In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites.

1981 ◽  
Vol 37 (2) ◽  
pp. 738-747 ◽  
Author(s):  
S Venkatesan ◽  
B Moss
Keyword(s):  
Vaccinia Virus ◽  
In Vitro Transcription ◽  
Virus Genome

scholarly journals Vaccinia Virus F1L Protein Is a Tail-Anchored Protein That Functions at the Mitochondria To Inhibit Apoptosis

2005 ◽  
Vol 79 (2) ◽  
pp. 1084-1098 ◽  
Author(s):  
Tara L. Stewart ◽  
Shawn T. Wasilenko ◽  
Michele Barry
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Transmembrane Domain ◽  
In Vitro Transcription ◽  
Translation System ◽  
Reading Frame ◽  
Protease Digestion ◽  
Necessary And Sufficient ◽  
Virus Survival

ABSTRACT Members of the poxvirus family encode multiple immune evasion proteins, including proteins that regulate apoptosis. We recently identified one such protein, F1L, encoded by vaccinia virus, the prototypic member of the poxvirus family. F1L localizes to the mitochondria and inhibits apoptosis by interfering with the release of cytochrome c, the pivotal commitment step in the apoptotic cascade. Sequence analysis of the F1L open reading frame revealed a C-terminal motif composed of a 12-amino-acid transmembrane domain flanked by positively charged lysines, followed by an 8-amino-acid hydrophilic tail. By generating a series of F1L deletion constructs, we show that the C-terminal domain is necessary and sufficient for localization of F1L to the mitochondria. In addition, mutation of lysines 219 and 222 downstream of the C-terminal transmembrane domain resulted in altered localization of F1L to the endoplasmic reticulum. Using F1L protein generated in an in vitro transcription-translation system, we found that F1L was posttranslationally inserted into mitochondria and tightly associated with mitochondrial membranes as demonstrated by resistance to alkaline extraction. Sensitivity to protease digestion showed that the N terminus of F1L was exposed to the cytoplasm. Utilizing various F1L deletion constructs, we found that F1L localization to the mitochondria was necessary to inhibit apoptosis, since constructs that no longer localized to the mitochondria had reduced antiapoptotic ability. Our studies show that F1L is a new member of the tail-anchored protein family that localizes to mitochondria during virus infection and inhibits apoptosis as a means to enhance virus survival.

In vitro transcription and replication of the mumps virus genome

1993 ◽  
Vol 128 (1-2) ◽  
pp. 177-183 ◽  
Author(s):  
K. Takeuchi ◽  
K. Tanabayashi ◽  
K. Okazaki ◽  
M. Hiahiyama ◽  
A. Yamada
Keyword(s):  
In Vitro Transcription ◽  
Virus Genome ◽  
Mumps Virus ◽  
Transcription And Replication

scholarly journals A New Vaccinia Virus Intermediate Transcription Factor

1998 ◽  
Vol 72 (8) ◽  
pp. 6880-6883 ◽  
Author(s):  
Patrick Sanz ◽  
Bernard Moss
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Late Stage ◽  
Virus Genome ◽  
Cellular Protein ◽  
Encoded Proteins ◽  
Capping Enzyme ◽  
Specific Factors

ABSTRACT Transcription of the vaccinia virus genome is mediated by a virus-encoded multisubunit DNA-dependent RNA polymerase in conjunction with early-, intermediate-, and late-stage-specific factors. Previous studies indicated that two virus-encoded proteins (capping enzyme and VITF-1) and one unidentified cellular protein (VITF-2) are required for specific transcription of an intermediate promoter template in vitro. We have now extensively purified an additional virus-induced intermediate transcription factor with a native mass of approximately 100 kDa.

scholarly journals Characterization of the Vaccinia Virus H3L Envelope Protein: Topology and Posttranslational Membrane Insertion via the C-Terminal Hydrophobic Tail

2000 ◽  
Vol 74 (16) ◽  
pp. 7508-7517 ◽  
Author(s):  
Flávio G. da Fonseca ◽  
Elizabeth J. Wolffe ◽  
Andrea Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Amorphous Material ◽  
In Vitro Transcription ◽  
Proteinase K ◽  
Translation System ◽  
Membrane Insertion ◽  
Hydrophobic Domain ◽  
Viral Particles ◽  
Hydrophobic Tail

ABSTRACT The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodominant membrane component of virus particles. Biochemical and microscopic studies demonstrated that the H3L protein was expressed late in infection, accumulated in the cytoplasmic viral factory regions, and associated primarily with amorphous material near immature virions and with intracellular virion membranes. Localization of the H3L protein on the surfaces of viral particles and anchorage via the hydrophobic tail were consistent with its extraction by NP-40 in the absence of reducing agents, its trypsin sensitivity, its reactivity with a membrane-impermeable biotinylation reagent, and its immunogold labeling with an antibody to a peptide comprising amino acids 247 to 259. The H3L protein, synthesized in a coupled in vitro transcription/translation system, was tightly anchored to membranes as determined by resistance to Na2CO3 (pH 11) extraction and cytoplasmically oriented as shown by sensitivity to proteinase K digestion. Further studies demonstrated that membrane insertion of the H3L protein occurred posttranslationally and that the C-terminal hydrophobic domain was necessary and sufficient for this to occur. These data indicated that the H3L protein is a member of the C-terminal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their maturation.

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