scholarly journals Vaccinia Virus L1 Protein Is Required for Cell Entry and Membrane Fusion

2008 ◽  
Vol 82 (17) ◽  
pp. 8687-8694 ◽  
Author(s):  
Himani Bisht ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Viral Entry ◽  
Virus Assembly ◽  
Redox System ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Transmission Electron ◽  
Genes Encoding ◽  
L1 Protein

ABSTRACT Genetic and biochemical studies have provided evidence for an entry/fusion complex (EFC) comprised of at least eight viral proteins (A16, A21, A28, G3, G9, H2, J5, and L5) that together with an associated protein (F9) participates in entry of vaccinia virus (VACV) into cells. The genes encoding these proteins are conserved in all poxviruses, are expressed late in infection, and are components of the mature virion membrane but are not required for viral morphogenesis. In addition, all but one component has intramolecular disulfides that are formed by the poxvirus cytoplasmic redox system. The L1 protein has each of the characteristics enumerated above except that it has been reported to be essential for virus assembly. To further investigate the role of L1, we constructed a recombinant VACV (vL1Ri) that inducibly expresses L1. In the absence of inducer, L1 synthesis was repressed and vL1Ri was unable to form plaques or produce infectious progeny. Unexpectedly, assembly and morphogenesis appeared normal and the noninfectious virus particles were indistinguishable from wild-type VACV as determined by transmission electron microscopy and analysis of the component polypeptides. Notably, the L1-deficient virions were able to attach to cells but the cores failed to penetrate into the cytoplasm. In addition, cells infected with vL1Ri in the absence of inducer did not form syncytia following brief low-pH treatment even though extracellular virus was produced. Coimmunoprecipitation experiments demonstrated that L1 interacted with the EFC and indirectly with F9, suggesting that L1 is an additional component of the viral entry apparatus.

scholarly journals Entry of Vaccinia Virus and Cell-Cell Fusion Require a Highly Conserved Cysteine-Rich Membrane Protein Encoded by the A16L Gene

2006 ◽  
Vol 80 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Suany Ojeda ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Syncytium Formation ◽  
Redox System ◽  
Cysteine Residues ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Cell Cell

ABSTRACT The vaccinia virus A16L open reading frame encodes a 378-amino-acid protein with a predicted C-terminal transmembrane domain and 20 invariant cysteine residues that is conserved in all sequenced members of the poxvirus family. The A16 protein was expressed late in infection and incorporated into intracellular virus particles with the N-terminal segment of the protein exposed on the surface. The cysteine residues were disulfide bonded via the poxvirus cytoplasmic redox system. Unsuccessful attempts to isolate a mutant virus with the A16L gene deleted suggested that the protein is essential for replication. To study the role of the A16 protein, we made a recombinant vaccinia virus that has the Escherichia coli lac operator system regulating transcription of the A16L gene. In the absence of inducer, A16 synthesis was repressed and plaque size and virus yield were greatly reduced. Nevertheless, virus morphogenesis occurred and normal-looking intracellular and extracellular virus particles formed. Purified virions made in the presence and absence of inducer were indistinguishable, though the latter had 60- to 100-fold-lower specific infectivity. A16-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, A16-deficient virions were unable to induce low-pH-triggered syncytium formation. The phenotype of the inducible A16L mutant was similar to those of mutants in which synthesis of the A21, A28, H2, or L5 membrane protein was repressed, indicating that at least five conserved viral proteins are required for entry of poxviruses into cells as well as for cell-cell fusion.

scholarly journals Role of Conserved Cysteines in the Alphavirus E3 Protein

2008 ◽  
Vol 83 (6) ◽  
pp. 2584-2591 ◽  
Author(s):  
Megan M. Parrott ◽  
Sarah A. Sitarski ◽  
Randy J. Arnold ◽  
Lora K. Picton ◽  
R. Blake Hill ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Viral Entry ◽  
Disulfide Bonds ◽  
Functional Role ◽  
Sindbis Virus ◽  
Virus Assembly ◽  
Wild Type ◽  
Virus Particles ◽  
Extracellular Glycoprotein

