scholarly journals Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

1987 ◽  
Vol 166 (3) ◽  
pp. 668-677 ◽  
Author(s):  
H Volkmer ◽  
C Bertholet ◽  
S Jonjić ◽  
R Wittek ◽  
U H Koszinowski
Keyword(s):  
Vaccinia Virus ◽  
Antigen Expression ◽  
Recombinant Vaccinia Virus ◽  
Virus Genome ◽  
Site Directed Mutagenesis ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Early Protein ◽  
Infected Cells ◽  
Essential Function

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

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 Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

1984 ◽  
Vol 4 (10) ◽  
pp. 2214-2223 ◽  
Author(s):  
K T Jeang ◽  
M S Cho ◽  
G S Hayward
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Regulatory Region ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Mouse Cell ◽  
Cellular Protein ◽  
Immediate Early ◽  
Early Protein ◽  
Infected Cells

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

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 Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

Viruses ◽  
2014 ◽  
Vol 6 (2) ◽  
pp. 808-831 ◽  
Author(s):  
Annette Fink ◽  
Julia Büttner ◽  
Doris Thomas ◽  
Rafaela Holtappels ◽  
Matthias Reddehase ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Epitope ◽  
Cd8 T Cell ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
T Cell Epitope ◽  
Upstream Gene ◽  
Early Protein

scholarly journals Vaccinia Virus Telomeres: Interaction with the Viral I1, I6, and K4 Proteins

2001 ◽  
Vol 75 (21) ◽  
pp. 10090-10105 ◽  
Author(s):  
Joseph DeMasi ◽  
Shan Du ◽  
David Lennon ◽  
Paula Traktman
Keyword(s):  
Complex Formation ◽  
Vaccinia Virus ◽  
Affinity Purification ◽  
Virus Genome ◽  
Specific Protein ◽  
Telomeric Sequence ◽  
Genome Replication ◽  
Mobility Shift ◽  
Infected Cells ◽  
Necessary And Sufficient

ABSTRACT The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the “flip” isoform and two with the “flop” isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation.

scholarly journals High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells

2003 ◽  
Vol 77 (13) ◽  
pp. 7281-7290 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Genetic Recombination ◽  
Virus Genome ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Plaque Purification ◽  
Infected Cells ◽  
Clone And Express ◽  
Recombination Reactions ◽  
Multiple Pcr

ABSTRACT Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 106 PFU/μg of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.

scholarly journals The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription.

1986 ◽  
Vol 58 (1) ◽  
pp. 59-66 ◽  
Author(s):  
U H Koszinowski ◽  
G M Keil ◽  
H Volkmer ◽  
M R Fibi ◽  
A Ebeling-Keil ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Early Protein

scholarly journals A nonstructural polypeptide encoded by immediate-early transcription unit 1 of murine cytomegalovirus is recognized by cytolytic T lymphocytes.

1987 ◽  
Vol 166 (1) ◽  
pp. 289-294 ◽  
Author(s):  
U H Koszinowski ◽  
G M Keil ◽  
H Schwarz ◽  
J Schickedanz ◽  
M J Reddehase
Keyword(s):  
Regulatory Protein ◽  
Antigen Expression ◽  
Transcription Unit ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Target Cells ◽  
Cytolytic T Lymphocytes ◽  
Gene Encoding ◽  
Dna Coding ◽  
Early Transcription

We have constructed target cells by cotransfection of the MHC gene Ld and fragments of murine cytomegalovirus (MCMV) DNA coding for nonstructural immediate-early (IE) proteins. Transfectants were tested by using CTL clone IE1 with specificity for an IE epitope presented in association with Ld. Data show that clone IE1 recognizes a product of the ie1 transcription unit of MCMV, and that its specificity is shared by approximately 25% of polyclonal IE-specific CTL. The results provide the first definite evidence that expression of a herpes virus IE gene encoding a regulatory protein gives rise to antigen expression detectable by specific CTL.

scholarly journals An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA

1992 ◽  
Vol 73 (3) ◽  
pp. 499-506 ◽  
Author(s):  
K. Munch ◽  
M. Messerle ◽  
B. Plachter ◽  
U. H. Koszinowski
Keyword(s):  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Early Protein ◽  
Acidic Region

scholarly journals Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression

2018 ◽  
Vol 26 ◽  
pp. 204020661876319 ◽  
Author(s):  
Koh-Hei Yamada ◽  
Ryuichi Majima ◽  
Toyofumi Yamaguchi ◽  
Naoki Inoue
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Early Gene ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Viral Attachment ◽  
Varicella Zoster ◽  
Infected Cells

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6–(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing β-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A “time of addition” experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.

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