scholarly journals Vaccinia Virus Entry into Cells Is Dependent on a Virion Surface Protein Encoded by the A28L Gene

2004 ◽  
Vol 78 (5) ◽  
pp. 2357-2366 ◽  
Author(s):  
Tatiana G. Senkevich ◽  
Brian M. Ward ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Surface Protein ◽  
Syncytium Formation ◽  
Recombinant Vaccinia Virus ◽  
Productive Infection ◽  
Cell Penetration ◽  
Cytopathic Effects ◽  
Virion Surface ◽  
Entry Block

ABSTRACT The A28L gene of vaccinia virus is conserved in all poxviruses and encodes a protein that is anchored to the surface of infectious intracellular mature virions (IMV) and consequently lies beneath the additional envelope of extracellular virions. A conditional lethal recombinant vaccinia virus, vA28-HAi, with an inducible A28L gene, undergoes a single round of replication in the absence of inducer, producing IMV, as well as extracellular virions with actin tails, but fails to infect neighboring cells. We show here that purified A28-deficient IMV appeared to be indistinguishable from wild-type IMV and were competent to synthesize RNA in vitro. Nevertheless, A28-deficient virions did not induce cytopathic effects, express early genes, or initiate a productive infection. Although A28-deficient IMV bound to the surface of cells, their cores did not penetrate into the cytoplasm. An associated defect in membrane fusion was demonstrated by the failure of low pH to trigger syncytium formation when cells were infected with vA28-HAi in the absence of inducer (fusion from within) or when cells were incubated with a high multiplicity of A28-deficient virions (fusion from without). The correlation between the entry block and the inability of A28-deficient virions to mediate fusion provided compelling evidence for a relationship between these events. Because repression of A28 inhibited cell-to-cell spread, which is mediated by extracellular virions, all forms of vaccinia virus regardless of their outer coat must use a common A28-dependent mechanism of cell penetration. Furthermore, since A28 is conserved, all poxviruses are likely to penetrate cells in a similar way.

scholarly journals Vaccinia Virus A28L Gene Encodes an Essential Protein Component of the Virion Membrane with Intramolecular Disulfide Bonds Formed by the Viral Cytoplasmic Redox Pathway

2004 ◽  
Vol 78 (5) ◽  
pp. 2348-2356 ◽  
Author(s):  
Tatiana G. Senkevich ◽  
Brian M. Ward ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Disulfide Bonds ◽  
Surface Membrane ◽  
Viral Proteins ◽  
Recombinant Vaccinia Virus ◽  
Cell Penetration ◽  
Synthesis And Processing ◽  
Virus Replication Cycle ◽  
Virion Surface

ABSTRACT We report the initial characterization of the product of the vaccinia virus A28L gene, which is highly conserved in all sequenced poxviruses. Our studies showed that the A28 protein is expressed at late times during the virus replication cycle and is a membrane component of the intracellular mature virion. An N-terminal hydrophobic sequence, present in all poxvirus A28 orthologs, anchors the protein in the virion surface membrane so that most of it is exposed to the cytoplasm. The cytoplasmic domain contains four conserved cysteines, which form two intramolecular disulfide bonds. Disulfide bond formation depended on the expression of three viral proteins, E10, A2.5, and G4, which together comprise a conserved cytoplasmic redox pathway. A28 is the third identified substrate of this pathway; the others are the L1 and F9 proteins. We constructed a conditional-lethal recombinant vaccinia virus with an inducible A28L gene. The recombinant virus was propagated in the presence of inducer but was unable to replicate and spread in its absence. During a single round of an abortive infection in the absence of inducer, the synthesis and processing of viral proteins, assembly of intra- and extracellular virions, and formation of actin tails occurred normally. In another paper (T. Senkevich, B. M. Ward, and B. Moss, J. Virol. 78:2357-2366, 2004), we have demonstrated that virions assembled without A28 cannot carry out a second round of infection because they are defective in cell penetration.

