Evolution

TWiM #233: Antivirals Made by Bacteria

Podcast and Annotation Information

  • Annotation by Angelika Jordan, Kristin Kruger, Paola Colon, Trenity Abbs, Rebecca Seipelt-Thiemann, and Maia Larios-Sanz
  • Podcast audio by TWiM: Listen to TWiM #233 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson, Grace Helle, Angelika Jordan, Kristin Kruger, Paola Colon, Trenity Abbs: Access Podcast Transcripts
  • Papers Discussed:
    • Wilder W, director. 1938. Jezebel [motion picture]. Warner Brothers. 104 minutes.
    • Bernheim A, Millman A, Ofir G, Meitav G, Avraham C, Shomar H, Rosenberg MM, Tal N, Melamed S, Amitai G, Sorek R. 2021. Prokaryotic viperins produce diverse antiviral molecules. Nature 589(7840):120-124. doi: 10.1038/s41586-020-2762-2

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:06 minutes

The Most Interesting Things (according to students)

Considering the impact of epidemics and pandemics on society, and how it is still a relevant problem (Yellow Fever in 1853, COVID in 2020). Also interesting – considering watching movies that touch on science-related topics and identifying what they get right/wrong.

n/a

1.2. Main paper; discussion starts at 18:33 minutes

The Most Interesting Things (according to students)

Bacteria produce similar anti-viral molecules as we do!

This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. A version of this paper is available on PubMed.

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_ME)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.
  • Fundamental statement 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental statement 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • Fundamental statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define vector and identify the vector involved in Yellow Fever contagion.
  • Recall effective measures for controlling Yellow Fever.
 S L
  • Deduce the inaccuracy in the dramatic experimental vaccination procedure.
 S H
  • Recall how nucleotide derivatives, such as didehydroCTP, are different from nucleotides, such as CTP.
  • Identify how viperins were shown to inhibit viral replication.
 M L
  • Design an experiment to identify the viperin genes in a newly sequenced genome.
  • Compare and contrast how chain terminator nucleotides affect nucleotide synthesis processes.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • n/a

4.2. Main Paper

  • Molecular Cloning (25:21–27:26): This is a broad array of techniques that involve making copies of DNA and manipulating DNA, also known as recombinant DNA techniques.  Here, the researchers used cloning techniques to express putative viperin genes in E. coli.
  • Plaque Assay (26:30–27:33):  This technique uses a bacterial lawn inoculated with putative phage or various dilutions of known phage.  Here the researchers used it to evaluate viperin effect on phage replication.
  • Mutational Analysis (27:31): This involves making changes (mutations) to DNA.  Here, mutational analysis was used to change key cysteine residues in viperin gene products to determine their importance in the activity of these proteins.
  • Phylogenetic Trees (28:20–30:59): This is a visual representation of similarities in sequences.  This bioinformatic analysis was used to study the evolutionary relatedness of eukaryotic and prokaryotic viperins.
  • Mass Spectrometry (35:00–35:47): The scientists use mass spectrometry, which can quantify and identify molecules based on size and charge, to identify key products of the viperin products, including ddhCTP and other modified nucleotides.
  • RNA Sequencing (39:51–40:11): Also known as transcriptomics, this technique involves sequencing all the RNA from a population of cells.  The researchers used this to determine whether the viperin expression inhibits transcription.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Contagion (3:27): During the yellow fever epidemic, the people had the incorrect idea of how disease was contracted, through touch and air.  It is really transmitted by insects.
  • Pasteur, Koch, and Germ Theory (3:30): They discuss that movie takes place before development of germ theory, and that people had no idea how infectious disease spread.
  • Quarantine (5:19): Affected people in movie are being sent to isolated spaces to avoid disease spread.
  • Yellow Fever (5:19): The first human virus that was identified in 1906. Occurred during the build of the Panama Canal. The workers were infected due to the bite of the Aedes aegypti mosquito. Those who were bitten by mosquitoes died until the vaccine was created in 1947. Yellow fever damages the liver, giving jaundice to those infected.

