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Molecular Pathogenesis: Viral Protein Synthesis

Author & Instructor Details

  • Instructor: Dr. Fred Krebs, Ph.D.
  • Position: Associate Professor
  • Department: Department of Microbiology & Immunology, Drexel University College of Medicine
  • Email: fck23@drexel.edu

Course Information

  • Course Codes: MIIM-512: Molecular Pathogenesis I (Viral Pathogenesis), MIIM-540: Viruses and Viral Infections
  • Copyright: © 2024 by Drexel University. All rights reserved.
  • Credits: Content is source-dependent; references found in the expandable Credits list.

Introduction to Protein Synthesis

Overview

  • The essential topic of study is Protein Synthesis.
  • Viruses rely on host machinery for protein synthesis as they do not encode their own protein synthesis machinery.

Curriculum Structure

  • To complete the topic, students must:
    1. Read each section and take notes.
    2. Play available videos and audio recordings (if provided).
    3. Complete the "Check Your Understanding" assessment questions included throughout the presentation (not graded).
    4. Engage in the Questions-Problems-Suggestions discussion forum for clarifications.
  • Content organization within the topic:
    • 1. Eukaryotic Protein Synthesis
    • 2. Viral Translation Strategies
    • 3. Regulation of Translation During Viral Infection
    • 4. Completion of Topic

Eukaryotic Protein Synthesis

Anatomy of Messenger RNA (mRNA)

  • Eukaryotic mRNA is primarily monocistronic, implying:
    • Contains a single open reading frame (ORF).
    • Translation initiates at the AUG start codon nearest to the 5' end.
    • Translation occurs in a 5' to 3' direction until a termination codon is reached.
  • Unlike eukaryotic mRNAs, prokaryotic mRNAs can be polycistronic, allowing multiple proteins to be encoded.
  • Both types include untranslated regions (UTRs) that affect translational efficiency and mRNA stability.

The Eukaryotic Ribosome

  • Eukaryotic ribosomes are 80S and consist of:
    • 40S Small Subunit: Contains 18S rRNA and around 30 ribosomal proteins.
    • 60S Large Subunit: Contains 28S, 5.8S, and 5S rRNAs and about 50 ribosomal proteins.
    • Note: Ribosomal RNA constitutes approximately 85% of total cellular RNA.

Transfer RNAs (tRNA)

  • tRNA molecules are approximately 70 nucleotides in length.
  • Each tRNA has:
    • An amino acid attached to the 3’ CCA terminus.
    • An anti-codon loop that binds complementary codons on mRNA.
  • Aminoacyl-tRNA synthetases ensure that tRNA is charged with the correct amino acid matching its anticodon.

Phases of Translation

  • Translation has three main phases:
    1. Initiation:
    • Assembly of the initiation complex is a primary regulation point.
    • Involves cap-dependent and cap-independent mechanisms.
    1. Elongation:
    • Formation of peptide bonds between amino acids, following mRNA codon sequence.
    1. Termination:
    • Release of the completed protein and recycling of ribosomal subunits.
  • Energy required for various steps typically comes from GTP or ATP hydrolysis.

Initiation Phase

  • Cap-dependent Initiation:
    • Involves recognition of the capped 5' end of mRNA.
    • The cap consists of a methylated guanosine residue linked in an unusual 5' to 5' linkage.
    • eIF-4E directly binds the cap; functions with eIF-4G and eIF-4A to facilitate translation initiation.

Ternary Complex Formation

  • The ternary complex is formed with:
    • Methionyl-tRNA
    • Initiation factor eIF-2
    • GTP
  • Important for preparing the small ribosomal subunit for initiation.

Assembly of 43S Pre-initiation Complex

  • Assembles small ribosomal subunit with initiation factors and initiator methionyl-tRNA to form 43S pre-initiation complex.

Scanning for AUG Start Codon

  • The small ribosomal subunit scans the mRNA to locate the AUG codon.
  • Scanning involves energy consumption via ATP hydrolysis and requires the helicase activity of eIF-4A to handle secondary structures in the mRNA's 5' UTR.