scholarly journals Double Recombinant Vaccinia Virus: A Candidate Drug against Human Glioblastoma

Life ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1084
Author(s):  
Natalia Vasileva ◽  
Alisa Ageenko ◽  
Maria Dmitrieva ◽  
Anna Nushtaeva ◽  
Sergey Mishinov ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Human Cancer ◽  
Recombinant Vaccinia Virus ◽  
Recombinant Vaccinia ◽  
Human Glioblastoma ◽  
Human Cancer Cells ◽  
Macrophage Colony ◽  
Metastasis Growth ◽  
High Cytotoxic Activity

Glioblastoma is one of the most aggressive brain tumors. Given the poor prognosis of this disease, novel methods for glioblastoma treatment are needed. Virotherapy is one of the most actively developed approaches for cancer therapy today. VV-GMCSF-Lact is a recombinant vaccinia virus with deletions of the viral thymidine kinase and growth factor genes and insertions of the granulocyte–macrophage colony-stimulating factor and oncotoxic protein lactaptin genes. The virus has high cytotoxic activity against human cancer cells of various histogenesis and antitumor efficacy against breast cancer. In this work, we show VV-GMCSF-Lact to be a promising therapeutic agent for glioblastoma treatment. VV-GMCSF-Lact effectively decreases the viability of glioblastoma cells of both immortalized and patient-derived cultures in vitro, crosses the blood–brain barrier, selectively replicates into orthotopically transplanted human glioblastoma when intravenously injected, and inhibits glioblastoma xenograft and metastasis growth when injected intratumorally.

Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus

2000 ◽  
Vol 86 (11) ◽  
pp. 934-939 ◽  
Author(s):  
Yoshifumi Nishikawa ◽  
Yuko Kousaka ◽  
Shiya Fukumoto ◽  
Xuenan Xuan ◽  
Hideyuki Nagasawa ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Neospora Caninum ◽  
Surface Protein ◽  
Recombinant Vaccinia Virus ◽  
Recombinant Vaccinia

Recombinant vaccinia virus expression of Anaplasma marginale surface protein MSP-1a: effect of promoters, leader sequences and GPI anchor sequence on antibody response

Vaccine ◽  
1994 ◽  
Vol 12 (5) ◽  
pp. 465-471 ◽  
Author(s):  
Travis C. McGuire ◽  
Edward B. Stephens ◽  
Guy H. Palmer ◽  
Terry F. McElwain ◽  
Carol A. Lichtensteiger ◽  
...  
Keyword(s):  
Antibody Response ◽  
Vaccinia Virus ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Recombinant Vaccinia Virus ◽  
Gpi Anchor ◽  
Recombinant Vaccinia ◽  
Anchor Sequence ◽  
Virus Expression

scholarly journals Development of Cell-Based Assays for In Vitro Characterization of Hepatitis C Virus NS3/4A Protease Inhibitors

2005 ◽  
Vol 49 (4) ◽  
pp. 1381-1390 ◽  
Author(s):  
Victoria Chung ◽  
Anthony R. Carroll ◽  
Norman M. Gray ◽  
Nigel R. Parry ◽  
Pia A. Thommes ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Vaccinia Virus ◽  
Protease Inhibitors ◽  
Expression System ◽  
Recombinant Vaccinia Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Vaccinia

