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Modeling of the 5’ Nontranslated Region of Coxsackievirus B1

Modeling of the 5’ Nontranslated Region of Coxsackievirus B1

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Modeling of the 5’ Nontranslated Region of Coxsackievirus B1

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  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. 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 Enterovirus http://www.cdc.gov/ncidod/dvrd/revb/enterovirus/non-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 2 weeks Viral replication Acute inflammation & damage Inject CVB1 into newborn mice Acute infection Hind limb flexion deformity/gait 1-6 months Chronicdisease

  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

  13. Data Analysis

  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