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ABSTRACT: 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.
Virologica Sinica 06/2011; 26(3):156-70.
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ABSTRACT: Herein, we report the development of an antifiloviral screening system, based on a pseudotyping strategy, and its application in the discovery of a novel group of small molecules that selectively inhibit the Ebola and Marburg glycoprotein (GP)-mediated infection of human cells. Using Ebola Zaire GP-pseudotyped HIV particles bearing a luciferase reporter gene and 293T cells, a library of 237 small molecules was screened for inhibition of GP-mediated viral entry. From this assay, lead compound 8a was identified as a selective inhibitor of filoviral entry with an IC(50) of 30 μM. To analyze functional group requirements for efficacy, a structure-activity relationship analysis of this 3,5-disubstituted isoxazole was then conducted with 56 isoxazole and triazole derivatives prepared using "click" chemistry. This study revealed that while the isoxazole ring can be replaced by a triazole system, the 5-(diethylamino)acetamido substituent found in 8a is required for inhibition of viral-cell entry. Variation of the 3-aryl substituent provided a number of more potent antiviral agents with IC(50) values ranging to 2.5 μM. Lead compound 8a and three of its derivatives were also found to block the Marburg glycoprotein (GP)-mediated infection of human cells.
Journal of Medicinal Chemistry 01/2011; 54(3):765-81. · 4.80 Impact Factor
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ABSTRACT: On the basis of mutagenesis, biochemical, and structural studies, heptad repeat 1 of HIV gp41 (HR1) has been shown to play numerous critical roles in HIV entry, including interacting with gp120 in prefusion states and interacting with gp41 heptad repeat 2 (HR2) in the fusion state. Moreover, HR1 is the site of therapeutic intervention by enfuviritide, a peptide analogue of HR2. In this study, the functional importance of each amino acid residue in gp41 HR1 has been systematically examined by alanine scanning mutagenesis, with subsequent characterization of the mutagenic effects on folding (as measured by incorporation into virions), association with gp120, and membrane fusion. The mutational effects on entry can be grouped into three classes: (1) wild type (defined as >40% of wild-type entry), (2) impaired (defined as 5-40% of wild-type entry), and (3) nonfunctional (defined as <5% of wild-type entry). Interestingly, the majority of HR1 mutations (77%) exhibit impaired or nonfunctional entry. Surprisingly, effects of mutations on folding, association, or fusion are not correlated to heptad position; however, folding defects are most often found in the N-terminal region of HR1. Moreover, disruption of the gp41-gp120 interaction is correlated to the C-terminal region of HR1, suggesting that this region interacts most closely with gp120. In summary, the sensitivity of gp41 HR1 to alanine substitutions suggests that even subtle changes in the local environment may severely affect envelope function, thereby strengthening the notion that HR1 is an attractive site for therapeutic intervention.
Biochemistry 06/2010; 49(24):5057-65. · 3.42 Impact Factor
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Ying Guo,
Emily Rumschlag-Booms, Jizhen Wang,
Haixia Xiao,
Jia Yu,
Jianwei Wang,
Li Guo,
George F Gao,
Youjia Cao,
Michael Caffrey,
Lijun Rong
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ABSTRACT: Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to alpha2,3-linked SAs, while human influenza A viruses exhibit a preference for alpha2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.
We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both alpha2,6-linked and alpha2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.
Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.
Virology Journal 05/2009; 6:39. · 2.34 Impact Factor
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ABSTRACT: 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.
Virologica Sinica 04/2009; 24(2):121-135.
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ABSTRACT: Avian influenza viruses continue to threaten globally with pandemic potential. The first step in a potential pandemic is the ability of the virus to enter human cells which is mediated by the viral surface glycoprotein hemagglutinin (HA). Viral entry of influenza is dependent upon the processing of the HA0 polypeptide precursor protein into HA1 and HA2 which is mediated by host cellular proteases. The sequence of the cleavage site which is recognized by host proteases has been linked with pathogenesis of various influenza viruses. Here we examined the effects of cleavage site sequences between a highly pathogenic H5N1 strain and a low pathogenic H5N2 strain to determine their effects on viral entry. From this analysis we determined that at the level of viral entry, the only observed difference between the low and high pathogenic strains is their ability to be cleaved by host cellular proteases.
Virology Journal 02/2009; 6:76. · 2.34 Impact Factor
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ABSTRACT: The importance of the N-terminal region of HIV gp120 conserved domain 1 (gp120-C1) to envelope function has been examined by alanine-scanning mutagenesis and subsequent characterization of the mutagenic effects on viral entry; envelope expression, processing, and incorporation; and gp120 association with gp41. With respect to the wild-type gp120, mutational effects on viral entry fall into two classes: functional, as defined by >20% entry with respect to wild type, and impaired, as defined by <20% entry with respect to wild type. Based on Western blot analyses of cell lysates and virions, the entry impairment of W35A, V38A, Y39A, Y40A, G41A, V42A, and I52A is due primarily to disruption of envelope processing. The entry impairment of P43A and W45A is apparently due to a combination of effects on processing and incorporation into virions. In contrast, the entry impairment of V44A and F53A is primarily due to disruption of the gp120-gp41 interaction, which results in dissociation of gp120 from the virion. We present a model for gp120-C1 interactions with gp120-C5 and the gp41 disulfide loop in unprocessed gp160 and processed gp120/gp41.
Journal of Biological Chemistry 10/2008; 283(47):32644-9. · 4.77 Impact Factor
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ABSTRACT: One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.
Virology 03/2007; 358(1):79-88. · 3.35 Impact Factor
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ABSTRACT: Ebola virus infection is initiated by interactions between the viral glycoprotein GP1 and its cognate receptor(s), but little is known about the structure and function of GP1 in viral entry, partly due to the concern about safety when working with the live Ebola virus and the difficulty of manipulating the RNA genome of Ebola virus. In this study, we have used a human immunodeficiency virus-based pseudotyped virus as a surrogate system to dissect the role of Ebola virus GP1 in viral entry. Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with respect to protein expression, processing, viral incorporation, and viral entry has allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues. Furthermore, six amino acids in this region have been identified as critical residues for early events in Ebola virus entry, and among these, three are clustered and are implicated as part of a potential receptor-binding pocket. In addition, substitutions of some 30 residues in GP1 are shown to adversely affect GP1 expression, processing, and viral incorporation, suggesting that these residues are involved in the proper folding and/or overall conformation of GP. Sequence comparison of the GP1 proteins suggests that the majority of the critical residues for GP folding and viral entry identified in Ebola virus GP1 are conserved in Marburg virus. These results provide information for elucidating the structural and functional roles of the filoviral glycoproteins and for developing potential therapeutics to block viral entry.
Journal of Virology 05/2005; 79(8):4793-805. · 5.40 Impact Factor
