Presentation Title

Creating a Plant Expression Construct of Bacteriophage T7 RNA Polymerase for Expression of Rose rosette virus Genomic RNAs

Start Date

November 2016

End Date

November 2016

Location

HUB 302-#176

Type of Presentation

Poster

Abstract

Rose rosette virus (RRV) is a negative-sense RNA virus with seven known RNA segments (RNA1-7) and eight open reading frames (ORFs): P1-P7, with RNA 6 containing two ORFs: P6a and P6b. Our goal is to reconstitute infectious RRV from cloned DNA copies of the genome for future reverse genetics studies. Our approach is based on a reverse genetics system used for Bunyavirus—the most similar mammalian virus with an established reverse genetics system. This requires the creation of three types of constructs: RNA polymerase, genomic RNA-coding genes and protein-coding genes. The coding sequence of T7 RNA polymerase was amplified through PCR with in-frame addition of a nuclear localization signal, and cloned into pGEM-T with the Tobacco mosaic virus omega fragment for efficient translation. This construct will be transferred into a binary vector for transgenic expression in the model plant, Nicotiana benthamiana. T7 RNA polymerase expression will enable testing of RRV segment transcription. In order to produce a working virus, the RNA segments will be cloned in the form of cDNA under control of a T7 RNA polymerase promoter for transcription of genomic RNAs. Constructs to express viral proteins are being created in the binary vector pBIN61 with and without a carboxy-terminal epitope tag. Four constructs are ready to test for expression; four are in the process of being cloned. We aim to study the function of each gene through transient expression and manipulation in a reverse genetics system. Testing the T7 RNA polymerase for viral RNA expression is the first step toward development of plant virus reverse genetics systems for negative-sense RNA viruses and will enable further understanding and research of RRV in the future.

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Creating a Plant Expression Construct of Bacteriophage T7 RNA Polymerase for Expression of Rose rosette virus Genomic RNAs

HUB 302-#176

Rose rosette virus (RRV) is a negative-sense RNA virus with seven known RNA segments (RNA1-7) and eight open reading frames (ORFs): P1-P7, with RNA 6 containing two ORFs: P6a and P6b. Our goal is to reconstitute infectious RRV from cloned DNA copies of the genome for future reverse genetics studies. Our approach is based on a reverse genetics system used for Bunyavirus—the most similar mammalian virus with an established reverse genetics system. This requires the creation of three types of constructs: RNA polymerase, genomic RNA-coding genes and protein-coding genes. The coding sequence of T7 RNA polymerase was amplified through PCR with in-frame addition of a nuclear localization signal, and cloned into pGEM-T with the Tobacco mosaic virus omega fragment for efficient translation. This construct will be transferred into a binary vector for transgenic expression in the model plant, Nicotiana benthamiana. T7 RNA polymerase expression will enable testing of RRV segment transcription. In order to produce a working virus, the RNA segments will be cloned in the form of cDNA under control of a T7 RNA polymerase promoter for transcription of genomic RNAs. Constructs to express viral proteins are being created in the binary vector pBIN61 with and without a carboxy-terminal epitope tag. Four constructs are ready to test for expression; four are in the process of being cloned. We aim to study the function of each gene through transient expression and manipulation in a reverse genetics system. Testing the T7 RNA polymerase for viral RNA expression is the first step toward development of plant virus reverse genetics systems for negative-sense RNA viruses and will enable further understanding and research of RRV in the future.