Identification of the dietary supplement capsaicin as an inhibitor of Lassa virus entry

The limited treatment options for the increasing occurrence of Lassa hemorrhagic fever in West Africa poses an urgent need for the discovery and development of novel therapeutics. Dietary supplements, especially natural products that are edible and safe for human use, are a good source of drug discovery with potential for uncovering novel applications. In this study, we tested 40 natural products of dietary supplements and identified capsaicin, a common dietary supplement abundant in chili peppers, as an inhibitor of Lassa virus (LASV) entry with EC of 6.9-10.0 μmol/L using an HIV based pseudovirus platform. Capsaicin inhibits the entry of five LASV strains but not against the Old World arenavirus lymphocytic choriomeningitis virus (LCMV), showing a preferential activity against LASV. Capsaicin inhibits LASV entry by blocking the pH dependent viral fusion through affecting the stable signal peptide (SSP)-GP2 transmembrane (GP2) region of the LASV surface glycoprotein. Mutational study revealed the key residues Ala25, Val431, Phe434 and Val435 in SSP-GP2 region in capsaicin's antiviral effect. This study for the first time reveals a direct acting antiviral effect of capsaicin against the hemorrhagic fever causing LASV, providing detailed interaction hot spots in the unique SSP-GP2 interface of LASV glycoprotein that is crucial in fusion inhibition, and offering a new strategy in discovering and developing antivirals from natural products that are safe for human use.

Anti-HIV-1 activities of pine cone extract from Pinus kesiya var. langbianensis in vitro / 中草药

Objective: To investigate the anti-HIV-1 activity and mechanism of pine cone extracts from Pinus kesiya var. langbianensis in vitro. Methods: The cytotoxicity of pine cone extract from P. kesiya var. langbianensis was examined by MTT assay; Syncytium formation inhibition assay, p24 assay, and the HIV-1-induced MT-4 cell death assay were used to evaluate the anti-HIV-1 activity of pine cone extract from P. kesiya var. langbianensis. HIV DNA species detection assay, cell fusion assays, and time of addition assay were used to determine the mechanisms of the extracts. Results: Pine cone extract from P. kesiya var. langbianensis named SMS-F showed the good inhibition on HIV-1 in C8166 cells with TI value above 436.46 and 558.61, respectively. HIV-1 DNA species detection showed that ssDNA and late-RT were lower in the SMS-F treated group than those in the control group. The result implied that the mechanism of SMS-F might target the early stage of HIV-1 replication. SMS-F blocked the cell to cell fusion with EC50 value 71.03 μg/mL and could not inhibit the replication of HIV-1 after entering the cell, which confirmed SMS-F targeting the HIV entry stage. Conclusion: Pine cone extract from P. kesiya var. langbianensis shows the good inhibition on HIV-1 targeting the viral entry stage.

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry / 中国病毒学·英文版

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

The Mechanism of Henipavirus Fusion: Examining the Relationships between the Attachment and Fusion Glycoproteins / 中国病毒学·英文版

The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.

The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry / 中国病毒学·英文版

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.

The Importance of Heparan Sulfate in Herpesvirus Infection / 中国病毒学·英文版

Herpes simplex virus type-1 (HSV-1) is one of many pathogens that use the cell surface glycosaminoglycan heparan sulfate as a receptor.Heparan sulfate is highly expressed on the surface and extracellular matrix of virtually all cell types making it an ideal receptor.Heparan sulfate interacts with HSV-1 envelope glycoproteins gB and gC during the initial attachment step during HSV-1 entry.In addition,a modified form of heparan sulfate,known as 3-O-sulfated heparan sulfate,interacts with HSV-1 gD to induce fusion between the viral envelope and host cell membrane.The 3-O-sulfation of heparan sulfate is a rare modification which occurs during the biosynthesis of heparan sulfate that is carded out by a family of enzymes known as 3-O-sulfotransferases.Due to its involvement in multiple steps of the infection process,heparan sulfate has been a prime target for the development of agents to inhibit HSV entry.Understanding how heparan sulfate functions during HSV-1 infection may not only be critical for inhibiting infection by this virus,but it may also be crucial in the fight against many other pathogens as well.

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches / 中国病毒学·英文版

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.

In vivo characterization of the integrin beta3 as a receptor for Hantaan virus cellular entry

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha v beta3 integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta3 is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta3 integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta3 or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P<0.01) and 18.4 +/- 0.9 days (P<0.01), respectively. XT-199, a chemical blocker of the alpha v beta3 receptor also prolonged survival to 19.5 +/- 1.3 days (P<0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta1 antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta1 receptors as well as through beta3 receptors in vivo. Our data demonstrate that the beta3 receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta3 for cellular entry within an organism.