5.2. Main Paper

  • Interferon (18:31): Viral infection stimulates expression genes through inducing interferon.  These genes are known as interferon stimulated genes (ISG).
  • Chain Termination (20:19–25-14): These are nucleotide-like chemicals that stop DNA replication by inhibiting addition of nucleotides to the 3’ hydroxyl group of the previous nucleotide in a growing DNA chain.  They are used in sequencing DNA and as anti-viral therapeutics.
  • Defense Islands (25:00–25:30): The researchers found CRISPR-Cas and restriction modification systems in bacterial genomes near the viperin gene, so they termed the region defense islands.
  • Homologous Genes and Phylogenetics (28:20): Genes that are similar across species are homologs.  These can be used in phylogenetics to study the relationship of the genes/species.
  • Mechanisms of Gene Transfer (28:20–30:54): The podcasters discuss ways genes may be common in many species, including endosymbiotic theory and horizontal gene transfer.
  • Prokaryotic Gene Organization (32:10–32:45): The podcasters discussed the differences in gene organization in prokaryotes (polycistronic) and eukaryotes (monocistronic). The polycistronic nature of the defense islands suggests prokaryotic origin.

6. Podcast Questions

  1. A vector is a ______ and the vector for Yellow Fever is ________.
    1. A source of contaminated air particles; currents of putrid air
    2. A non-living transmission route for a pathogen; contaminated water
    3. An organism that carries a pathogen but is unaffected; a mosquito
    4. An organism that engulfs another organism; Naegleria fowleri
  2. What is the most effective way to control Yellow Fever?
    1. Remove standing water
    2. Insecticide treatments
    3. Water purification systems
    4. Air purification systems
  3. The podcasters discuss how the movie Contagion portrayed a very inaccurate experimental vaccination procedure. Why is this an issue?
    1. Different viruses can’t be used in vaccination procedures in place of other viruses.
    2. Different immunization routes can give different results for vaccination testing.
    3. Heat-killed bacteria do not induce widespread immunity to RNA viral infections.
    4. Testing virus inactivation in test tubes does not provide evidence of immunization.
  4. The researchers found that cells that had a viperin gene produced didehydro-CTP, which is a modified version of the nucleotide CTP, when they were infected with a virus.  How is didehydro-CTP different from CTP? [pick all that apply]
    1. Didehydro-CTP is a metabolite of CTP nucleotide.
    2. Only one is a substrate of the viperin enzyme.
    3. Only one is a toxin used in preventing blood clots.
    4. Only one can be used by nucleotide polymerases.
  5. The final experiments discussed were about the mechanism that viperins use to alter viral replication. What were the conclusions?
    1. Viperins produce variant nucleotides that inhibit capsid formation.
    2. Viperins produce variant nucleotides that inhibit viral translation.
    3. Viperins produce variant nucleotides that inhibit viral transcription.
    4. Viperins produce variant nucleotides that inhibit viral DNA replication.
  6. How could you find a viperin gene in a newly sequenced genome?
    1. Compare a viperin-containing genome to your new genome sequence.
    2. Culture the bacterium and look for the presence of di-dehydroCTP.
    3. Culture the bacterium and test for phage killing using plaque assays.
    4. Search the sequence of the new genome using a viperin gene sequence.
  7. Open-ended Question: Explain how chain terminators affect DNA and RNA strand elongation, and how this might affect both replication and transcription.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 2 and 4).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Define key features in a phylogenetic tree diagram.
  • Identify relationships among elements in a phylogenetic tree.
  • Evaluate the data to make conclusions about ancestral origins of sequences.
  • Design an experiment to biologically confirm a gene from a newly sequenced genome encodes a viperin.

Experimental Background (Bernheim et al, Figure 2)

A group of antiviral proteins called viperins, previously described in animals, have now been discovered to be both abundant and more diverse in prokaryotes. Bernheim et al. (2020) explores the origin of the eukaryotic group of viral defense proteins and characterizes the function of prokaryotic homologs. The researchers first wanted to identify how similar/related these genes are among the species where they are found.  They use viperin amino acid sequences to generate a phylogenetic tree (inside area of the figure), which is color coded by group (named on the outside of the ring).  Other features of note are displayed on the outer color ring, such as the type of ddh-nucleotide found using mass spectrometry (triangle) and presence of a nearby nucleotide kinase in the viperin genomic region (brown rectangles).

