Tricistronic Plasmid Expressing the T7 RNA Polymerase and Measles Virus N and P Proteins for Rescue of Measles Virus AIK-C Strain

: Up to now, several attenuated measles virus vaccine strains derived from the Edmonston B vaccine consisting of Schwarz/Moraten, Zagreb, and AIK-C have been introduced for the rescue of their relative viruses through reverse genetics. In most studies, the measles virus rescue was done by supplying a cell line that expresses T7 RNA polymerase and measles virus N and P proteins as accessory proteins. The present study aimed to evaluate the rescue efficiency of the recombinant measles virus AIK-C vaccine strain by using a tricistronic expression plasmid. In this study, the rescue of the recombinant measles virus AIK-C vaccine strain was performed by co-transfection of three plasmids, including the cloned antigenomic cDNA of measles virus, a tricistronic expression plasmid that contained T7 RNA polymerase and measles virus N and P genes, and measles virus L polymerase expression plasmid. To increase the rescue efficiency, the transfected HEK-293 cells were co-cultured with Vero cells. As a result, the use of tricistronic expression plasmid that concomitantly encoded three necessary genes for the rescue of the measles virus led to the viral cytopathic effect with high efficacy five days post-transfection. Finally, the co-culture of transfected HEK-293 cells with Vero cells showed a relatively fast induction of viral cytopathic effect.

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Bilayer stabilizing peptides and the inhibition of viral infection: antimeasles activity of carbobenzoxy-Ser-Leu-amide

Bioscience Reports ◽

10.1007/bf01116868 ◽

1987 ◽

Vol 7 (9) ◽

pp. 745-749 ◽

Cited By ~ 12

Author(s):

Richard M. Epand ◽

Thomas J. Lobl ◽

H. E. Renis

Keyword(s):

Phase Transition ◽

Transition Temperature ◽

Phase Transition Temperature ◽

Measles Virus ◽

Cytopathic Effect ◽

Hexagonal Phase ◽

Vero Cells ◽

Time Parameter ◽

Scanning Calorimetry ◽

Hplc Retention Time

A number of carbobenzoxy-dipeptide-amides raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine (stabilizes the bilayer). The potency of the peptides in stabilizing the bilayer phase is Z-Tyr-Leu-NH2>Z-Gly-Phe-NH2>Z-Ser-Leu-NH2>Z-Gly-Leu-NH2>Z-Gly-Gly-NH2. A linear correlation was found between the respective HPLC retention time parameter k′ for the peptide and the slope of the bilayer stabilization curve determined with model membranes by differential scanning calorimetry. One dipeptide, Z-Ser-Leu-NH2, reduces measles virus cytopathic effect (CPE) in Vero cells. The mechanism by which this peptide reduces the CPE is not known, although some peptides which raise the bilayer to hexagonal phase transition temperature of phospholipids inhibit membrane fusion.

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Measles Virus Attenuation Associated with Transcriptional Impediment and a Few Amino Acid Changes in the Polymerase and Accessory Proteins

Journal of Virology ◽

10.1128/jvi.72.11.8690-8696.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8690-8696 ◽

Cited By ~ 72

Author(s):

Makoto Takeda ◽

Atsushi Kato ◽

Fumio Kobune ◽

Hiroko Sakata ◽

Yan Li ◽

...

Keyword(s):

