Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

ABSTRACT The original annotation of the vaccinia virus (VACV) genome was limited to open reading frames (ORFs) of at least 65 amino acids. Here, we characterized a 35-amino-acid ORF (O3L) located between ORFs O2L and I1L. ORFs similar in length to O3L were found at the same genetic locus in all vertebrate poxviruses. Although amino acid identities were low, the presence of a characteristic N-terminal hydrophobic domain strongly suggested that the other poxvirus genes were orthologs. Further studies demonstrated that the O3 protein was expressed at late times after infection and incorporated into the membrane of the mature virion. An O3L deletion mutant was barely viable, producing tiny plaques and a 3-log reduction in infectious progeny. A mutant VACV with a regulated O3L gene had a similar phenotype in the absence of inducer. There was no apparent defect in virus morphogenesis, though O3-deficient virus had low infectivity. The impairment was shown to be at the stage of virus entry, as cores were not detected in the cytoplasm after virus adsorption. Furthermore, O3-deficient virus did not induce fusion of infected cells when triggered by low pH. These characteristics are hallmarks of a group of proteins that form the entry/fusion complex (EFC). Affinity purification experiments demonstrated an association of O3 with EFC proteins. In addition, the assembly or stability of the EFC was impaired when expression of O3 was repressed. Thus, O3 is the newest recognized component of the EFC and the smallest VACV protein shown to have a function.

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A Conserved Sequence within the H2 Subunit of the Vaccinia Virus Entry/Fusion Complex Is Important for Interaction with the A28 Subunit and Infectivity

Journal of Virology ◽

10.1128/jvi.00434-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6244-6250 ◽

Cited By ~ 22

Author(s):

Gretchen E. Nelson ◽

Timothy R. Wagenaar ◽

Bernard Moss

Keyword(s):

Amino Acid ◽

Virus Infection ◽

Glutamic Acid ◽

Vaccinia Virus ◽

Virus Entry ◽

Amino Acid Substitutions ◽

Null Mutant ◽

Negative Effects ◽

Conserved Sequence ◽

Amino Acid Segment

ABSTRACT The recently described vaccinia virus entry/fusion complex (EFC) comprises at least eight polypeptides that are conserved in all poxviruses. Neither the structure of the complex nor the roles of individual subunits are known. Here we provide evidence for an interaction between the H2 and A28 subunits in the context of a virus infection as well as in uninfected cells transfected with plasmids expressing the corresponding genes. We focused on a highly conserved 21-amino acid-segment in H2 that is flanked by cysteine residues. The effect of amino acid substitutions within the 21-amino-acid segment was determined by an infectivity complementation assay using a conditional H2-null mutant of vaccinia virus. Mutations that had no, moderate, or large negative effects on complementation were found. The latter group included glutamic acid substitutions of leucine and individual glycines and alanine substitution of both glycines within a LGYSG sequence. Mutations with the most pronounced effect on infectivity disrupted the interaction of H2 with A28 to the greatest extent in both infected and uninfected cells. These data indicate that the LGYSG sequence is important for the interaction of H2 with A28 and suggest that this sequence is buried within the EFC complex.

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Vaccinia virus L1 binds to cell surfaces and blocks virus entry independently of glycosaminoglycans

Virology ◽

10.1016/j.virol.2008.12.019 ◽

2009 ◽

Vol 385 (2) ◽

pp. 368-382 ◽

Cited By ~ 24

Author(s):

Chwan Hong Foo ◽

Huan Lou ◽

J. Charles Whitbeck ◽

Manuel Ponce-de-León ◽

Doina Atanasiu ◽

...

Keyword(s):

Vaccinia Virus ◽

Virus Entry ◽

Cell Surfaces

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Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.79.8.4744-4754.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4744-4754 ◽

Cited By ~ 70

Author(s):

Tatiana G. Senkevich ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Cell Fusion ◽

Essential Role ◽

Transmembrane Domain ◽

Virus Entry ◽

Recombinant Vaccinia Virus ◽

Low Ph ◽

Microscopic Appearance ◽

Recombinant Vaccinia ◽

Virus Morphogenesis

ABSTRACT The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.

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Vaccinia Virus Entry into Cells via a Low-pH-Dependent Endosomal Pathway

Journal of Virology ◽

10.1128/jvi.01053-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 8899-8908 ◽

Cited By ~ 120

Author(s):

Alan C. Townsley ◽

Andrea S. Weisberg ◽

Timothy R. Wagenaar ◽

Bernard Moss

Keyword(s):

Plasma Membrane ◽

Vaccinia Virus ◽

Virus Entry ◽

Low Ph ◽

Neutral Ph ◽

Concanamycin A ◽

Endosomal Acidification ◽

Wide Range ◽

Endosomal Pathway ◽

Ph Dependent

ABSTRACT Previous studies established that vaccinia virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of vaccinia virus-infected cells, a hallmark of viruses that enter by the endosomal route. Here, we demonstrate that entry of mature vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of vaccinia virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of vaccinia virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and virus fusion. The choice of two distinct pathways may contribute to the ability of vaccinia virus to enter a wide range of cells.

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Vaccinia Virus Envelope D8L Protein Binds to Cell Surface Chondroitin Sulfate and Mediates the Adsorption of Intracellular Mature Virions to Cells

Journal of Virology ◽

10.1128/jvi.73.10.8750-8761.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8750-8761 ◽

Cited By ~ 172

Author(s):

Jye-Chian Hsiao ◽

Che-Sheng Chung ◽

Wen Chang

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Chondroitin Sulfate ◽

Virus Entry ◽

Viral Envelope ◽

Binding Assays ◽

Viral Envelope Protein ◽

Virus Envelope ◽

Virus Growth ◽

Control Virus

ABSTRACT We previously showed that an envelope A27L protein of intracellular mature virions (IMV) of vaccinia virus binds to cell surface heparan sulfate during virus infection. In the present study we identified another viral envelope protein, D8L, that binds to chondroitin sulfate on cells. Soluble D8L protein interferes with the adsorption of wild-type vaccinia virions to cells, indicating a role in virus entry. To explore the interaction of cell surface glycosaminoglycans and vaccinia virus, we generated mutant viruses from a control virus, WR32-7/Ind14K (A27L+ D8L+) to be defective in expression of either the A27L or the D8L gene (A27L+D8L− or A27L− D8L+) or both (A27L− D8L−). The A27L+D8L+ and A27L− D8L+ mutants grew well in BSC40 cells, consistent with previous observations. However, the IMV titers of A27L+ D8L− and A27L− D8L− viruses in BSC40 cells were reduced, reaching only 10% of the level for the control virus. The data suggested an important role for D8L protein in WR32-7/Ind14K virus growth in cell cultures. A27L protein, on the other hand, could not complement the functions of D8L protein. The low titers of the A27L+ D8L− and A27L−D8L− mutant viruses were not due to defects in the morphogenesis of IMV, and the mutant virions demonstrated a brick shape similar to that of the control virions. Furthermore, the infectivities of the A27L+ D8L− and A27L−D8L− mutant virions were 6 to 10% of that of the A27L+ D8L+ control virus. Virion binding assays revealed that A27L+ D8L− and A27L− D8L− mutant virions bound less well to BSC40 cells, indicating that binding of viral D8L protein to cell surface chondroitin sulfate could be important for vaccinia virus entry.

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