7.1.2. Questions

  1. The researchers included additional features mapped along with the phylogenetic tree they generated, including which ddh-nucleotide the viperin generated.  This was determined using mass spectrometry.  Which ddh-nucleotide does vip60 in clade 3 make?
    1. ddhATP
    2. ddhCTP
    3. ddhGTP
    4. ddhUTP
  2. The eukaryotic viperin genes _____ monophyletic because _________.
    1. are; all members of the group that share a common recent ancestor are included
    2. are not; not all descendants of the common ancestor are included in the branch
    3. are; all the groups sharing a common ancestor and some that don’t are included
    4. are not; of the groups that share a common ancestor, some that don’t are included
  3. The Bacteroidetes viperins are present in clade 3.  Which clade’s viperins are most like the Bacteroidetes? What is your evidence?
    1. Clade 6; these have a similar branch number
    2. Clade 5; these have a similar branch depth
    3. Clade 4; these are nearer each other in the tree
    4. Clade 2; these split from the same tree branch
  4. The phylogenetic analysis shows evidence that eukaryotic viperins (Eukaryotes label in the outer ring)  were obtained by horizontal transfer.  Which clade is the likely origin of this HGT event?  What is the evidence?
    1. Spirochetes or Firmicutes; clade 1 has complex structures so they must be the origin.
    2. Archaea or Cyanobacteria; clade 2 shares a common origin with the eukaryotic viperins.
    3. Bacteroidetes; clade 3 is located nearest the eukaryotic viperins in the phylogenetic tree.
    4. Proteobacteria; clades 6 and 7 are most abundantly represented in the phylogenetic tree.
  5. How could you biologically confirm whether a sequence you found in a newly sequenced genome encodes a viperin?
    1. Compare a viperin-containing genome to your new genome sequence.
    2. Culture bacteria expressing the gene and look for didehydro-nucleotides.
    3. Culture the bacterium and test for phage killing using plaque assays.
    4. Translate the sequence to amino acids and compare to known viperins.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify conditions for expression of each inducible feature in the experiment.
  • Identify key features in line and bar graphs based on reporter gene assays.
  • Identify positive and negative controls for these experiments.
  • Interpret experimental data to draw conclusions regarding specific hypotheses.
  • Predict results for alternative mechanisms of action.

Experimental Background (Bernheim et al., Figure 4)

A group of antiviral proteins called viperins, previously described in animals, have now been discovered to be both abundant and more diverse in prokaryotes. Bernheim et al. (2020) explores the origin of the eukaryotic group of viral defense proteins and characterizes the function of prokaryotic homologs. The researchers had previously found that E. coli that expressed a viperin protein had two features: 1) they were less sensitive to viral infection, and 2) they produced di-deoxydro nucleotides, such as ddh-CTP, ddh-UTP, and/or ddh-GTP.  These nucleotides chemically lack the ability to extend a growing nucleotide chain.  Recall that both DNA replication and transcription require synthesis of nucleotide polymers.  In this experiment, Bernheim et al (2020) wanted to test determine whether or not viperins used a mechanism involving transcription, so they engineered several bacterial strains with a reporter system that has three essential components (panel a).  First, the presence of a chemical called IPTG in the medium induced expression of the phage T7 RNA polymerase.  Second, the presence of arabinose sugar in the medium induced expression of the viperin.  Third, the amount of functional T7 RNA polymerase is going to correspond to the level of transcribed RNA encoding the green fluorescent protein (GFP). They used this elaborately controlled system to determine the effect of viperin expression on T7 polymerase transcription by using no inducer (gray lines), IPTG only (green lines), or IPTG+arabinose (red lines) for the original MoA strain and strain engineered with viperins from human and 3 other clades.  GFP fluorescence over time is shown for no viperin (panel b) or each viperin (panels c-f), as well as a comparison of GFP RNA level, as measured using a transcriptomics technique called RNA sequencing (RNA-seq; panel g).

