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The Effect of Single Amino Acid Mutations on the Function of the RNA-Dependent RNA Polymerase (RdRp) of the Tomato Bushy Stunt Virus (TBSV)

Internship Report 2008 18 Pages

Biology - Genetics / Gene Technology

Excerpt

Table of Contents

1. INTRODUCTION
1.1 TOMATO BUSHY STUNT VIRUS RDRP
1.2 GOAL OF THIS WORK

2. MATERIALS AND METHODS
2.1 SITE-DIRECTED MUTAGENESIS
2.2 LINEARIZATION OF THE PLASMID AND PHENOL CHLOROFORM EXTRACTION
2.3 I N VITRO TRANSCRIPTION
2.4 INOCULATION ON TOBACCO PLANTS (N ICOTIANA BENTHIANA) AND INFECTION MONITORING
2.5 RNA ISOLATION
2.6 RT-PCR AND SEQUENCING
2.7 OVERVIEW OF EXPERIMENTS AND ORDER IN WHICH THEY WERE CONDUCTED

3. RESULTS
3.1 SITE-DIRECTED MUTAGENESIS AND SCREENING OF CLONES (EXAMPLES OF WORKFLOW)
3.2 LINEARIZATION AND IN VITRO TRANSCRIPTION
3.3 INOCULATION AND INFECTION MONITORING
3.4 RT-PCR AND SEQUENCING
3.5 ESTABLISHMENT OF A “HOME BREW” SITE-DIRECTED MUTAGENESIS PROTOCOL

4. DISCUSSION
4.1 MUTATION OF F1
4.2 MUTATION OF F2
4.3 MUTATION OF F3
4.4 ESTABLISHMENT OF A “HOME BREW” SITE-DIRECTED MUTAGENESIS PROTOCOL
4.5 OUTLOOK

5. REFERENCES

1. Introduction

1.1 Tomato Bushy Stunt Virus RdRp

The tomato bushy stunt virus (TBSV) is a plant virus and a member of Tombusviridae family. It has a ~4.8 kb, single-component plus-strand RNA genome that is directly translated into the two proteins p33 and p92 (Russo et al., 1994). Based on sequencing analysis, p92 was predicted to have RNA-dependent RNA polymerase (RdRp) activity (Haernet et al., 1990). The RdRp activities of p92 were intensively studied in vitro and in vivo (Rajendran et al., 2006, Panaviene et al., 2003, 2004 and 2005, Nagy and Pogany, 2000, Panavas et al., 2002a, 2002b, Panavas and Nagy, 2003a, 2005, Panavas et al., 2003, Rajendran and Nagy 2003, 2004 ). It has previously been demonstrated that both p33 and p92 are necessary for tombusvirus replication.

RdRps function as the catalytic subunit in concert with host proteins and sometimes viral proteins in the replication of the viral genome, and they are required for the replication of all positive-strand RNA viruses (Quadt et al., 1993; de Graf and Jaspars, 1994; Buck 1996; Lai 1998).

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Figure 1: Pathways of information flow for RNA viruses (taken from www.mcb.uct.ac.za)

Despite the increasing number of studies on the characterization of RdRp activity and structure, the precise molecular mechanisms remain unclear. RNA replication in positive-sense, single-stranded (ss) RNA viruses is initiated at or near the 3´end of the template, using either a primer-dependent or primer-independent (de novo) mechanism. The de novo initiation consists in the addition of a nucleotide triphosphate (NTP) to the 3´OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product.

Generally, polymerase shapes resemble a semi-closed right hand and are made of three subdomains: fingers, palm and thumb. Currently, there are eight conserved RdRp motifs identified (Koonin 1991; Poch et al., 1989). Four of these eight conserved motifs are now known to be present in all classes of polymerases and reside in the catalytic portion of the “palm” domain (Hansen et al., 1997 and Ollis et al., 1985). The motifs of the palm subdomain are named A, B, C and D, plus a fifth motif E, unique to reverse transcriptases (RTs) and RdRps (Poch et al., 1989).

The conserved motif F, which this work is about, was identified first in Totiviridae (Bruenn, 1993 and Routhier et al., 1998). A systematic bioinformatic approach to identifying the evolutionarily conserved regions of proteins has verified the universality of this conserved motif in RNA-dependent RNA polymerases (Bruenn, 2003) (see figure 2).

