scholarly journals Vaccinia Virus Protein F12 Associates with Intracellular Enveloped Virions through an Interaction with A36

2008 ◽  
Vol 83 (4) ◽  
pp. 1708-1717 ◽  
Author(s):  
Sara C. Johnston ◽  
Brian M. Ward
Keyword(s):  
Amino Acids ◽  
Vaccinia Virus ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Recombinant Vaccinia Virus ◽  
Virus Protein ◽  
Viral Egress ◽  
Plaque Phenotype ◽  
Infected Cells

ABSTRACT Vaccinia virus is the prototypical member of the family Poxviridae. Three morphologically distinct forms are produced during infection: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular enveloped virions (EEV). Two viral proteins, F12 and A36, are found exclusively on IEV but not on IMV and EEV. Analysis of membranes from infected cells showed that F12 was only associated with membranes and is not an integral membrane protein. A yeast two-hybrid assay revealed an interaction between amino acids 351 to 458 of F12 and amino acids 91 to 111 of A36. We generated a recombinant vaccinia virus that expresses an F12, which lacks residues 351 to 458. Characterization of this recombinant revealed a small-plaque phenotype and a subsequent defect in virus release similar to a recombinant virus that had F12L deleted. In addition, F12 lacking residues 351 to 458 was unable to associate with membranes in infected cells. These results suggest that F12 associates with IEV through an interaction with A36 and that this interaction is critical for the function of F12 during viral egress.

scholarly journals Interactions between Vaccinia Virus IEV Membrane Proteins and Their Roles in IEV Assembly and Actin Tail Formation

1999 ◽  
Vol 73 (4) ◽  
pp. 2863-2875 ◽  
Author(s):  
Sabine Röttger ◽  
Friedrich Frischknecht ◽  
Inge Reckmann ◽  
Geoffrey L. Smith ◽  
Michael Way
Keyword(s):  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Protein S ◽  
Recombinant Vaccinia Virus ◽  
Virus Protein ◽  
Particle Assembly ◽  
Severe Reduction ◽  
Specific Proteins ◽  
Infected Cells ◽  
Virus Strains

ABSTRACT The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the ΔB5R and ΔF13L deletion strains, but not ΔA34R, were still able to induce actin tails. The ΔA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from ΔA34R- and ΔA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.

scholarly journals Fowlpox Virus Encodes a Bcl-2 Homologue That Protects Cells from Apoptotic Death through Interaction with the Proapoptotic Protein Bak

2007 ◽  
Vol 81 (20) ◽  
pp. 11032-11045 ◽  
Author(s):  
Logan Banadyga ◽  
Jenna Gerig ◽  
Tara Stewart ◽  
Michele Barry
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Recombinant Vaccinia Virus ◽  
Virus Protein ◽  
Necrosis Factor Alpha ◽  
Fowlpox Virus ◽  
Proapoptotic Protein ◽  
Infected Cells ◽  
Factor Alpha ◽  
Transient Overexpression

ABSTRACT Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.

Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.

1988 ◽  
Vol 62 (1) ◽  
pp. 176-180 ◽  
Author(s):  
S L Hu ◽  
G D Plowman ◽  
P Sridhar ◽  
U S Stevenson ◽  
J P Brown ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Recombinant Vaccinia Virus ◽  
Human Melanoma ◽  
Recombinant Vaccinia

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.

Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus

1993 ◽  
Vol 61 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Mary-Helen Binger ◽  
Denis Hug ◽  
Gilbert Weber ◽  
Eugene Schildknecht ◽  
Markus Hümbelin ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Surface Antigen ◽  
Recombinant Vaccinia Virus ◽  
Eimeria Tenella ◽  
Recombinant Vaccinia

Plasmodium falciparum:Heterologous Synthesis of the Transmission-Blocking Vaccine Candidate Pfs48/45 in Recombinant Vaccinia Virus-Infected Cells

1998 ◽  
Vol 90 (2) ◽  
pp. 165-174 ◽  
Author(s):  
Richard L.B. Milek ◽  
Antoine A.F. DeVries ◽  
Will F.G. Roeffen ◽  
Henk Stunnenberg ◽  
Peter J.M. Rottier ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Vaccine Candidate ◽  
Recombinant Vaccinia Virus ◽  
Recombinant Vaccinia ◽  
Transmission Blocking ◽  
Infected Cells ◽  
Transmission Blocking Vaccine

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 Novel Acylguanidine-Based Inhibitor of HIV-1

2016 ◽  
Vol 90 (20) ◽  
pp. 9495-9508 ◽  
Author(s):  
Philip Mwimanzi ◽  
Ian Tietjen ◽  
Scott C. Miller ◽  
Aniqa Shahid ◽  
Kyle Cobarrubias ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Antiretroviral Drugs ◽  
Virion Release ◽  
Viral Egress ◽  
Pharmacological Studies ◽  
Infected Cells ◽  
New Treatments ◽  
Hiv 1

ABSTRACTThe emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibitsin vitroreplication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24Gagproduction was unaffected, but virion release (measured as extracellular p24Gag) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity.IMPORTANCENew inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

scholarly journals Characterization of Two Putative Protein Translocation Components in the Apicoplast of Plasmodium falciparum

2009 ◽  
Vol 8 (8) ◽  
pp. 1146-1154 ◽  
Author(s):  
Ming Kalanon ◽  
Christopher J. Tonkin ◽  
Geoffrey I. McFadden
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Trafficking ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Diatom Algae ◽  
Trophozoite Stage ◽  
Replacement Strategy ◽  
Putative Protein

ABSTRACT Protein trafficking to the stroma of the apicoplast of Plasmodium falciparum requires translocation across several membranes. To further elucidate the mechanisms responsible, we investigated two proteins: P. falciparum Tic22 (PfTic22), a putative component of the translocon of the inner chloroplast membrane; and PfsDer1-1, one of two homologues of the P. falciparum symbiont-derived Der1 (sDer1) protein, a putative component of an endoplasmic reticulum-associated degradation (ERAD) complex in the periplastid membrane. We constructed parasites expressing hemagglutinin (HA)-tagged PfTic22 and PfsDer1-1 under the control of their endogenous promoters using the 3′ replacement strategy. We show that both PfTic22-HA and PfsDer1-1-HA are expressed predominantly during the trophozoite stage of the asexual replication cycle, which corresponds to the most dynamic stages of apicoplast activity. Although both proteins localize to the periphery of the apicoplast, PfTic22-HA is a membrane-associated protein while PfsDer1-1-HA is an integral membrane protein. Phylogenetic analysis indicates that PfsDer1-1 is one of two Der1 paralogues predicted to localize to the apicoplast in P. falciparum and that it has orthologues in diatom algae, supporting the chromalveolate hypothesis. These observations are consistent with putative roles for PfTic22 and PfsDer1-1 in protein translocation into the apicoplast of P. falciparum.

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