scholarly journals Interaction between Vaccinia Virus Extracellular Virus Envelope A33 and B5 Glycoproteins

2006 ◽  
Vol 80 (17) ◽  
pp. 8763-8777 ◽  
Author(s):  
Beatriz Perdiguero ◽  
Rafael Blasco
Keyword(s):  
Vaccinia Virus ◽  
Protein Interactions ◽  
Transmembrane Domain ◽  
Recombinant Vaccinia Virus ◽  
Protein Protein Interactions ◽  
Virus Envelope ◽  
Extracellular Virus ◽  
Vaccinia Viruses ◽  
Extracellular Form ◽  
Infected Cells

ABSTRACT The extracellular form of vaccinia virus acquires its outer envelope by wrapping with cytoplasmic membranes that contain at least seven virus-encoded proteins, of which four are glycoproteins. We searched for interactions between the vaccinia virus A33 glycoprotein and proteins A34, A36, B5, F12, and F13. First, when myc epitope-tagged A33 was expressed in combination with other envelope proteins, A33 colocalized with B5 and A36, suggesting that direct A33-B5 and A33-A36 interactions occur in the absence of infection. A recombinant vaccinia virus (vA33Rmyc) was constructed by introduction of the myc-tagged A33 version (A33myc) into A33-deficient vaccinia virus. A33myc partially restored plaque formation and colocalized with enveloped virions in infected cells. Coimmunoprecipitation experiments with extracts of vA33Rmyc-infected cells confirmed the existence of a physical association of A33 with A36 and B5. Of these, the A33-B5 interaction is a novel finding, whereas the interaction between A33 and A36 has been previously characterized. A collection of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A33. Both the cytoplasmic domain and most of the extracellular domain, but not the transmembrane domain, of the B5 protein were dispensable for binding to A33. Mutations in the extracellular portions of B5 and A33 that enhance extracellular virus release did not affect the interaction between the two. In contrast, substituting the B5 transmembrane domain with that of the vesicular stomatitis virus G glycoprotein prevented the association with A33. Immunofluorescence experiments on virus mutants indicated that B5 is required for efficient targeting of A33 into enveloped virions. These results point to the transmembrane domain of B5 as the major determinant of the A33-B5 interaction and demonstrate that protein-protein interactions are crucial in determining the composition of the virus envelope.

scholarly journals Vaccinia Virus A34 Glycoprotein Determines the Protein Composition of the Extracellular Virus Envelope

2007 ◽  
Vol 82 (5) ◽  
pp. 2150-2160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
María M. Lorenzo ◽  
Rafael Blasco
Keyword(s):  
Vaccinia Virus ◽  
Protein Composition ◽  
Recombinant Vaccinia Virus ◽  
Virus Envelope ◽  
Enveloped Virus ◽  
Extracellular Virus ◽  
Transmembrane Glycoprotein ◽  
C Terminus ◽  
Vaccinia Viruses ◽  
Infected Cells

ABSTRACT The outer envelope of the extracellular form of vaccinia virus contains five virus-encoded proteins, F13, A33, A34, A56, and B5, that, with the exception of A56, are implicated in virus egress or infectivity. A34, a type II transmembrane glycoprotein, is involved in the induction of actin tails, the release of enveloped virus from the surfaces of infected cells, and the disruption of the virus envelope after ligand binding prior to virus entry. To investigate interactions between A34 and other envelope proteins, a recombinant vaccinia virus (vA34RHA) expressing an epitope-tagged version of A34 (A34HA) was constructed by appending an epitope from influenza virus hemagglutinin to the C terminus of A34. Complexes of A34HA with B5 and A36, but not with A33 or F13, were detected in vA34RHA-infected cells. A series of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A34. Both the cytoplasmic and the transmembrane domains of B5 were dispensable for binding to A34. Most of the extracellular domain of B5, which contains four short consensus repeats homologous to complement control proteins, was sufficient for A34 interaction, indicating that both proteins interact through their ectodomains. Immunofluorescence experiments on cells infected with A34-deficient virus indicated that A34 is required for efficient targeting of B5, A36, and A33 into wrapped virions. Consistent with this observation, the envelope of A34-deficient virus contained normal amounts of F13 but decreased amounts of A33 and B5 with respect to the parental WR virus. These results point to A34 as a major determinant in the protein composition of the vaccinia virus envelope.

scholarly journals Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5

2012 ◽  
Vol 93 (4) ◽  
pp. 733-743 ◽  
Author(s):  
María M. Lorenzo ◽  
Juana M. Sánchez-Puig ◽  
Rafael Blasco
Keyword(s):  
Vaccinia Virus ◽  
Protein Interactions ◽  
Intracellular Transport ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Virus Transmission ◽  
Cytoplasmic Tail ◽  
Type I ◽  
Virus Envelope ◽  
Extracellular Virus

The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein–protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

scholarly journals Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

1986 ◽  
Vol 6 (9) ◽  
pp. 3191-3199 ◽  
Author(s):  
C J Langford ◽  
S J Edwards ◽  
G L Smith ◽  
G F Mitchell ◽  
B Moss ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Poor Response ◽  
Subcellular Location ◽  
Epidemiological Data ◽  
Recombinant Vaccinia Virus ◽  
Live Virus ◽  
Recombinant Vaccinia ◽  
Wide Range ◽  
Vaccinia Viruses ◽  
Infected Cells

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity

1986 ◽  
Vol 6 (9) ◽  
pp. 3191-3199
Author(s):  
C J Langford ◽  
S J Edwards ◽  
G L Smith ◽  
G F Mitchell ◽  
B Moss ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Poor Response ◽  
Subcellular Location ◽  
Epidemiological Data ◽  
Recombinant Vaccinia Virus ◽  
Live Virus ◽  
Recombinant Vaccinia ◽  
Wide Range ◽  
Vaccinia Viruses ◽  
Infected Cells

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

scholarly journals The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

2006 ◽  
Vol 80 (17) ◽  
pp. 8402-8410 ◽  
Author(s):  
Ruzan A. Izmailyan ◽  
Cheng-Yen Huang ◽  
Shamim Mohammad ◽  
Stuart N. Isaacs ◽  
Wen Chang
Keyword(s):  
Life Cycle ◽  
Vaccinia Virus ◽  
Transmembrane Domain ◽  
Recombinant Vaccinia Virus ◽  
Host Cells ◽  
Virus Growth ◽  
Enveloped Virus ◽  
Virus Life Cycle ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-β-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L+) or lacking (G3L−) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L+ and G3L− IMV bound to HeLa cells; however, G3L− IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Analysis of Vaccinia Virus−Host Protein−Protein Interactions: Validations of Yeast Two-Hybrid Screenings

2009 ◽  
Vol 8 (9) ◽  
pp. 4311-4318 ◽  
Author(s):  
Leiliang Zhang ◽  
Nancy Y. Villa ◽  
Masmudur M. Rahman ◽  
Sherin Smallwood ◽  
Donna Shattuck ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Protein Interactions ◽  
Host Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Two Hybrid

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 209-218 ◽  
Author(s):  
Mirriam G. J. Tacken ◽  
Peter J. M. Rottier ◽  
Arno L. J. Gielkens ◽  
Ben P. H. Peeters
Keyword(s):  
Disease Virus ◽  
Protein Interactions ◽  
Infectious Bursal Disease Virus ◽  
Viral Proteins ◽  
Infectious Bursal Disease ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Bursal Disease ◽  
Infected Cells

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein–protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1–VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1–VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1–VP3 complex is involved in replication and packaging of the IBDV genome.

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