Non‐replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase

Sutter, Gerd; Ohlmann, Marion; Erfle, Volker

doi:10.1016/0014-5793(95)00843-x

Cited by 214 publications

(140 citation statements)

References 18 publications

Supporting

Mentioning

138

Contrasting

Order By: Relevance

“…The B cell-derived cell line B95a (25) was used as host, thus limiting replication in nonlymphoid cells that may affect virulence. These cells were transfected with plasmids expressing the three proteins of the CDV polymerase complex (N, P, and L) and a full-length CDV genomic plasmid by using Lipofectamine as detailed (26), and infected with a modified vaccine Ankara virus expressing the T7 RNA polymerase (27). After 3 days, the B95a cells were overlaid on VerodogSLAMtag cells that served as indicator: within 24-48 h, syncytia were detected and transferred back to B95a cells to obtain virus stocks.…”

Section: Methodsmentioning

confidence: 99%

Tropism illuminated: Lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system

Messling

Milošević

Cattaneo

2004

Proc. Natl. Acad. Sci. U.S.A.

177

200

View full text Add to dashboard Cite

The immunosuppressive properties of morbilliviruses including measles and canine distemper virus (CDV) are well known, but the host cells supporting infection are poorly characterized. To identify these cells, a recombinant CDV expressing green fluorescent protein was produced by reverse genetics based on a wild-type strain lethal for ferrets. This recombinant virus fully retained virulence and blazed three lymphocyte-based pathways through the immune system of its host: first, it infected rapidly and massively circulating B and T cells; second, it took over and damaged secondary lymphatic organs including spleen, lymph nodes, and gut-associated and mucosal lymphoid tissues; third, it infected most thymocytes. In contrast, replication in epithelial cells was initially not detectable, but substantial before host death. Thus, CDV initially infects lymphocytes and massively replicates therein, thereby causing immunosuppression and preparing systemic invasion and host escape.virus ͉ measles ͉ immunosuppression

show abstract

Section: Methodsmentioning

confidence: 99%

Tropism illuminated: Lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system

Messling

Milošević

Cattaneo

2004

Proc. Natl. Acad. Sci. U.S.A.

177

200

View full text Add to dashboard Cite

show abstract

“…5. The T7 vaccinia virus system was used in BHK-21 cells for protein expression (9). For transfection of plasmid DNA (3 g), SuperFect was applied (Qiagen, Hilden, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…The metabolically labeled proteins were further analyzed by coradioimmunoprecipitation (CoRIP), SDS͞PAGE, and autoradiography. Protein expression throughout this study occurred in BHK-21 cells by using the vaccinia virus modified virus Ankara T7 RNA polymerase (T7pol) expression system (9). Coprecipitation was observed between Jiv-C-flag and GST-NS2͞D as well as GST-NS2͞E (Fig.…”

