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Modeling of the 5’ Nontranslated Region of Coxsackievirus B1 Wade Schulz Biology Colloquium Spring 2003.

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Presentation on theme: "Modeling of the 5’ Nontranslated Region of Coxsackievirus B1 Wade Schulz Biology Colloquium Spring 2003."— Presentation transcript:

1 Modeling of the 5’ Nontranslated Region of Coxsackievirus B1 Wade Schulz Biology Colloquium Spring 2003

2 Introduction  Coxsackievirus  RNA Secondary Structures  Computer Modeling  Analyzing Results  Enzymatic Probing

3 Enterovirus  Small virus made of protein and RNA  Poliovirus, coxsackievirus, echovirus –3 Forms of Poliovirus –23 Coxsackie A viruses –6 Coxsackie B viruses –28 Echoviruses –4 other Enteroviruses  Second most common viral infection –Led by rhinovirus (common cold) ssRNA(+) Protein coat polio_entero.htm

4 Associated Infections  Aseptic meningitis, encephalitis  Myocarditis, Dilated Cardiomyopathy  Type I diabetes  Amyotrophic Lateral Sclerosis (ALS)  Post-Polio Syndrome  Chronic Fatigue Syndrome

5 Coxsackievirus B1  Two types used in experiment  1.24 –Wild type virus –Causes acute infection as well as chronic disease  2.17 –Mutated form of virus –Causes acute infection but no chronic disease

6 Mouse Model Chronic disease Acute infection 2 weeks 1-6 months Hind limb flexion deformity/gait Viral replication Acute inflammation & damage Inject CVB1 into newborn mice

7 Mutational Change  What changes does the mutation cause in the viral structure  Does the structural change affect regulation

8 RNA Secondary Structures  Caused by bonding between bases on RNA  Creates stems and loops in RNA –Double-stranded stem –Single-stranded loop

9 Computer Modeling  MFold –Created by Michael Zuker –Uses algorithms to compute lowest energy models to create structures –Also known as a “squiggles” plot  GeneQuest –Part of Lasergene Suite –Only provides folding for one energy level

10 Computer Modeling  RNA sequence is entered into database  Folding conditions are set –37°C, energy increments  Server folds based on Zuker or other algorithms

11 Data Analysis  Stems and loops were identified  Beginning and ending nucleotides were recorded  Most common stems are assumed to exist  Enzymatic probing done to determine actual structure

12 Data Analysis 1.24 NCR kcal/mole kcal/mole StartEndLengthStartEndLength Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem

13 Data Analysis 2.17 NCR kcal/mole kcal/mole StartEndLengthStartEndLength Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem Stem

14 Stem Changes  Repeated stem changes were noticed between 1.24 and 2.17 structures  Change was noted in stem where mutation was present

15 Enzymatic Probing: Primer Extension  Use enzymes to cleave RNA at specific points (single strands) –RNase T1 - Guanosine –RNase U2 - Adenosine –RNase A - Pyrimidines  RNA then placed in gel to determine lengths

16 Primer Extension and Gel Analysis Hsue, et al.

17 Conclusions 1. Presumptive evidence from computer modeling that 1.24 and 2.17 forms differ 2. Stem and loop just upstream of translational start may function in regulation and tropism


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