Nagy et al. (2003) suggested that the motif F is located in region two of the three RNA-binding regions in TBSV p92. This motif was also found in the RNA-binding region of NS5B of HCV which is closely related to the TBSV RdRp (Koonin, 1991)

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Figure 2: Similarity peaks in four groups of RdRps

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Figure 3: Alignment of the conserved motif F of different Tombusvirudae (ClustalW, PAM 250, edited in GeneDoc), consensus shown at the bottom, similarity groups enabled (physiochemical similar amino acids are marked with *)

1.2 Goal of this Work

Although the alignment results of RdRp (O´Reilly and Kao, 1998) and some biochemical studies of different RdRps might be used as primary data for the prediction of the Tombusviridae RdRps structure and its functions, biochemical studies of the TBSV RdRp and other Tombusvirus RdRps, especially ones concerning the motif F, are not available. Therefore we will attempt to determine the essential amino acids in the motif F of TBSV p92. The idea we have in mind for approaching this goal is to mutate single amino acids in the motif F of p92 and then infect plants with this mutated form of the virus. The infectivity of the virus will be used as an assay for the activity of p92 because the virus cannot replicate without a functional p92. For example, when a plant becomes infected by a mutated clone, this shows that the mutation does not stop the virus from replicating. However, if an amino acid is changed, that results in a non-infectious virus clone, which shows that the amino acid cannot be interchanged. As mutation occurs very frequently in virus replication, we will check for reverse mutations in the virus in infected plants. This may occur when the mutation introduced into p92 only partially inhibits the polymerase.

2. Materials and Methods

2.1 Site-Directed Mutagenesis

A full length (4,8kb) cDNA clone of TBSV was used (TBSV statice-BS3 (Galetzka et al., 2000)) as starting material for the mutagenesis. The template plasmid was amplified in its whole length by PCR using complementary mutagenic primers containing mismatches to the template plasmid through which the desired mutations were introduced into p92. Amplification was carried out with the proofreading enzyme Pfu polymerase. The resulting PCR product was digested with Dpn1 for 2h at 37°C to eliminate the parental, unmutated plasmid, which unlike the newly synthesized one was still methylated as it was previously amplified in a methylating E. coli strain (Inv-alpha). Dpn1 digests only methylated DNA by cutting it at the restriction site GATC. This site is very frequent so that digestion with Dpn1 results in degradation of methylated DNA. After Dpn1 digestion, 5µl of the PCR product were transformed into competent E. coli cells (Inv-alpha) by heatshock transformation.

The mutagenic primers were designed so that a successful mutation would either introduce or eliminate a restriction site in the template plasmid. This allowed for a first screening of the colonies resulting from transformation. A screening step was needed because unmutated plasmid was still found in 10% to 30% of the resulting clones in spite of Dpn1 digestion. In the first check for successful mutations, eight colonies were picked and grown in 2ml of TY-medium at 37°C overnight. Plasmid preparation was then performed the next day using a standard protocol. Restriction digestion with the “marker restriction enzyme” was carried out to determine whether or not the restriction site existed. Clones that were shown to have the desired mutation in this step were sequenced at the mutation site to check for successful mutation on sequence level. Sequencing was performed by 4base-Lab (www.4base-lab.de, Reutlingen). Successfully mutated clones confirmed by restriction digestion and sequencing were grown in 7ml of TYmedium overnight, and plasmid was isolated using DNA spin columns (NucleoSpin Plasmid kit, Macherey- Nagel).

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Figure 4: Overview of site-directed mutagenesis process. Picture taken from the Stratagene manual

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Details

Pages
18
Year
2008
ISBN (eBook)
9783640994304
ISBN (Book)
9783640995141
File size
884 KB
Language
English
Catalog Number
v177660
Institution / College
Johannes Gutenberg University Mainz – Alplanta - Plant Research Institue
Grade
1,0
Tags
tomato bushy stunt virus TBSV virology virus plant molecular genetics mutagenesis site directed mutagenesis plasmid PCR RNA dependent RNA polymerase RdRp in vitro transcription

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Title: The Effect of Single Amino Acid Mutations on the Function of the RNA-Dependent RNA Polymerase (RdRp) of the Tomato Bushy Stunt Virus (TBSV)