HIV entry and accessory genes
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HIV entry and accessory genes
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[post_date] => 2024-12-23 17:53:37
[post_date_gmt] => 2024-12-23 22:53:37
[post_content] => Practice Passage (Question 1-6)
*This passage is the property of Khan Academy and has been reformatted into an AAMC-style interface in their entirety by MedLife Mastery. MedLife Mastery does not endorse and is not an affiliate of Khan Academy.
The cellular entry of human immunodeficiency virus 1 (HIV-1) involves the adhesion of the virus to CD4+ T cells, the fusion of the cell and viral membranes, and the subsequent delivery of the viral core into the cytoplasm. The initial binding of virions to the target cell is usually mediated by the HIV-1 envelope (Env) protein, a heavily glycosylated trimer of gp120–gp41 heterodimers. Its attachment to the target cell can be relatively nonspecific, with Env interacting with negatively charged cell-surface heparan sulfate proteoglycans, or it can result from more specific interactions. The second step of virus entry entails the binding of Env to its primary receptor, the host protein CD4. Entry also requires coreceptor binding, which is thought to be the trigger that activates the membrane fusion potential of Env. HIV-1 strains can be broadly classified based on their coreceptor usage, but all require binding to the CCR5 and/or CXR5 host cell coreceptor for entry.
Restriction factors are proteins on or in the host cell that can help inhibit cellular entry, as well as other stages of the viral life cycle, including replication, assembly, and release. To counteract these host defense mechanisms, HIV has evolved various accessory genes that encode proteins capable of antagonizing restriction factors.
Researchers evaluating the role of HIV-1 accessory genes in cell infectivity generated four HIV-1 packaging vectors with single mutations in the vif, vpr, vpu, or nef accessory genes and used them to transfect lymphoid CD4 and non-lymphoid CD4 cells. Cells were cotransfected using an inLuc-mR reporter plasmid, a plasmid containing a luciferase gene with expression that is dependent on HIV-1 replication. Scientists analyzed cell-coculture infection, in which the target cells are cultured together with other virus-infected cells, as well as cell-free infection, in which target cells are exposed to purified or concentrated virus particles (virions) that have been removed from infected cells. In both cases, the cells were lysed after three days, and luciferase activity was measured. The results are shown in Figure 1.
Figure 1 Relative luciferase activity in lymphoid and non-lymphoid cells following cell-coculture infection and cell-free infection; * and ** indicate significant differences from wt results at a p < 0.05 and p < 0.01, respectively
Information obtained from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405824/; research data adapted from https://www.mdpi.com/1999-4915/11/5/390
[post_title] => HIV entry and accessory genes
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[question] => The names of gp120 and gp41 are derived from their molecular weights, which are approximately 120 and 41 kDa, respectively. According to this, what is the total molecular weight of the fully assembled Env protein as defined by the passage?
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[answer] => 3
[description] => Reason for Correct Answer:
The passage defines Env as a heavily glycosylated “trimer of gp120–gp41 heterodimers.”
A heterodimer would consist of two different proteins – in this case, gp120 and gp41.
Each of these heterodimers will weigh about 120 + 41 = 161 kDa.
If Env is a trimer of these, it contains three of these heterodimers.
Accordingly, the Env protein weighs about 3 x 161 kDa = 483 kDa. (You can simplify your calculations by doing 3 x 160 = 480, and picking the closest answer.)
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[answers] => Array
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[0] => Array
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[each_answer] => A. 161 kDa
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[each_answer] => B. 322 kDa
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[each_answer] => C. 483 kDa
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[3] => Array
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[each_answer] => D. 966 kDa
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[1] => Array
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[quiz_unique_key] => 1403770772
[question] => Much of the weight of the gp proteins comes from their glycosylated portions. If gp120 consists of 480 amino acids and gp41 consists of 345 amino acids, then what percentage of the gp120–gp41 heterodimer’s 160 kDa weight is attributable to its protein component?
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[answer] => 2
[description] => Reason for Correct Answer:
The total weight of the gp120–gp41 heterodimer is about 160 kDa, as stated in the question and discussed in the previous explanation. In this question, you have to determine what percentage of that comes from the protein component, given the number of amino acids.
The average weight of an amino acid is approximately 110 Da (daltons). When estimating the molecular weight of a protein, you can use the average amino acid weight because many different amino acids are present in proteins.
Together, the gp120 and gp41 proteins have 480 + 345 = 825 amino acids.
This means that the approximate weight of the protein component is 825 amino acids x 110 Da/amino acid = 90,750 Da. (Alternatively, you could simplify the calculation by doing 825 x 100 Da/amino acid = 82,500, which you could round up, knowing that it will be a slight underestimate.)
This means that the weight percent of gp120–gp41 protein components is approximately (91 kDa/160 kDa) x 100 = 57% (or, if you simplified the calculation, >83 kDa/160 kDa x 100 = a little over 50%**).
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[answers] => Array
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[each_answer] => A. 25%
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[each_answer] => B. 57%
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[each_answer] => C. 75%
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[each_answer] => D. 91%
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[2] => Array
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[quiz_unique_key] => 1403770772
[question] => Which residues on Env are most likely to interact with the cell surface on initial, non-specific attachment to the cell, according to the information in the passage?