Section: Ns2 Encompassesmentioning

confidence: 99%

Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis

Lackner

Thiel

Tautz

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Replication of positive-strand RNA viruses involves translation of polyproteins which are proteolytically processed into functional peptides. These maturation steps often involve virus-encoded autoproteases specialized in generating their own N or C termini. Nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus represents such an enzyme. Bovine viral diarrhea virus NS2 creates in cis its own C terminus and thereby releases an essential viral replication factor. As a unique feature, this enzyme requires for proteolytic activity stoichiometric amounts of a cellular chaperone termed Jiv (J-domain protein interacting with viral protein) or its fragment Jiv90. To obtain insight into the structural organization of the NS2 autoprotease, the basis for its restriction to cis cleavage, as well as its activation by Jiv, we dissected NS2 into functional domains. Interestingly, an N-terminal NS2 fragment covering the active center of the protease, cleaved in trans an artificial substrate composed of a C-terminal NS2 fragment and two downstream amino acids. In the authentic NS2, the 4 Cterminal amino acids interfered with binding and cleavage of substrates offered in trans. These findings strongly suggest an intramolecular product inhibition for the NS2 autoprotease. Remarkably, the chaperone fragment Jiv90 independently interacted with protease and substrate domain and turned out to be essential for the formation of a protease͞substrate complex that is required for cleavage. Thus, the function of the cell-derived protease cofactor Jiv in proteolysis is regulation of protease͞substrate interaction, which ultimately results in positioning of active site and substrate peptide into a cleavage-competent conformation.hepacivirus ͉ J-domain chaperone ͉ pestivirus ͉ protease cofactor T he pestiviruses, together with the flaviviruses and hepatitis C virus, form the family Flaviviridae (1). Replication of these positive-strand RNA viruses involves translation of one polyprotein that is processed by cellular and virus-encoded proteases (2). These processing events generate the proteins required for viral RNA replication and the production of infectious progeny.Recently in nonstructural protein 2 (NS2) of the pestivirus bovine viral diarrhea virus (BVDV) an autoprotease was identified (3). In the polyprotein of BVDV strain NCP7, NS2 encompasses amino acids 1137-1589 and thus has a length of 453 aa (4). The N terminus of NS2 is generated by cellular signal peptidases, and the protein remains associated to intracellular membranes, presumably at the endoplasmatic reticulum. The N-terminal half of NS2 is highly hydrophobic and therefore likely to be involved in membrane association. However, so far no experimental data on membrane topology or protein structure are available, in part because of its biochemical properties as well as its toxicity for bacteria. A recent study revealed that NS2 is a cysteine protease (3); the obtained data indicated a catalytic role for histidine at position 1447 (H1447) and cysteine at pos...

show abstract

“…The T7 RNA polymerase rFPV, together with the recently developed T7 RNA polymerase recombinants of the restricted host-range MVA strain of VV (Wyatt et al, 1995;Sutter et al, 1995), provides both an additional tool and an alternative to vTF7-3 for transient expression allowing study of RNA or protein products, The rFPV described here has the T7 RNA polymerase under control of the VV P7.5 early/late promoter, as in the MVA recombinant by Sutter et al (1995) and in vTF7-3. In the other MVA recombinant (Wyatt et al, 1995) it is under control of the VV Ptl late promoter.…”

Section: Amount Of Catmentioning

confidence: 99%

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus

Britton¹,

Green²,

Kottier³

et al. 1996

Journal of General Virology

View full text Add to dashboard Cite

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5-The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA polymerase offers an alternative to the widely used vaccinia virus vTF7-3, or the recently developed modified vaccinia virus Ankara (MVA) T7 RNA polymerase recombinant, a highly attenuated strain with restricted host-range. Recombinant fowlpox viruses have the advantage that as no infectious virus are produced from mammalian cells they do not have to be used under stringent microbiological safety conditions. Recombinant vaccinia viruses (rVV) have been produced expressing a variety of bacteriophage DNA-dependent RNA polymerases: T7 RNA polymerase (Fuerst et al., 1986), T3 RNA polymerase (Rodriguez et al., 1990) and SP6 RNA polymerase (Usdin et al., 1993). Cells infected with these rVVs can express biologically active products transiently from genes under the control of the appropriate RNA polymerase promoter. The most utilized system is an rVV expressing T7 RNA polymerase, vTF7-3. Translation of the T7 transcripts was inefficient, as only 5-10 % were capped, but inclusion of an internal ribosome entry signal (IRES) sequence, from encephalomyocarditis virus, at the 5' end of the T7-derived RNA transcripts, resulted in mRNAs that were CAP-independent and efficiently translated (Elroy-Stein et al., 1989).In addition to transient expression, a gene under the control of the T7 RNA polymerase promoter can also be integrated into the genome of a second rVV allowing high level expression of the gene product in cells dually infected with vTF7-3 and the second rVV (Fuerst et al., 1987). The vTF7-3 system has been utilized for the expression of RNA per se, e.g. a defective RNA (D-

show abstract

Non‐replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase

Cited by 214 publications

References 18 publications

Tropism illuminated: Lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system

Tropism illuminated: Lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system

Dissection of a viral autoprotease elucidates a function of a cellular chaperone in proteolysis

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus

Contact Info

Product

Resources

About