Modeling of the 5 Nontranslated Region of Coxsackievirus B1

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Modeling of the 5 Nontranslated Region of
Coxsackievirus B1

• Wade Schulz
• Biology Colloquium
• Spring 2003

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Introduction

• Coxsackievirus
• RNA Secondary Structures
• Computer Modeling
• Analyzing Results
• Enzymatic Probing

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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)

http//www.cdc.gov/ncidod/dvrd/revb/enterovirus/no
n-polio_entero.htm
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Associated Infections

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

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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

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

• What changes does the mutation cause in the viral
  structure
• Does the structural change affect regulation

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RNA Secondary Structures

• Caused by bonding between bases on RNA
• Creates stems and loops in RNA
• Double-stranded stem
• Single-stranded loop

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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

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Computer Modeling

• RNA sequence is entered into database
• Folding conditions are set
• 37C, energy increments
• Server folds based on Zuker or other algorithms

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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

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Data Analysis
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Data Analysis
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Stem Changes

• Repeated stem changes were noticed between 1.24
  and 2.17 structures
• Change was noted in stem where mutation was
  present

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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

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Primer Extension and Gel Analysis
Hsue, et al.
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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