SB-BB

Passage 3

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a viral enzyme that mediates the integration of viral DNA (vDNA) into the host chromosome. IN is a 288-residue protein composed of three domains: the N-terminal domain, the catalytic core domain (CCD), and the C-terminal domain. The CCD contains three highly conserved residues (D64, D116, and E152), and mutation of any of these three residues abolishes IN activity.

Integration of vDNA into the host chromosome proceeds by two well-characterized steps, both of which are catalyzed by IN. In the host cell cytoplasm, each end of a newly synthesized vDNA segment is bound at a specific long terminal repeat (LTR) nucleotide sequence by one IN dimer. Upon binding, IN catalyzes cleavage of a GT dinucleotide from each 3PRIME vDNA terminus. Still bound to either end of the vDNA segment, the IN dimers join to form a tetramer, which then binds host cell protein LEDGF at the CCD. This vDNA–IN–LEDGF complex enters the nucleus, where LEDGF tethers it to host chromatin. Then, IN catalyzes the insertion of the vDNA into the host genome.

Small molecule IN inhibitors show promise as antiviral drugs against HIV-1. One class of these inhibitors are LEDGINs, which bind IN and shift its oligomerization equilibrium toward an inactive tetramer that cannot bind vDNA. Another class of small molecule inhibitors are short-length oligodeoxynucleotides (ODNs), whose sequences mimic LTRs. ODNs are competitive inhibitors of vDNA for binding to IN.

Questions

18. What bond is cleaved by IN during the first reaction of integration?

  1. P–C
  2. P–P
  3. P–H
  4. P–O

19. A vDNA sequence encoding a protein is inserted into a host genome by IN. The protein is translated from the hypothetical mRNA sequence shown.

5’–GGCAACUGACUA–3’

Based on the passage, the segment of the original viral genome that encoded this protein had what nucleotide sequence?

  1. 5'–GGCAACUGACUA–3'
  2. 5'–TAGTCAGTTGCC–3'
  3. 5'–CCGTTGACTGAT–3'
  4. 5'–UAGUCAGUUGCC–3'

20. Which experiment would best provide data to support the mechanism by which an ODN inhibits IN activity?

  1. Keep IN concentration constant and measure the initial velocity of the reaction at increasing vDNA concentrations in the presence and absence of the ODN.
  2. Keep vDNA concentration constant and measure the initial velocity of the reaction at increasing concentrations of IN in the presence and absence of the ODN.
  3. Keep ODN and IN concentration constant and measure initial velocity of the reaction at increasing concentrations of vDNA.
  4. Keep IN and vDNA concentrations constant and measure initial velocity of the reaction at increasing concentrations the ODN.

21. Which amino acid substitution for the conserved residue at position 64 is LEAST likely to affect the enzymatic function of IN?

  1. Asparagine
  2. Glutamate
  3. Lysine
  4. Valine

22. An inactive tetramer of IN is expected to have approximately what molecular weight?

  1. 16 kDa
  2. 32 kDa
  3. 64 kDa
  4. 128 kDa

23. To determine if a small molecule acts like a LEDGIN with respect to IN, a researcher plans to incubate purified IN both with and without the small molecule and then perform a Western blot to detect IN in each sample. Under which condition(s) should the gel electrophoresis step be performed?

  1. Denaturing
  2. Reducing
  3. Native
  1. I only
  2. II only
  3. III only
  4. I and II only

Answers

Question 18 Solution: The correct answer is D. This is a Biology question that falls under the content category “Transmission of genetic information from the gene to the protein.” The answer to this question is D because in the first reaction of integration, integrase cleaves a dinucleotide from the 3' end of a strand of viral DNA. This involves the cleavage of a P–O bond. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the structure of DNA.
Question 19 Solution: The correct answer is A. This is a Biology question that falls under the content category “The structure, growth, physiology, and genetics of prokaryotes and viruses.” The answer to this question is A because according to the passage, viral DNA integrated into a host cell genome by integrase would originate from a retrovirus. mRNA transcribed from retroviral DNA is either used to synthesize viral proteins, or used as the RNA genome for progeny viruses. Thus, the sequence of the nucleotide in the original viral genome will be the same as that of the transcribed mRNA. It is a Scientific Reasoning and Problem Solving question because you are asked to recall that HIV is a retrovirus with an RNA genome and use your understanding of reverse transcriptase to use a given transcribed mRNA sequence to determine the original viral genome sequence.
Question 20 Solution: The correct answer is A. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is A because according to the passage, ODNs are competitive inhibitors of viral DNA for integrase. Competitive inhibition can be determined through rate experiments by applying the principles of the Michaelis–Menten equation. By keeping enzyme concentration constant, varying substrate concentration, and either including or excluding the inhibitor, the effect of the inhibitor on the Vmax and apparent Km of the reaction can be determined. A competitive inhibitor will increase the apparent Km and not affect the Vmax. It is a Reasoning about the Design and Execution of Research question because you are asked to evaluate various experiments and select the one that will produce data relevant to the question being investigated.
Question 21 Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because the conserved residue at position 64 in integrase is aspartate, which has a negatively charged side chain. Substitution with glutamate, which also has a negatively charged side chain, is least likely to affect integrase function. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall the properties of the different amino acids.
Question 22 Solution: The correct answer is D. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is D because the passage indicates that an integrase monomer is composed of 288 amino acids, which will have an approximate molecular weight of 32 kDa (the average molecular weight of an amino acid is 110 Da). Thus, a tetramer will have an approximate molecular weight of 128 kDa. It is a Scientific Reasoning and Problem Solving question because you are asked to recall a characteristic of amino acids and, using a calculation, apply that information to solve a problem.
Question 23 Solution: The correct answer is C. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is C because if the intent is to confirm that a small molecule induces the formation of integrase tetramers from integrase dimers, it is necessary to visualize the proteins in their native state. Use of a denaturing agent will disrupt the interactions between monomers. Use of a reducing agent only will disrupt any disulfide bonds. It is a Reasoning about the Design and Execution of Research question because you are asked to choose the experimental conditions that should be used in order to successfully gather data to answer a given question.