7.2.2. Questions

  1. The model of inducible elements is shown in panel a.  What do the small maroon hexagons represent?
    1. T7 polymerase
    2. Green fluorescent protein
    3. ddh-nucleotides
    4. Viperin
  2. The model of inducible elements is shown in panel a.  Which of the following will be produced in a medium containing only IPTG? [pick all that apply]
    1. T7 polymerase
    2. Green fluorescent protein
    3. ddh-nucleotides
    4. Viperin
  3. As a control, green fluorescent protein production is quantified for bacteria engineered to express an unrelated protein, MoaA (panel b) in the presence of arabinose sugar, instead of a viperin protein (panels c-f).  Thus, panel b is entirely experimental controls.  Match each media condition to its control type and reason for inclusion.
Condition Control/explanation
____ No inducer A. negative control; shows that neither the non-viperin MoaA protein nor the sugar inhibit GFP expression
____ IPTG B. positive control; shows GFP expression occurs and also its level
____ IPTG + arabinose C. negative control; shows GFP expression  is dependent on inducer IPTG
  1. What do the results comparing MoaA (panel b) and human viperin (panel c) allow you to conclude?  What is your evidence?
    1. Viperin enhances GFP expression; green line is high in both panels b and c
    2. Viperin expression is dependent on IPTG; gray line in both panels b and c
    3. Viperin expression is high; in panel c, green line is high and red line is low
    4. Viperin stops GFP expression; red line is low in panel c, but high in panel b
  2. From these data (panels b-f), which viperins likely stop viral replication using the same mechanism?  What is your evidence?
    1. human viperin; green line has a higher variability compared to the red
    2. Vip8 (clade 4); green line has the lowest variability compared to the red
    3. Vip9 (clade 7); green line has a higher variability compared to the red
    4. Vip60 (clade 3); green line shows the highest GFP expression of all
    5. All inhibit GFP expression; red line is low and green line is high for all

Use this text for the next three questions:

To test whether the effects the researchers observed for GFP expression/fluorescence were due to inhibiting transcription or translation, they also quantified the levels of GFP RNA in the same bacteria used in panels b-f that were grown in medium with IPTG and arabinose (panel g).

  1. Match the variable type (dependent or independent) and the data’s axis (x-axis or y-axis) to the description for this bar graph (panel g). [D = dependent variable; In = Independent variable; X = x-axis; Y = y-axis]
    1. _________ Medium type
    2. _________ % GFP RNA
    3. _________ Expressed protein type
    4. _________ % GFP Protein
  2. Which bacteria produce GFP mRNA in these conditions (IPTG and arabinose)? Based on these data, which biological process is likely blocked in viperin-induced viral inhibition? [pick all that apply]
    1. MoaA strain; transcription
    2. human viperin strain; translation
    3. Vip8 (clade 4) strain; transcription
    4. Vip9 (clade 7) strain; translation
    5. Vip60 (clade 3) strain; transcription
  3. The investigators demonstrated that the prokaryotic viperins pVip8, pVip9 and pVip60 act via a nucleotide-modifying process. If the mechanism had targeted DNA replication to inhibit viral replication, rather than transcription or translation, how would you expect the GFP RNA abundance results (panel g) to be different?
    1. The bar for the MoaA would look very similar to the bar for human viperin expression.
    2. The bar for the viperin-expressing strains would be similar to the human viperin bar.
    3. The bar for the MoaA would look similar to the bar for the viperin-expressing strains.
    4. The bar for the viperin-expressing strains would be similar to the MoaA strain’s bar.

8. Paper Information and Licensing

8.1. Snippet content

  • Wilder W, director. 1938. Jezebel [motion picture]. Warner Brothers. 104 minutes.

8.2. Main paper

  • Bernheim A, Millman A, Ofir G, Meitav G, Avraham C, Shomar H, Rosenberg MM, Tal N, Melamed S, Amitai G, Sorek R. 2021. Prokaryotic viperins produce diverse antiviral molecules. Nature 589(7840):120-124. doi: 10.1038/s41586-020-2762-2
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.  A version of this paper is available on PubMed.

License

Icon for the Creative Commons Attribution 4.0 International License

Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

Share This Book