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NAME (in all caps) ____________________________________ Student ID ___________________ MIC 102 WS3 Page 1 of 4 Last updated: 5/26/20 WORKSHEET 3 20 points. Submit by 10:30 AM Tuesday, 6/2/20, to Canvas > Gradescope > Worksheet 3. OBJECTIVES: to familiarize you with key features of the SARS-CoV-2 virus such that you can understand—and educate others on—its replication cycle, treatment, and genome changes. TO DO: Read and follow the instructions posted on Canvas > Assignments > Worksheet 3. ACADEMIC INTEGRITY AFFIRMATION: I hereby verify that the work I submit is the result of my individual effort. FULL NAME (SIGNATURE) _________________________________________ BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, (+)-sense, single-strand RNA virus that causes coronavirus disease 2019 (COVID-19). The coronavirus family is named for the crown-like spikes on the outer surface of the virus and includes viruses that infect many species of animals, including camels, cattle, bats, and cats. The SARS-CoV-2 virus appears to have “spilled” out of bats, its primary animal host, and into humans in December of 2019 in Wuhan, China. COVID-19 symptoms appear 2-14 days after exposure and include fever, dry cough, shortness of breath, chills, muscle pain, headache, diarrhea, and loss of taste or smell. The virus spreads from person- to-person, primarily through droplets that are released when a person coughs or sneezes, but also through indirect contact with secretions. It is highly contagious and spread primarily by symptomatic individuals but also by individuals who are asymptomatic. Researchers are working at a frantic pace to design an effective vaccine and determine whether people who recover from COVID-19 can be re-infected. Coronaviruses have remarkably low mutation rates for RNA viruses. Their replicative enzyme has low fidelity (accuracy) but they have acquired the gene for a 3’-to-5’ exonuclease (ExoN) that removes mismatches during and after RNA synthesis. QUESTIONS TO ANSWER: 1. (5 pts) Fill in the missing terms in the replication cycle of SARS-CoV, a virus that is very similar to SARS-CoV-2 with respect to its replication. A. During attachment, the SARS-CoV ___________________ [name specific viral ligand] binds receptor molecules on the ___________________ [name specific structure] of the host cell. NAME (in all caps) ____________________________________ Student ID ___________________ MIC 102 WS3 Page 2 of 4 Last updated: 5/26/20 B. During penetration and uncoating, the virion enters the cell by ___________________ and then releases its genome into the cytoplasm of the cell through ___________________ [name process]. C. During biosynthesis, the ___________________ [positive- and/or negative] – sense genome is replicated using the viral ______________________ [name specific enzyme] to produce ___________________ [positive- and/or negative] – sense strands. This activity will also produce ___________________ [name specific macromolecule] that will be translated to produce viral structural proteins. D. As a precursor to assembly, viral surface proteins are translated in the ___________________ [name host cell compartment]. These surface proteins will then assemble with other viral proteins and a copy of the genome. E. During release, infectious virions leave the host cell by the process of ___________________ [name cell process]. ANTIVIRAL THERAPY 2. (2 pts) The most promising broad-spectrum class of antiviral drugs to treat RNA virus infections are nucleoside analogs. These are synthetic, chemically-modified bases that are incorporated into the replicating viral genome by low-fidelity polymerases. The incorporation of the nucleoside analog typically interferes with synthesis by the polymerase or the resultant mutation disrupts viral genes and replication. Unfortunately, the SARS-CoV-2 ExoN protein renders most nucleoside analogs ineffective as a treatment against this particular RNA virus. Explain why the proofreading activity of ExoN reduces the ability of nucleoside analogs to inhibit viral replication. 3. (3 pts) Happily, the antiviral remdesivir was demonstrated to reduce SARS-CoV-2 replication. Host enzymes convert it into the compound GS-441524 which, upon incorporation into the nascent strand by the viral polymerase, terminates RNA synthesis. (continue to next page…) NAME (in all caps) ____________________________________ Student ID ___________________ MIC 102 WS3 Page 3 of 4 Last updated: 5/26/20 A. Compare the structure of the analog GS-441524 (below) to the structure of the molecule it is mimicking, adenosine (adenine + ribose), and list three differences between them. B. Is GS-441524 a competitive or non-competitive inhibitor of the viral polymerase? Briefly explain your answer. SCENARIO: Your neighbor is very worried by a news headline claiming that the SARS-CoV-2 virus has changed and is easier to spread than the original virus. Knowing that you are in a microbiology class, she asks you for advice on how concerned to be, and how to avoid getting infected. You locate the original research paper and learn that: (1) researchers have sequenced the genomes of thousands of SARS-CoV-2 viruses that were isolated from COVID-19 cases worldwide, (2) collectively, these genomes contain multiple nucleotide sequence variations from the early isolates from Wuhan, (3) one particular sequence variant has a mutation in the gene for the viral ligand protein, (4) this variant is the dominant virus spreading through Europe, and (5) the research paper was not reviewed by other scientists before it was uploaded to a quick-share website and reported by the press. Consider the following to inform the advice you will give. 4. (1 pt) Which stage in viral replication is most likely to be impacted by a mutation in the viral ligand protein? 5. (2 pt) What role does this stage (above) play in transmission (infection of a new host)? https://pubchem.ncbi.nlm.nih.gov/compound/44468216 https://pubchem.ncbi.nlm.nih.gov/compound/Adenosine NAME (in all caps) ____________________________________ Student ID ___________________ MIC 102 WS3 Page 4 of 4 Last updated: 5/26/20 6. (2 pts) Is a missense mutation guaranteed to impact the function of the protein and, therefore, the phenotype of the virus? Briefly explain your answer. 7. (2 pts) Does the increased frequency of a particular sequence variant prove the variant has increased transmissibility? Explain your answer. (Consider: if an outbreak is initiated by a single infected individual, how many variants would you expect to isolate from new cases?) 8. (1 pt) Why is it important—and standard procedure—for other experts in the field of study to critically evaluate the methods, results, and conclusions in a manuscript before it is published and presented to the general public as fact, particularly during an epidemic? 9. (2 pts) How concerned should your neighbor be about the reported transmissibility of the mutated strain, and how can she avoid getting infected in light of this new data?