ABSTRACT A recombinant vaccinia virus, expressing the NS3-to-NS5 region of the N clone of hepatitis C virus (HCV), was generated and utilized both in a gel-based assay and in an enzyme-linked immunosorbent assay (ELISA) to evaluate the pyrrolidine-5,5-trans-lactams, a series of inhibitors of the HCV NS3/4A protease. The absolute levels of processed, mature HCV nonstructural proteins in this system were found to decrease in the presence of the trans-lactams. Monitoring of this reduction enabled end points and 50% inhibitory concentrations to be calculated in order to rank the active compounds according to potency. These compounds had no effect on the transcription or translation of the NS3-5 polyprotein at concentrations shown to inhibit NS3/4A protease, and they were shown to be specific inhibitors of this protease. The ELISA, originally developed using the vaccinia virus expression system, was modified to utilize Huh-7 cells containing an HCV replicon. Results with this assay correlated well with those obtained with the recombinant vaccinia virus assays. These results demonstrate the utility of these assays for the characterization of NS3/4A protease inhibitors. In addition, inhibitors of other viral targets, such as polymerase and helicase, can be evaluated in the context of the replicon ELISA.

scholarly journals Expression of normal and truncated forms of human endoglin

1999 ◽  
Vol 339 (3) ◽  
pp. 579-588 ◽  
Author(s):  
Ulla RAAB ◽  
Beatriz VELASCO ◽  
Pedro LASTRES ◽  
Ainhoa LETAMENDÍA ◽  
Carmela CALÉS ◽  
...  
Keyword(s):  
Surface Protein ◽  
Recombinant Vaccinia Virus ◽  
Expression Vectors ◽  
Soluble Form ◽  
Cysteine Residues ◽  
Hereditary Haemorrhagic Telangiectasia ◽  
Coding Region ◽  
Transmembrane Glycoprotein

Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281-Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330-Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1.

scholarly journals Altered Plaque Formation by Recombinant Vaccinia Virus Expressing Simian Immunodeficiency Virus Nef

1998 ◽  
Vol 72 (6) ◽  
pp. 5291-5295 ◽  
Author(s):  
Miles K. Yamanaka ◽  
Tilahun Yilma
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Simian Immunodeficiency Virus ◽  
Recombinant Vaccinia Virus ◽  
Cellular Protein ◽  
Plaque Formation ◽  
Site Directed Mutagenesis ◽  
Immunodeficiency Virus ◽  
High Virus

ABSTRACT The nef gene of primate lentiviruses encodes a myristoylated protein that is important for pathogenicity and the maintenance of high virus loads. A deletion in nef leads to a significant reduction of the pathogenicity of simian immunodeficiency virus (SIV) in macaques. At the cellular and biochemical levels, Nef has been shown to down-regulate CD4 and major histocompatibility complex class I molecules and to interact with cellular protein kinases. The importance of these activities for Nef function remains uncertain. We have prepared vaccinia virus recombinants expressing different alleles of SIV nef. When grown on TK− 143 cells, recombinants constructed with thenef allele from SIVmac1A11 produced typical plaques while recombinants expressing the nef allele from SIVmac239-R1 gave rise to plaques with altered morphology. By using chimeric Nef proteins and site-directed mutagenesis, the amino acid responsible for altered plaque formation was mapped to a leucine at residue 211. In vitro phosphorylation of immunoprecipitates prepared from cells infected with the vaccinia virus recombinants resulted in labeled proteins of 62 and 90 kDa. The recombinants differed in the ability to stimulate phosphorylation, and the leucine at residue 211 was again found to be the determining amino acid. These results might help elucidate the role of nef in the pathogenesis of SIV.

scholarly journals Nanoparticle composite TPNT1 is effective against SARS-CoV-2 and influenza viruses

2020 ◽  
Author(s):  
Sui-Yuan Chang ◽  
Kuo-Yen Huang ◽  
Tai-Ling Chao ◽  
Han-Chieh Kao ◽  
Yu-Hao Pang ◽  
...  
Keyword(s):  
Viral Entry ◽  
Opportunistic Infections ◽  
Food Additives ◽  
Syncytium Formation ◽  
Influenza Viruses ◽  
Metal Nanoparticle ◽  
Cytopathic Effects ◽  
H5n1 Influenza ◽  
Transmission Electron

Abstract A metal nanoparticle composite TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 can inhibit six major clades of SARS-CoV-2 with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to ACE2 receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and Tamiflu-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.

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