Inhibition of Cell-Free Human T-Cell Leukemia Virus Type 1 Infection at a Postbinding Step by the Synthetic Peptide Derived from an Ectodomain of the gp21 Transmembrane Glycoprotein

To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.     

Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins

The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role.     

Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64null AcMNPV budded virions. The combined use of gp64null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.     

Processing of the Borna Disease Virus Glycoprotein gp94 by the Subtilisin-Like Endoprotease Furin

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.     

Characterization of Borna disease virus p56 protein, a surface glycoprotein involved in virus entry.

Borna disease virus (BDV) is a nonsegmented negative-stranded (NNS) RNA virus, prototype of a new taxon in the Mononegavirales order. BDV causes neurologic disease manifested by behavioral abnormalities in several animal species, and evidence suggests that it may be a human pathogen. To improve our knowledge about the biology of this novel virus, we have identified and characterized the product of BDV open reading frame IV (BVp56). Based on sequence features, BVp56 encodes a virus surface glycoprotein. Glycoproteins play essential roles in the biology of NNS RNA viruses. Expression of BVp56 resulted in the generation of two polypeptides with molecular masses of about 84 and 43 kDa (GP-84 and GP-43). GP-84 and GP-43 likely correspond to the full-length BVp56 gene and to its C terminus, respectively. Endoglycosidase studies demonstrated that both products were glycosylated and that this process was required for the stabilization of newly synthesized products. Moreover, our results suggested that GP-43 is generated by cleavage of GP-84 by a cellular protease. Subcellular localization studies demonstrated that GP-84 accumulates in the ER, whereas GP-43 reaches the cell surface. Both BVp56 products were found to be associated with infectious virions, and antibodies to BVp56 had neutralizing activity. Our findings suggest that BVp56 exhibits a novel form of processing for an animal NNS RNA virus surface glycoprotein, which might influence the assembly and budding of BDV.     

p38: A novel protein that associates with the vesicular stomatitis virus glycoprotein

The vesicular stomatitis virus glycoprotein (VSV G) is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. The cytoplasmic domain of VSV G contains information for several intracellular sorting steps including efficient export from the ER, basolateral delivery, and endocytosis. In order to identify proteins that potentially interact with the polypeptide sorting motifs in the VSV G tail, the carboxy-terminal 27 amino acids of VSV G were used as bait in a yeast two-hybrid system. The protein identified most frequently in the screen is a novel protein of 38 kDa, p38. In the present work, the initial molecular and biochemical characterization of p38 is described. Preliminary evidence suggests that p38 may interact transiently with endoplasmic reticulum (ER) membranes, and thus may affect VSV G and other cargo movement at the step of ER to Golgi traffic.     

Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.     

表达尼帕病毒G囊膜糖蛋白重组牛痘病毒的研究

采用牛痘病毒WR株,构建了表达哺乳动物密码子优化的NiV G蛋白基因的重组病毒rWR-NiV-G。Westernblot证实大小为66kDa的重组G蛋白在rWR-NiV-G感染的Hela细胞中获得表达;采用兔抗NiV高免血清间接免疫荧光检测重组痘病毒表达G蛋白显示出良好的特异免疫反应原性。rWR-NiV-G感染NiV敏感的BHK细胞系,并与NiV融合蛋白F共同表达,可形成强烈细胞融合现象。rWR-NiV-G感染免疫BALB/c小鼠,可诱导显著的NiV G蛋白特异体液免疫反应。以原核表达NiV G蛋白片段为包被抗原,间接ELISA检测rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体,具有良好的敏感性和特异性。同时,rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体可有效中和NiV囊膜蛋白F和G介导的伪型VSV重组病毒侵入NiV易感宿主细胞的感染性。结果表明,重组牛痘病毒表达的NiV G蛋白有良好的免疫原性和生物学活性功能,为进一步深入研究NiV G蛋白生物学功能、免疫原性及重组活载体疫苗研究奠定了重要基础。     

Biochemical and functional analysis of the Borna disease virus G protein.

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.     

Identification of Functional Domains in the 14-Kilodalton Envelope Protein (A27L) of Vaccinia Virus

The mechanism of entry of vaccinia virus (VV) into cells is still a poorly understood process. A 14-kDa protein (encoded by the A27L gene) in the envelope of intracellular mature virus (IMV) has been implicated in virus-cell attachment, virus-cell fusion, and virus release from cells. We have previously described the structural organization of the VV 14-kDa protein, consisting of a triple-stranded coiled-coil region responsible for oligomer formation and a predicted Leu zipper-like third alpha helix with an important role in the interaction with a 21-kDa membrane protein (encoded by the A17L gene) thought to anchor the 14-kDa protein to the envelope of IMV (M.-I. Vázquez, G. Rivas, D. Cregut, L. Serrano, and M. Esteban, J. Virol. 72:10126-10137, 1998). To identify the functional domains important for virus entry and release, we have generated VV recombinants containing a copy of the A27L gene regulated by the lacI operator-repressor system of Escherichia coli (VVIndA27L) in the thymidine kinase locus and a mutant form of the A27L gene in the hemagglutinin locus but expressed constitutively under the control of an early-late VV promoter. Cells infected with a VV recombinant that expresses a mutant 14-kDa form lacking the first 29 amino acids at the N terminus failed to form extracellular enveloped virus (EEV). Fusion-from-without assays with purified virus confirmed that the fusion process was mediated by the 14-kDa protein and the fusion domain to be contained within amino acids 29 to 43 of the N-terminal region. Competitive inhibition of the infection process with soluble heparin and synthetic peptides and in vitro experiments with purified mutant proteins identified the heparin binding domain within amino acids 21 to 33, suggesting that this domain is involved in virus-cell binding via heparan sulfate. Thus, the N terminus of the 14-kDa protein contains a heparin binding domain, a fusion domain, and a domain responsible for interacting with proteins or lipids in the Golgi stacks for EEV formation and virus spread.     

Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.     

Open Reading Frame III of Borna Disease Virus Encodes a Nonglycosylated Matrix Protein

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.     

Functional Murine Leukemia Virus Vectors Pseudotyped with the Visna Virus Envelope Show Expanded Visna Virus Cell Tropism

Pseudotype virus vectors serve as a powerful tool for the study of virus receptor usage and entry. We describe the development of murine leukemia virus (MuLV) particles pseudotyped with the visna virus envelope glycoprotein and encoding a green fluorescent protein reporter as a tool to study the expression of the visna virus receptor. Functional MuLV/visna virus pseudotypes were obtained when the cytoplasmic tail of the visna virus envelope TM protein was truncated to 3, 7, or 11 amino acids in length. MuLV/visna virus particles were used to transduce a panel of cell types from various organisms, including sheep, goat, human, hamster, mouse, monkey, and quail. The majority of the cells examined were susceptible to MuLV/visna pseudotype viruses, supporting the notion that the visna virus cellular receptor is a widely expressed protein found in many species. Of 16 different cell types tested, only mouse embryo fibroblast NIH 3T3 cells, hamster ovary CHO cells, and the human promonocyte cell line U937 cells were not susceptible to transduction by the pseudotyped virus. The production of functional MuLV/visna virus pseudotypes has provided a sensitive, biologically relevant system to study visna virus cell entry and envelope-receptor interactions.     

Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus.

Mixed infection of a cell by vesicular stomatitis virus (VSV) and retroviruses results in the production of progeny virions bearing the genome of one virus encapsidated by the envelope proteins of the other. The mechanism for the phenomenon of pseudotype formation is not clear, although specific recognition of a viral envelope protein by the nucleocapsid of an unrelated virus is presumably involved. In this study, we used Moloney murine leukemia virus (MoMLV)-based retroviral vectors encoding the gene for neomycin phosphotransferase to investigate the interaction between the VSV G protein and the retroviral nucleocapsid during the formation of MoMLV(VSV) pseudotypes. Our results show that VSV G protein can be incorporated into the virions of retrovirus in the absence of other VSV-encoded proteins or of retroviral envelope protein. Infection of hamster cells by MoMLV(VSV) pseudotypes gave rise to neomycin phosphotransferase-resistant colonies, and addition of anti-VSV serum to the virus preparations completely abolished the infectivity of MoMLV(VSV) pseudotypes. It should be possible to use existing mutants of VSV G protein in the system described here to identify the signals that are important for the formation of MoMLV(VSV) pseudotypes.     

Mutational Analysis of the Putative Fusion Domain of Ebola Virus Glycoprotein

Ebola viruses contain a single glycoprotein (GP) spike, which functions as a receptor binding and membrane fusion protein. It contains a highly conserved hydrophobic region (amino acids 524 to 539) located 24 amino acids downstream of the N terminus of the Ebola virus GP2 subunit. Comparison of this region with the structural features of the transmembrane subunit of avian retroviral GPs suggests that the conserved Ebola virus hydrophobic region may, in fact, serve as the fusion peptide. To test this hypothesis directly, we introduced conservative (alanine) and nonconservative (arginine) amino acid substitutions at eight positions in this region of the GP2 molecule. The effects of these mutations were deduced from the ability of the Ebola virus GP to complement the infectivity of a vesicular stomatitis virus (VSV) lacking the receptor-binding G protein. Some mutations, such as Ile-to-Arg substitutions at positions 532 (I532R), F535R, G536A, and P537R, almost completely abolished the ability of the GP to support VSV infectivity without affecting the transport of GP to the cell surface and its incorporation into virions or the production of virus particles. Other mutations, such as G528R, L529A, L529R, I532A, and F535A, reduced the infectivity of the VSV-Ebola virus pseudotypes by at least one-half. These findings, together with previous reports of liposome association with a peptide corresponding to positions 524 to 539 in the GP molecule, offer compelling support for a fusion peptide role for the conserved hydrophobic region in the Ebola virus GP.     

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45-50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.     

Characterization of the Bas-Congo Virus Glycoprotein and Its Function in Pseudotyped Viruses

Bas-Congo virus (BASV) is a novel rhabdovirus recently identified from a patient with acute hemorrhagic fever in the Bas-Congo province of the Democratic Republic of Congo (DRC). Here we show that the BASV glycoprotein (BASV-G) can be successfully used to pseudotype glycoprotein-deficient vesicular stomatitis virus (VSV), allowing studies of BASV-G-driven membrane fusion and viral entry into target cells without replication-competent virus. BASV-G displayed broad tissue and species tropism in vitro, and BASV-G-mediated membrane fusion was pH dependent. The conformational changes induced in BASV-G by acidification were fully reversible and did not lead to inactivation of the viral fusion protein. Our data combined with comparative sequence similarity analyses suggest that BASV-G shares structural and functional features with other rhabdovirus glycoproteins and falls into the group of class III viral fusion proteins. However, activation of BASV-G-driven fusion required a lower pH and higher temperatures than did VSV-G-mediated fusion. Moreover, in contrast to VSV-G, mature BASV-G in VSV pseudotypes consists of a mixture of high-mannose and complex glycans that enables it to bind to certain C-type lectins, thereby enhancing its attachment to target cells. Taken together, the results presented in this study will facilitate future investigations of BASV-G-mediated cell entry and its inhibition in the absence of an infectious cell culture assay for BASV and at lower biosafety levels. Moreover, serology testing based on BASV-G pseudotype neutralization can be used to uncover the prevalence and importance of BASV as a potential novel human pathogen in the DRC and throughout Central Africa.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).