Joining of 60S Ribosomal Subunit

  • Recognition of the start codon leads to GTP hydrolysis, allowing the 60S subunit to join, forming the 80S complex.

Circularization of mRNA

  • The circularization process, facilitated by eIF-4G, enhances the initiation phase and facilitates re-initiation of translation.

Cap-independent Translation Initiation

  • Some mRNAs can initiate translation via Internal Ribosome Entry Sites (IRES), a mechanism more common in viral mRNAs.
  • Types of IRES:
    • Categorized based on sequence and structure.

Elongation Phase

Peptide Bond Formation

  • During elongation, amino acids are linked through peptide bonds based on mRNA codon sequence.
  • The reaction is catalyzed by 28S rRNA of the large ribosomal subunit, exemplifying catalytic RNA (ribozyme).

Translocation

  • The tRNA moves from the P-site to the E-site, while a new tRNA enters the A-site, requiring eEF-2 and GTP.

Termination Phase

  • Occurs when a termination codon enters the A-site.
  • The release factor eRF-1 recognizes termination codons (UAA, UGA, UAG) and facilitates the release of the newly synthesized protein from the ribosome.
  • Ribosomal subunits are recycled for future translation cycles.

Viral Translation Strategies

Overview

  • Study of translation variations by viruses reveals how they adapt mechanisms to maximize protein coding from limited genomes.

5' End Recognition in Viral mRNAs

  • RNA viruses face the challenge of marking their mRNAs for translation without a 5' cap, utilizing VPg to mimic cap function, recruiting necessary translation factors.
  • IRES usage bypasses the need for caps, allowing direct binding to the ribosomal machinery.

Preferential Translation of IRES-containing mRNAs

  • IRES incorporation allows some viruses, like poliovirus, to selectively initiate translation even when cellular proteins are inhibited.
  • The poliovirus exploits the cleavage of eIF-4G to prioritize its translation over that of cellular mRNAs.

Key Mechanisms for Maximizing Protein Synthesis

Polyprotein Synthesis

  • Polyproteins are translated from a single ORF and later processed into multiple individual proteins.

Leaky Scanning

  • Scanning ribosomes sometimes bypass the first AUG to initiate translation at downstream AUGs, producing truncated proteins or different reading frames.

Reinitiation and Suppression of Termination

  • Ribosomes can terminate and then immediately reinitiate translation for proteins in successive ORFs; suppression of termination allows continuation into downstream ORFs, producing fused proteins.

Ribosomal Frameshifting

  • Ribosomal frameshifting occurs when the ribosome shifts reading frames during translation, leading to the production of alternative protein products.

Regulation of Translation During Viral Infection

Overview

  • Viral infections initiate complex interactions at the translation level, where cells attempt to prevent virus replication via various regulatory mechanisms.
  • Key focus areas include: 1) Regulation of eIF-2 activities; 2) Regulation of eIF-4E, the cap-binding protein.

Phosphorylation of eIF2-α

  • Phosphorylation leads to a down-regulation of translation, primarily by inhibiting the recycling of eIF-2B, needed for new initiation.
  • Protein kinases (PKR, PERK, GCN2, HRI) are activated in response to stressors, particularly viral infections.

Viral Defenses Against eIF2-α Kinases

  • Viruses evolve strategies to counteract phosphorylation, ensuring their survival and replication.
  • Mechanisms include inhibiting kinase actions or binding dsRNA, which prevents PKR activation.

Viral Modulation of eIF-4F Activity

  • Viruses can manipulate initiation factors such as eIF-4G and eIF-4E to favor translation of viral mRNA over cellular mRNA.
  • Cleavage of eIF-4G by viral proteases marks a significant approach to prioritizing viral translation.

Summary of Regulation

  • Key points include regulation at initiation stages of translation under cellular stress, kinases involved, and how viruses effectively promote their mRNA translation despite host defenses.