Interpreting Adenovirus Transcription Map

Adenovirus 3D Model
Adenovirus Transcription Map
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Penton Protein
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Virus as a Vector for Gene Therapy

Viral particles are designed in a way that their structures possess the ability to protect the viral genome from damage during host-to-host transmission, and deliver the viral genome into the appropriate site within the host cell if the infection conditions are ideal. Scientists take advantage of the viral machinery as a genome delivery vehicle to develop the viral vector for application in gene therapy. In gene therapy, genetically engineered virus functions as the carrier/vector to deliver a functional exogenous gene into a patient's body (i.e., a person with a particular gene deficiency) to relieve disease symptoms
(Figure 1 a,b).


Understanding Viral Transcription Map Fosters the Development of Gene Therapy

Mastering the viral transcription map significantly helps scientists to understand how to insert a desired therapeutic gene into the viral genome. In one example, adenovirus is used as a vector to deliver the functional clotting factor VIII gene to treat hemophilia A since the patients with this disease are unable to produce the wild type factor VIII by themselves (Pierri N, 2013, Engleberg et al., 2012). It is highly impossible to design a functional viral vector without knowing the accurate information on the location and the function of the adenovirus E1 gene region.
(If you are interested to see this viral vector design solution, go to the interactive section of this page > choose “All genes” > turn “explore” ON > click the “plus” icon for adenovirus genome).
Thus, by utilizing this interactive web source (i.e., the second visual summary for ViraLiterate), I hope you will acquire the basic knowledge of understanding the viral transcription map and will understand how to relate this knowledge to the biomedical engineering field, for instance, in gene therapy.

Case Study: Adenovirus Transcription Map

In this interactive website for teaching visual literacy on viral transcription map, I use the transcription map of adenovirus serotype 5 (Ad5) as an example because it has great potential for gene therapy applications. This transcription map is adapted and modified from the Fundamentals of Molecular Virology, 2nd Edition (Acheson, 2011). The type 5 adenovirus genome is a linear double-stranded DNA molecule about 35000 base-pairs in length. The total length of the genome is divided into 100 map units, and each map unit equals to ~350 base-pairs. (See the interactive section > explore ON > viral genome > "plus" icon for more details).
Adenovirus genes with related biological functions tend to be clustered at certain regions of the viral genome and expressed from a common promoter as the clustering of genes provide an energy-saving mechanism for gene expression. It is also a method of regulating gene expressions at different infection stages - i.e., early (E), intermediate (I), and late (L) stages (Engleberg et al., 2012). Examples of the clustering genes are all late expressed genes (orange), and all early expressed E2 genes (blue). All the arrows shown in the transcription map are viral mRNAs transcribed from their corresponding genes.
Take a late RNA as an example (Figure 2), the vertical bar of the arrow represents the methyl cap as well as the transcription initiation site at 5’ end of mRNA. The arrowhead represents 3’ end of mRNAs with polyadenylation, as well as the direction of transcription. Viral-associated RNA (VA RNA) is the only exception which does not contain arrowhead nor vertical bar since it is a non-translated mRNA used to suppress the host immune response (Figure 2) . Notice that most arrows are illustrated as broken lines with gaps, which correspond to the removed introns (Figure 2). By hovering over each of the arrows in the interactive section, all regions with highlighted yellow strokes belong to the exons of the same processed mRNA transcript (Figure 2). Introns removal are facilitated by the complicated RNA splicing mechanisms (please review ViraLiterate module 2 for more details).

Eventually, all these mature mRNAs will be translated into different proteins used to build the progeny virus.

Adenovirus 3D Model and Design Inspiration

The 3D model of the adenovirus capsid in the interactive section (entry code: 6CGV) is obtained from the RCSB Protein Data Bank (website: PDB). The fiber Knob protein PDB entry code is 6HCN whereas the 3D structure of the shaft and tail portions of the fiber protein are not solved yet (Figure 3). All the viral proteins and the viral DNA molecule were built in Maya using molecular Maya. The overall design was inspired by the biomedical visualization website called Visual Science.

References

Textbooks:
1. Acheson, N. (2011). Fundamentals of molecular virology. 2nd ed. Hoboken, NJ: John Wiley & Sons, p.277.
2. Flint, S. (2015). Principles of virology. 4th ed. Washington, DC: ASM Press.
3. Engleberg, N., Dermody, T., DiRita, V. and Schaechter, M. (2012). Schaechter's mechanisms of microbial disease. 5th ed. philadelphia, PA 19103: Wolters Kluwer, pp.402-410.
4. Knipe, D. and Howley, P. (2015). Fields Virology. 6th ed. Philadelphia: Wolters Kluwer.


Journal articles:
1. Lee, C., Bishop, E., Zhang, R., Yu, X., Farina, E., Yan, S., Zhao, C., Zeng, Z., Shu, Y., Wu, X., Lei, J., Li, Y., Zhang, W., Yang, C., Wu, K., Wu, Y., Ho, S., Athiviraham, A., Lee, M., Wolf, J., Reid, R. and He, T. (2017). Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes & Diseases, 4(2), pp.43-63.
2. Reddy, V. and Nemerow, G. (2014). Structures and organization of adenovirus cement proteins provide insights into the role of capsid maturation in virus entry and infection. Proceedings of the National Academy of Sciences, 111(32), pp.11715-11720.
3. Pierri N, B. (2013). Adenoviral Vectors for Hemophilia Gene Therapy. Journal of Genetic Syndromes & Gene Therapy, 01(S1), pp.1-13.
4. LEE, T., MATTHEWS, D. and BLAIR, G. (2005). Novel molecular approaches to cystic fibrosis gene therapy. Biochemical Journal, 387(1), pp.1-15.


