KP Ramirez Week 9
Former Question: Are their differences in HIV-1 diversity or divergence between participants with high CD4 T cell variability within the study (between visits) as compared to participants with linear ‘progression’ (defined as CD4 T cell counts which fall rapidly, or linearly, over time (slope ~ -1)?
Former Prediction: We predict that participants with high variability in T cell count between visits will show a lower HIV-1 diversity and divergence than participants with linear progression. This is predicted under two assumptions
- (1) High diversity and divergence of HIV-1 variants indicates a more rapidly progressing virus (and thus a steadily falling CD4 T cell count)
- (2) high variability in T-Cell count will indicate a participant’s immune system was able to manage this virus better than a participant with a steadily falling CD4 counts. If we do see high diversity in participants with high variability in T cell count between visits, we predict that these will be predominantly synonymous mutations as opposed to non-synonymous mutations (which we would expect to see with linear progressors).
- Linear Progressors: (slope: -1) Subject: 4, 10
- High Variability between visits: Subject 12, 8
- (Low Variability between visits: 5
Article: Early viral load and CD4+ T cell count, but not percentage of CCR5+ or CXCR4+ CD4+ T cells, are associated with R5-to-X4 HIV type 1 virus evolution.*
- Pancera M, Majeed S, Ban YE, Chen L, Huang CC, Kong L, Kwon YD, Stuckey J, Zhou T, Robinson JE, Schief WR, Sodroski J, Wyatt R, and Kwong PD. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1166-71. DOI:10.1073/pnas.0911004107 |
HIV Structure Research Project
Today you will begin your HIV gp120 Structure Research Project.
- For this project, you can choose to work with the same sequences you used for the HIV Evolution Project, or you may choose different sequences. You will reframe your question from the HIV Evolution Project to make it a structure→function question. Instead of looking at how the evolution of variation of the viral DNA sequence affects the different patient groups, you will look at how variations in the viral sequence affect the structure and, therefore, function of the virus. To answer your question, you will need to do the following:
- Convert your DNA sequences into protein sequences.
- How will you do this?
- How will you know that it was done correctly?
- Perform a multiple sequence alignment on the protein sequences.
- Are there more or fewer differences between the sequences when you look at the DNA sequences versus the protein sequences?
- How do you account for this?
- Which of the procedures from Chapter 6 that you ran on the entire gp120 sequence are applicable to the V3 fragment you are working with now?
- How are they applicable?
- Chapter 11 contains procedures to use for working with protein 3D structures. Find the section on "Predicting the Secondary Structure of a Protein Sequence" and perform this on both the entire gp120 sequence and on the V3 fragment that we are now working with. You will compare the predictions with the actual structures.
- Download the structure files for the papers we read in journal club from the NCBI Structure Database.
- These files can be opened with the Cn3D software site that is installed on the computers in the lab (this software is free, so you can download it and use it at home, too.) Familiarize yourself with the software features (rendering and coloring) with both the gp120 peptide and ternary complex structures. (The Dummies book has some information on this program as well). Answer the following:
- Find the N-terminus and C-terminus of each (poly)peptide structure.
- Locate all the secondary structure elements. Do these match the predictions you made above?
- Locate the V3 region and figure out which sequences from your alignment are present in the structures and which sequences are absent.
- Once you have oriented yourself, analyze whether the amino acid changes that you see in the multiple sequence alignment would affect the 3D structure and explain why you think this.
- The journal club papers we read are quite old already for a fast-moving field. Using the Web of Science (or PubMed or Structure) databases, find at least one more recent publication that has a structure of gp120 (V3) in it and download the structure file to view. What additional information has been learned from this new paper?
- Your presentation will be formatted similarly to the previous HIV Evolution Project. In this case, you will want to work on creating structure figures that illustrate what result you are trying to show.
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