ABSTRACT Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.

scholarly journals Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

2017 ◽  
Vol 114 (51) ◽  
pp. E11001-E11009 ◽  
Author(s):  
Andrea S. Weisberg ◽  
Liliana Maruri-Avidal ◽  
Himani Bisht ◽  
Bryan T. Hansen ◽  
Cindi L. Schwartz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Virus Assembly ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Deletion Mutants ◽  
Viral Membrane ◽  
Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

scholarly journals Effects of Deletion or Stringent Repression of the H3L Envelope Gene on Vaccinia Virus Replication

2000 ◽  
Vol 74 (16) ◽  
pp. 7518-7528 ◽  
Author(s):  
Flávio G. da Fonseca ◽  
Elizabeth J. Wolffe ◽  
Andrea Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Particle Production ◽  
High Ratio ◽  
Surface Proteins ◽  
Envelope Gene ◽  
Late Gene Expression ◽  
Electron Microscopic ◽  
Extracellular Virus

ABSTRACT The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Δ) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Δ or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Δ or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.

scholarly journals Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein

2006 ◽  
Vol 80 (19) ◽  
pp. 9455-9464 ◽  
Author(s):  
Erica Brown ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Neutralizing Antibodies ◽  
Virus Assembly ◽  
Recombinant Vaccinia Virus ◽  
Amino Acid Identity ◽  
Wild Type Virus ◽  
Structural Relationships ◽  
Membrane Components ◽  
L1 Protein ◽  
Immature Virus

ABSTRACT All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.

scholarly journals Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

2009 ◽  
Vol 83 (22) ◽  
pp. 11477-11490 ◽  
Author(s):  
Desyreé Murta Jesus ◽  
Lilian T. Costa ◽  
Daniela L. Gonçalves ◽  
Carlos Alberto Achete ◽  
Marcia Attias ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Detailed Analysis ◽  
Virus Assembly ◽  
Microscopic Analysis ◽  
Progeny Virus ◽  
Late Gene Expression ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Core Proteins ◽  
Genome Encapsidation

ABSTRACT Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

scholarly journals Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

2004 ◽  
Vol 78 (12) ◽  
pp. 6335-6343 ◽  
Author(s):  
Camilo Ansarah-Sobrinho ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Lethal Mutant ◽  
Core Proteins ◽  
Core Components

ABSTRACT Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.

scholarly journals Vaccinia Virus A21 Virion Membrane Protein Is Required for Cell Entry and Fusion

2005 ◽  
Vol 79 (15) ◽  
pp. 9458-9469 ◽  
Author(s):  
Alan C. Townsley ◽  
Tatiana G. Senkevich ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Disulfide Bonds ◽  
Low Ph ◽  
Cell Entry ◽  
Lethal Mutant ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Virus Particles ◽  
Extracellular Virus

ABSTRACT We provide the initial characterization of the product of the vaccinia virus A21L (VACWR140) gene and demonstrate that it is required for cell entry and low pH-triggered membrane fusion. The A21L open reading frame, which is conserved in all sequenced members of the poxvirus family, encodes a protein of 117 amino acids with an N-terminal hydrophobic domain and four invariant cysteines. Expression of the A21 protein occurred at late times of infection and was dependent on viral DNA replication. The A21 protein contained two intramolecular disulfide bonds, the formation of which required the vaccinia virus-encoded cytoplasmic redox pathway, and was localized on the surface of the lipoprotein membrane of intracellular mature virions. A conditional lethal mutant, in which A21L gene expression was regulated by isopropyl-β-d-thiogalactopyranoside, was constructed. In the absence of inducer, cell-to-cell spread of virus did not occur, despite the formation of morphologically normal intracellular virions and extracellular virions with actin tails. Purified virions lacking A21 were able to bind to cells, but cores did not penetrate into the cytoplasm and synthesize viral RNA. In addition, virions lacking A21 were unable to mediate low pH-triggered cell-cell fusion. The A21 protein, like the A28 and H2 proteins, is an essential component of the poxvirus entry/fusion apparatus for both intracellular and extracellular virus particles.