Websites:
1. Konstantinov, I. (2014). [online] Visual-science.com. Available at: https://www.visual-science.com/projects/adenovirus/web-application/ [Accessed 27 Mar. 2019].
2. Viralzone.expasy.org. (2019). Adenoviridae ~ ViralZone page. [online] Available at: https://viralzone.expasy.org/4?outline=all_by_species [Accessed 27 Mar. 2019].
3. Abmgood.com. (2019). Cell Culture - Adenovirus Techniques | ABM Inc.. [online] Available at: https://www.abmgood.com/marketing/knowledge_base/cell_culture_adenovirus_techniques.php [Accessed 2 Apr. 2019].
4. Bank, R. (2019). RCSB PDB: Homepage. [online] Rcsb.org. Available at: https://www.rcsb.org/ [Accessed 2 Apr. 2019].

Type 5 adenovirus contains a linear double-stranded DNA genome about 35000 base-pairs in length with two terminal proteins attached to the 5’ end of each DNA strand, with both ends of the genome containing inverted terminal repeats (ITR) sequence of 100 base-pairs (Figure: viral genome). ITR and terminal proteins are involved in viral DNA replication (see ViraLiterate module 1 for more details).

Before you read the next section, I suggest that you close the pop-up window and first to read through the supplementary materials (i.e. lower section) of this website.

In order to construct an adenovirus vector for gene therapy, a recombined E1-deleted adenovirus genome need to be designed to prevent the viral replication in human/host cells since E1 genes are responsible for the subsequent viral genes expressions (Figure: viral vector design solution).

  1. The first step is to construct a plasmid that contains modified genomic adenovirus DNA without the E1 gene region.
  2. Secondly, a gene of interest (for instance, clotting factor VIII gene used to treat hemophilia A) will be inserted into the transfer plasmid.
  3. Transfer plasmid and the modified adenovirus plasmid are transformed into E.coli for homologous recombination to occur, replacing the E1 gene with our gene of interest.
  4. When enough clones of recombined adenovirus plasmid are obtained, the clones will be linearized with certain enzymes.
  5. Eventually, 293 helper cells are transfected with recombinant adenovirus DNA to produce progeny viral vectors. The 293 helper cells can provide all the missing E1 viral proteins that are needed for the amplification and packaging of the genome as well as assembly of new virions.
  6. Thus, the progeny viral vectors have intact viral structures with our gene of interest but lack the functional E1 genes. When they are injected into the patient, they only can express the inserted therapeutic gene instead of the viral genes needed for virus self-replication.

Finally, I want you to find the viral genome by clicking the highlighted schematic viral genome > find the corresponding highlighted part shown in the 3D model on your left.

Every late RNA transcript shares a tripartite leader exon sequence as shown in the figure, and the remaining part of this transcript is the main body exon sequence for penton protein. (Figure: RNA transcript). The gaps between these exons sequences are the removed introns, which are generated by using alternative polyadenylation and alternative splicing (see ViraLiterate module 2 for more details).

Eventually the processed penton mRNA transcribed during the late infectious stage will be exported from the nucleus into the cytoplasm for translation, and the translated penton proteins will be imported back to the nucleus for progeny viruses’ assembly. (Figure: pathway for penton protein synthesis)

Finally, I want you to click the highlighted penton RNA orange arrow > find the corresponding part shown in the 3D model on your left.

Every late RNA transcript shares a tripartite leader exon sequence as shown in the figure, and the remaining part of this transcript is the main body exon sequence for hexon protein. (Figure: RNA transcript). The gaps between these exons sequences are the removed introns, which are generated by using alternative polyadenylation and alternative splicing (see ViraLiterate module 2 for more details).

Eventually the processed hexon mRNA transcribed during the late infectious stage will be exported from the nucleus into the cytoplasm for translation, and the translated hexon proteins will be imported back to the nucleus for progeny viruses’ assembly. (Figure: pathway for hexon protein synthesis)

Finally, I want you to click the highlighted hexon RNA orange arrow > find the corresponding part shown in the 3D model on your left.

Every late RNA transcript shares a tripartite leader exon sequence as shown in the figure, and the remaining part of this transcript is the main body exon sequence for fiber protein. (Figure: RNA transcript). The gaps between these exons sequences are the removed introns, which are generated by using alternative polyadenylation and alternative splicing (see ViraLiterate module 2 for more details).

Eventually the processed fiber mRNA transcribed during the late infectious stage will be exported from the nucleus into the cytoplasm for translation, and the translated fiber proteins will be imported back to the nucleus for progeny viruses’ assembly. (figure: pathway for fiber protein synthesis)

Finally, I want you to click the highlighted fiber RNA orange arrow > find the corresponding part shown in the 3D model on your left.

The analyses of the human viruses have increased the volume and complexity of scientific data visualizations and they cannot be easily explained with simple images. However, students are expected to interpret these sophisticated figures present in scientific research papers, and they are not taught how to do it.

The viral transcription map is an example of the type of visualization (Acheson, 2011):

This interactive website was made to help students develop visual literacy skills to understand this type of data visualization.

Biomedical Communications Faculty Supervisors:
Prof. Derek Ng, BSc, MScBMC, PhD
Prof. Michael Corrin, BFA, BA, Hons BSc, MScBMC, CMI

Content Advisor:
Dr. Martha Brown, BSc, MSc, PhD. Department of Molecular Genetics, University of Toronto.

Client:
Dr. Martha Brown
Prof. Jodie Jenkinson, BA, MScBMC, PhD, FAMI

Project Leader, Illustrator & UI/UX Designer:
Shawn Yue Liu

Program Developers:
Xiongjie
Chenshiming