Amino Acid ◽

Vero Cell ◽

Cell Line ◽

Measles Virus ◽

Vero Cells ◽

Functional Difference ◽

Pathogenic Potential ◽

P Proteins

ABSTRACT Measles virus (MV) isolated in B95a cells, a marmoset B-cell line, retains full pathogenicity for cynomolgus monkeys, while its derivative obtained by adaptation to the growth in Vero cells, a monkey kidney cell line, loses the pathogenic potential (F. Kobune, H. Sakata, and A. Sugiura, J. Virol. 64:700–705, 1990). Here, we show with a pair of strains, a fresh isolate (9301B) in B95a cells and its Vero cell-adapted form (9301V), that the in vivo attenuation parallels the decrease of replication and syncytium-inducing capabilities in the original B95a cells and that these in vitro phenotypes are attributable to impediment of transcription, which is already obvious at the level of primary transcription catalyzed by the virion-associated RNA polymerase. On the other hand, cell fusion assays detected no functional difference between the glycoproteins of the two viruses. Essentially the same transcriptional impediment with reduced syncytium induction following Vero cell adaptation was found with two other pairs of strains that had been similarly prepared. Nucleotide sequence comparison between the 9301B and 9301V viruses revealed that a few (at most five) amino acid changes, which sporadically took place in the polymerase (L and P proteins) and/or accessory V and C proteins, were responsible for the in vitro and in vivo attenuation through adaptation to growth in Vero cells.

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Assay of human interferon in Vero cells by several methods

Journal of Clinical Microbiology ◽

10.1128/jcm.9.4.471-475.1979 ◽

1979 ◽

Vol 9 (4) ◽

pp. 471-475

Author(s):

P C Ferreira ◽

M L Peixoto ◽

M A Silva ◽

R R Golgher

Keyword(s):

Vesicular Stomatitis Virus ◽

Sindbis Virus ◽

Reduction Method ◽

Cytopathic Effect ◽

Vero Cells ◽

Human Interferon ◽

Inhibition Assay ◽

Vesicular Stomatitis ◽

Viral Cytopathic Effect

Four methods for the assay of human interferon in Vero cells were compared based on the inhibition of viral cytopathic effect (CPE) in tubes, the inhibition of CPE in microplates, the reduction of plaques, and the inhibition of quantitative hemadsorption. For inhibition of CPE, Sindbis virus, vesicular stomatitis virus, poliovirus type 2, and vaccinia virus were used for challenge. In the plaque reduction method, Sindbis virus, vesicular stomatitis virus, and poliovirus were employed, and Newcastle disease virus was used in the quantitative hemadsorption assay. Sindbis virus was most susceptible to interferon in those tests measuring inhibition of CPE, but vesicular stomatitis virus was as sensitive in the plaque reduction method. Highest titers of interferon were recorded in microplates, especially with Sindbis virus as the challenge agent, followed by the quantitative inhibition assay. The CPE inhibition method was the simplest, and the quantitative hemadsorption assay was the most rapid to perform. Reproducibilities, as shown by the coefficient of variation, were 15, 39, and 59% for plaque reduction, CPE inhibition in tubes, and CPE inhibition in microplates, respectively.

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An interaction between Gβγ and RNA polymerase II regulates transcription in cardiac fibroblasts

10.1101/415935 ◽

2018 ◽

Author(s):

Shahriar M. Khan ◽

Ryan D. Martin ◽

Sarah Gora ◽

Celia Bouazza ◽

Jace Jones-Tabah ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Transcriptional Response ◽

Type I ◽

Hek 293 ◽

293 Cells ◽

Insight Into

SUMMARYGβγ subunits are involved in many different signalling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gβγ, we investigated the functional role of Gβγ signalling in regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. Following activation of the angiotensin II type I receptor in these cells, Gβγ dimers interact with RNA polymerase II (RNAPII). Our findings suggest that Gβ1recruitment to RNAPII negatively regulates the fibrotic transcriptional response, which can be overcome by strong fibrotic stimuli. The interaction between Gβγ subunits and RNAPII expands the role for Gβγ signalling in cardiac fibrosis. The Gβγ-RNAPII interaction was regulated by signaling pathways in HEK 293 cells that diverged from those operating in cardiac fibroblasts. Thus, the interaction may be a conserved feature of transcriptional regulation although such regulation may be cell-specific.

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Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

Journal of Virology ◽

10.1128/jvi.79.16.10420-10428.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10420-10428 ◽

Cited By ~ 70

Author(s):

Gjon Blakqori ◽

Friedemann Weber

Keyword(s):

Rna Polymerase ◽

Reverse Genetics ◽

Nonstructural Protein ◽

Viral Proteins ◽

Vero Cells ◽

T7 Rna Polymerase ◽

Wild Type Virus ◽

La Crosse Virus ◽

Additional Support ◽

Bunyamwera Virus

ABSTRACT La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3′ ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 108 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

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