa changes were located within the McKrae gL sequence, which were preserved in the H129 isolate, but differed for the F strain. Viral glycoproteins gD and gK and the membrane protein UL20 were conserved between McKrae and F strains.ConclusionsThe results indicate that the observed entry phenotype of the McKrae strain is most likely due to a combination of increased binding to heparan sulfate receptors and enhanced virus entry via gB-mediated fusion of the viral envelope with plasma membranes.