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[answer] => 1
[description] => Reason for Correct Answer:
The passage states that the envelope glycoprotein (Env) interacts with “negatively charged cell-surface heparan sulfate proteoglycans” upon initial, non-specific attachment.
You should expect positive residues on Env to interact with negatively charged heparan sulfate proteoglycans.
Here is a table of the amino acids:
https://commons.wikimedia.org/wiki/File:Amino_acids.png
Positively charged arginine (Arg) or lysine (Lys) residues can interact with the negatively charged sulfate groups of heparan sulfate through charge–charge (electrostatic) interactions.
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[answers] => Array
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[0] => Array
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[each_answer] => A. Arginine and lysine
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[1] => Array
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[each_answer] => B. Glutamine and asparagine
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[each_answer] => C. Cysteine and proline
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[each_answer] => D. Methionine and threonine
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[quiz_unique_key] => 1403770772
[question] => In this study, what is the purpose of cotransfecting the cells with the inLuc-mR reporter plasmid?
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[answer] => 3
[description] => Reason for Correct Answer:
The inLuc-mR reporter plasmid contains the luciferase gene, according to the passage.
Luciferase is an enzyme found in bioluminescent organisms that catalyzes a chemical reaction that produces light. It is used as a molecular tool in research to track biological processes by engineering cells to produce luciferase and measuring the intensity of the light emitted.
Figure 1 uses relative luminescence as a marker of relative HIV-1 infection of target cells under varying conditions (graphed as ‘relative luciferase activity’).
Thus, the luciferase activity of the “HIV-1 replication-dependent” reporter plasmid is used to quantify relative HIV-1 infection of target cells. The luciferase gene of the reported plasmid is interrupted by an intron; this intron prevents the expression of luciferase in transfected cells. However, infected cells can splice this intron out of the reporter gene, allowing for the expression of the luciferase protein.
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[each_answer] => A. To verify that cell transfection was working properly
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[each_answer] => B. To increase the availability of target cells for infection
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[each_answer] => C. To quantify relative HIV-1 infection of target cells
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[each_answer] => D. To normalize cell infection data
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[quiz_unique_key] => 1403770772
[question] => The results in Figure 1 support which conclusion?
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[answer] => 2
[description] => Reason for Correct Answer:
The passage states that “cell-coculture infection” entails target cells being cultured together with other virus-infected cells, versus “cell-free expression, in which target cells are exposed to purified or concentrated virus particles.” This means that the cell-coculture condition gauges the cell–cell transmission of the virus.
Figure 1 shows the results of cell-coculture infection and cell-free infection for lymphoid and non-lymphoid cells in the presence of various gene mutations.
For non-lymphoid cells, the vpr mutation did not inhibit infection (for either transmission route), compared to wild type, and the vif mutation only significantly impacted transmission in the cell-free case, so Choice C cannot be correct.
Mutation of the nef gene did NOT impact cell–cell transmission in non-lymphoid cells, so Choice A cannot be correct.
Mutation of the vpu gene resulted in increased infectivity of HIV-1 in the cell coculture setting for both lymphoid and non-lymphoid cells.
This suggests that a functional vpu gene and Vpu protein may inhibit cell–cell HIV-1 transmission in both non-lymphoid and lymphoid cells. This suggests B is true and D is not.
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[each_answer] => A. Mutation of the nef gene significantly impacts cell–cell HIV-1 transmission in both non-lymphoid and lymphoid cells.
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[each_answer] => B. A functional vpu gene may inhibit cell–cell HIV-1 transmission in both non-lymphoid and lymphoid cells.
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[each_answer] => C. Vpr and vif proteins are required for HIV-1 replication in non-lymphoid cells, regardless of the transmission route.
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[each_answer] => D. The Vpu protein increases HIV-1 replication in cell cocultures in non-lymphoid cells.
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[quiz_unique_key] => 1115843717
[question] => Which of the following statements is consistent with the results presented in Figure 1?
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[answer] => 1
[description] => Reason for Correct Answer:
The data in Figure 1 shows that the vpr mutation decreases relative infectivity in both modes of transmission in lymphoid cells.
The data shows that the vpu mutation can actually increase the infectivity of HIV at least in the cell-coculture setting.
Antagonists decrease the activity of their targets; therefore, they should mimic the effects of the mutations. This means that Vpr antagonists will counter infection and Vpu antagonists will promote infection; so, Choices B and C are incorrect.
Figure 1 suggests that more accessory genes are essential for the infection of lymphoid cells than non-lymphoid cells (in either mode of transmission), as more gene knockouts lead to loss of infection in these cells:
As discussed in the passage, restriction factors are host proteins that defend against viruses by counteracting (‘restricting’) viral replication, and accessory genes help the virus to evade restriction factors.
Therefore, the data suggest that lymphoid cells express a wider range of restriction factors, and more accessory proteins must act to evade these restriction factors.
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[each_answer] => A. Lymphoid cells express a broader range of restriction factors than non-lymphoid cells.
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[each_answer] => B. Vpr antagonists promote HIV-1 infection in lymphoid cells.
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[each_answer] => C. Vpu antagonists are potential antivirals for the treatment of HIV-1 infection.
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[each_answer] => D. The vpr gene is more critical to one mode of transmission than the other.
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Figure 1
