Virology and RNA Synthesis

Introduction and Context

• Unique Water Bottle: Features a biohazard symbol, discouraging theft.
• Virology Education: Highlights the importance of public understanding of virology.
• Historical Context:
  • 1935: Wendell Stanley crystallized tobacco mosaic virus, believed protein was genetic material.
  • 1944: DNA identified as genetic material.
  • 1952: Hershey Chase experiment showed DNA is the genetic material of viruses.
  • 1956: RNA in tobacco mosaic virus shown to be genetic material.
  • 1959: RNA found in various animal viruses, considered genetic material.

RNA in Viruses

• Types of Viral RNA:
  • Double-stranded RNA
  • Single-stranded negative-sense RNA
  • Single-stranded positive-sense RNA
  • Retroviruses (RNA with DNA intermediate)

RNA Synthesis Experiments

• Polio Virus Experiment:
  • Infect cells with polio, make cell extracts at different times.
  • Use radioactive triphosphates to measure RNA synthesis.
  • Found viral origin of RNA synthesis, not cellular.

RNA Polymerase and Viral Replication

• RNA-Dependent RNA Polymerase (RdRP):
  • Found in particles of negative-strand RNA viruses.
  • Essential for replication of negative and double-strand RNAs.
  • Plus-strand RNA viruses do not carry polymerase in the particle.

Structure of Polymerases

• Signatures of RNA Polymerases:
  • Gly-asp-asp (GDD) motif is a signature for RNA polymerase.
• Polymerase Structure:
  • Palm, fingers, and thumb domains.
  • Conserved motifs across different classes of polymerases.
  • Active site involves two metal (Mg) catalysis mechanism.

RNA Synthesis Mechanism

• Templated vs. Non-Templated Synthesis
  • Templated synthesis: Follows a template for accurate replication.
  • Non-templated synthesis: Occurs due to polymerase stuttering.
• Polymerase Initiation:
  • Denovo: Without a primer required.
  • Primer-dependent: Requires a starting RNA or DNA molecule.

Virus-Specific Replication Strategies

• Poliovirus:
  • Plus-strand RNA, uses protein-linked primer (vpg).
  • Replication involves circularization of RNA template.
• Alphaviruses:
  • Produce subgenomic RNAs for protein synthesis.
• Coronaviruses:
  • Plus-strand RNA that makes nested subgenomic RNAs.

Negative-Strand RNA Viruses

• Replication Process:
  • Viral genome is not mRNA; requires switch from mRNA synthesis to genome synthesis.
• Example: VSV:
  • Negative strand RNA requires polymerase in particle.
  • Subgenomic mRNAs made through start-stop polymerase activity.
  • Nucleocapsid protein controls switch to genome synthesis.

Double-Stranded RNA Viruses

• Reovirus:
  • Double-strand RNA, polymerase in core.
  • Genome never leaves the core; mRNAs synthesized within.

Mutation and Recombination

• RNA Virus Mutation:
  • High error rate due to lack of error correction.
  • Nido viruses have exonuclease for error correction.
• Recombination:
  • Polymerase can switch templates, creating recombinant RNAs.
  • Specific amino acids in polymerase regulate recombination frequency.

Conclusion

• Understanding of RNA synthesis in viruses is crucial for developing antivirals and vaccines.
• Next lecture will focus on RNA synthesis using DNA as a template.