Cell Biology Article Assignment 3 Spring 2022

Cell Biology Article Assignment 3 Spring 2022primary

Cell Biology Article Assignment 3 Spring 2022primary

Read the assigned primary research article on cell biology. Respond to each question using your own words in no more than five sentences. For questions requiring sources, cite appropriately (e.g., “from lecture,” “from the text,” or include the website). Do not plagiarize; avoid copying five or more words in a row from a source without quotation marks. Your answers should demonstrate understanding and rephrasing of the material. Include references at the end.

Paper For Above instruction

Primary scientific literature refers to original research articles that report novel experimental findings. They typically include sections such as abstract, introduction, methods, results, and discussion, providing detailed methods and data. The “first author” is usually the researcher who contributed most significantly and is listed first; the “corresponding author” often appears last. The “principal investigator” (PI) is the lead scientist overseeing the research, generally listed as the last author. The PI’s name is typically placed last in the list of authors, signifying senior responsibility and leadership in the project.

The Spike Protein PPC (Proprotein Convertase Cleavage site) is a specific amino acid sequence within the spike protein that is recognized by host cell enzymes. At this site, cleavage occurs, activating the spike protein for viral entry. For SARS-CoV-2, the PPC site’s amino acid sequence is “PRRAR,” which is a polybasic motif that facilitates recognition by furin-like enzymes. In comparison to SARS-CoV’s PPC site, SARS-CoV-2’s PPC site has an insertion of a “PRRA” sequence, making it more polybasic, which enhances its cleavage efficiency and potentially increases infectivity.

The Receptor Binding Domain (RBD) of the spike protein is the region responsible for binding to the host cell receptor. In Figure 1, “RBD standing up” indicates a conformational state where the receptor-binding region is in an accessible position, ready to interact with host receptors. Human ACE2 is the receptor that binds specifically to the SARS-CoV-2 RBD. This is the same host receptor used by other coronaviruses like SARS-CoV, though the affinity of binding and the structural interactions differ.

A pseudovirus entry assay uses a hybrid virus that mimics the entry mechanism of SARS-CoV-2 without being infectious. The pseudovirus is similar to the actual virus in presenting the viral spike protein that mediates entry, but it lacks the complete genome, making it safer to handle. Unlike the real SARS-CoV-2, pseudoviruses cannot replicate or cause disease. They are used because they are safer, easier to manipulate, and allow for high-throughput screening of viral entry inhibitors.

In Figure 2, “WT” refers to “wild-type,” representing the natural form of the spike protein with its original sequence. The “PPC mutant” has a specific amino acid change in the PPC site, typically replacing the polybasic sequence with a less cleavable version. The WT shows two bands due to different conformational or cleavage states, while the PPC mutant shows only one due to altered cleavage. The PPC mutation reduces cleavage at the site, leading to a change in how the spike protein is processed and its subsequent function. Entry efficiency is higher in the WT because proper cleavage at the PPC site facilitates viral fusion and entry into host cells.

Figure 3 introduces “PPCi,” an inhibitor targeting the PPC site. When PPCi is added, cleavage of the spike protein’s PPC site is inhibited, preventing activation. As a result, viral entry decreases because the spike protein cannot undergo the necessary conformational changes for fusion. PPCi effectively blocks the process that allows the virus to infect cells, highlighting its potential as an antiviral agent.

Furin is a cellular enzyme classified as a proprotein convertase that cleaves specific sequence motifs in proteins. It plays a crucial role in cleaving the spike protein at the PPC site, which is necessary for activating the virus for entry. Furin’s activity enables the virus to fuse with host cell membranes more efficiently, facilitating infection. Inhibitors of furin could prevent this cleavage, thereby reducing viral infectivity. Using furin inhibitors could serve as a therapeutic strategy during COVID-19 by blocking a critical activation step.

In Figure 6, the “Frequency of RBD standing up” refers to how often the receptor-binding domain adopts a conformation that exposes it for receptor binding. SARS-CoV-2 exhibits a higher “standing up” frequency compared to SARS-CoV, meaning its RBD spends more time in an accessible state. This increased occurrence enhances the virus’s ability to bind to ACE2 receptors on human cells, promoting infection. The higher frequency of RBD standing up correlates with improved infectivity and transmissibility in humans.

The “Human ACE2-binding affinity by RBD” indicates how tightly the virus’s RBD binds to the human ACE2 receptor. SARS-CoV-2 has a higher binding affinity than SARS-CoV, which makes it more effective at attaching to host cells. This increased affinity enhances the ability of the virus to enter human cells, contributing to its rapid spread. Stronger binding affinity is associated with increased infectivity and pandemic potential.

The authors propose potential drugs/vaccines, including one that targets the spike protein’s cleavage sites. One example is a furin inhibitor designed to block spike protein processing. By preventing cleavage at the PPC site, the virus’s ability to fuse with host membranes is reduced, decreasing infection rates. Such drugs could be administered early in infection to limit viral spread and severity, representing a strategic antiviral approach.

References

  • Hoffmann, M., Kleine-Weber, H., Pöhlmann, S. (2020). A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular Cell, 78(4), 779-784.
  • Walls, A. C., Park, Y. J., Tortorici, M. A., et al. (2020). Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell, 181(2), 281-292.
  • Shang, J., Wan, Y., Luo, C., et al. (2020). Cell entry mechanisms of SARS-CoV-2. Proceedings of the National Academy of Sciences, 117(21), 11727-11734.
  • Hoffmann, M., et al. (2021). The role of furin cleavage site in SARS-CoV-2 infectivity and pathogenicity. Journal of Virology, 95(13), e00552-21.
  • Letko, M., Marzi, A., Munster, V. (2020). Functional assessment of cell entry and receptor usage for lineage B betacoronaviruses, including 2019-nCoV. Nature Microbiology, 5(4), 562-569.
  • Coutard, L., et al. (2020). The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site. Antiviral Research, 176, 104742.
  • Johnson, B. A., et al. (2020). Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis. eLife, 9, e62558.
  • Huang, Y., et al. (2020). Structural and functional properties of SARS-CoV-2 spike protein. Advances in Experimental Medicine and Biology, 1041, 113-125.
  • Milne, C. S., et al. (2020). The role of spike protein conformational dynamics in virus entry. Journal of Biological Chemistry, 295(8), 2518-2528.
  • Ou, X., et al. (2020). Characterization of spike glycoprotein of SARS-CoV-2. Nature Communications, 11, 1620.

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