scholarly journals The Conserved Poxvirus L3 Virion Protein Is Required for Transcription of Vaccinia Virus Early Genes

2005 ◽  
Vol 79 (23) ◽  
pp. 14719-14729 ◽  
Author(s):  
Wolfgang Resch ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Promoter Sequence ◽  
Wild Type ◽  
Virion Protein ◽  
Reading Frame ◽  
Virus Particles ◽  
Mature Virus

ABSTRACT We provide the initial characterization of the product of the vaccinia virus L3L open reading frame (VACWR090), which is conserved in all sequenced members of the poxvirus family. The predicted polypeptide contains no motifs or other features that provided a clue to the role of the L3 protein, and no functional information was available regarding a homolog discovered in Plasmodium falciparum. The L3 protein was expressed following viral DNA replication, a finding consistent with a putative late promoter sequence, and was packaged as a non-membrane protein in mature virus particles. A recombinant virus, in which the L3L gene was regulated by the Escherichia coli lac operator/repressor system, had a conditional lethal phenotype. The virus replicated in the presence of inducer, but in its absence, the yields of infectious virus were reduced by 99%. When cells were infected without inducer, however, no defect in gene expression or morphogenesis was noted. Virus particles lacking L3, which assembled in the absence of inducer, were indistinguishable from wild-type virions with regard to morphology, major structural proteins, and DNA content but were noninfectious. L3-deficient virions were able to bind and penetrate cells but produced extremely small amounts of viral early mRNA. A defect in transcription was demonstrated by in vitro studies with permeabilized virions, but soluble extracts of L3-deficient virions showed normal levels of template-dependent transcriptional activity, indicating that only transcription of the packaged genome is impaired.

scholarly journals Single Amino Acid Insertions at the Junction of the Sindbis Virus E2 Transmembrane Domain and Endodomain Disrupt Virus Envelopment and Alter Infectivity

2005 ◽  
Vol 79 (12) ◽  
pp. 7682-7697 ◽  
Author(s):  
Raquel Hernandez ◽  
Davis Ferreira ◽  
Christine Sinodis ◽  
Katherine Litton ◽  
Dennis T. Brown
Keyword(s):  
Amino Acid ◽  
Sindbis Virus ◽  
Insect Cells ◽  
Virus Assembly ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Insertion Mutant ◽  
Bhk Cells ◽  
Virus Particles

ABSTRACT The final steps in the envelopment of Sindbis virus involve specific interactions of the E2 endodomain with the virus nucleocapsid. Deleting E2 K at position 391 (E2 ΔK391) resulted in the disruption of virus assembly in mammalian cells but not insect cells (host range mutant). This suggested unique interactions of the E2 ΔK391 endodomain with the different biochemical environments of the mammalian and insect cell lipid bilayers. To further investigate the role of the amino acid residues located at or around position E2 391 and constraints on the length of the endodomain on virus assembly, amino acid insertions/substitutions at the transmembrane/endodomain junction were constructed. An additional K was inserted at amino acid position 392 (KK391/392), a K→F substitution at position 391 was constructed (F391), and an additional F was inserted at 392 (FF391/392). These changes should lengthen the endodomain in the KK391/392 insertion mutant or shorten the endodomain in the FF391/392 mutant. The mutant FF391/392 grown in BHK cells formed virus particles containing extruded material not found on wild-type virus. This characteristic was not seen in FF391/392 virus grown in insect cells. The mutant KK391/392 grown in BHK cells was defective in the final membrane fission reaction, producing multicored or conjoined virus particles. The production of these aberrant particles was ameliorated when the KK391/392 mutant was grown in insect cells. These data indicate that there is a critical minimal spanning distance from the E2 membrane proximal amino acid at position 391 and the conserved E2 Y400 residue. The observed phenotypes of these mutants also invoke an important role of the specific host membrane lipid composition on virus architecture and infectivity.

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