BIOL368/F14:Isabel Gonzaga Week 9

Defining Your HIV Structure Research Project

This research project will be completed in conjunction with Nicole Anguiano and Chloe Jones.

Question

How does the structure of the V3 protein region affect the HIV status (diagnosed, progressing or non-trending) of the patient?

Hypothesis

We hypothesize that diagnosed groups will express greater variability in the V3 region in their protein structure, in comparison to the non-trending groups. Initial comparisons show that diagnosed groups and progressing groups expressed greater genetic variability than non-trending groups. These changes may affect the third variable region, affecting the host's ability to adapt to the changes and generate sufficient immune response.

Subject Data

According to the BEDROCK HIV Sequence Data Table, I was able to determine which of the subjects used within my study actually developed aids. All 3 AIDS diagnosed were confirmed with the disease by their final visit. In the AIDS progressing groups, subjects developed AIDS within 1 year after their final visit. The Non-Trending groups all maintained high CD4 T Cell Counts above the threshold, even after the study was conducted. Sequences were for each visit and subject were chosen using a Random Integer Generator, to eliminate selection bias.

The following sequences was taken from the BEDROCK HIV Problem Space Database, from the Markham et al. (1998) study.
Table 1: Sequences analyzed

Group	Subject	Visit	Sequences
AIDS Diagnosed	3 10 15	1 6 1 6 1 4	1, 2, 4 3, 4, 5 3, 6, 7 2, 4, 8 2, 3, 4 5, 8, 10
AIDS Progressing	7 8 14	1 5 1 7 1 9	2, 3, 9 2, 8, 9 1, 4, 5 1, 6, 7 2, 3, 4 9, 10, 11
No Trend	5 6 13	1 5 1 9 1 5	1, 3, 8 4, 5, 2 1, 2, 3 6, 7, 9 1, 3, 4 3, 5, 4

Protein sequences for each data set were taken from BEDROCK HIV Problem Space and converted to the following .txt files using word processor programs:

• AIDS Diagnosed Sequences
• AIDS Progressing Sequences
• [[Media:|No Trend Sequences]].
  

DNA sequences for each data were also taken from BEDROCK HIV Problem Space and converted to the following .txt files: [[Media:|AIDS Diagnosed Sequences]], AIDS Progressing Sequences, [[Media:|No Trend Sequences]].

Protein Sequence Multiple Sequence Alignment

ClustalW was performed for each group category from Visit 1 under the Biology Workbench Protein tools. Rootless phylogenetic trees were analyzed, and the multiple sequence alignment was conducted. This alignment was used to determine diversity within each category at each amino acid residue. ClustalW was also performed for each group category from the final visit.

Multiple Sequence alignment was also used to compare differences between amino acid sequences and DNA sequences. The DNA sequences for each clone was uploaded onto Biology Workbench under the 'nucleic tools' tab. ClustalW was performed for each group category three times: for visit 1, final visit and both visits combined.

There seem to be fewer differences between the amino acid sequences compared to the DNA sequences. This is likely due to the redundancy of the degenerate genetic code (ie. different combinations of DNA sequences form different codons that code for the same amino acid residue).

Protein sequence alignments were downloaded as a .txt file for further analysis. The alignments can be found here:

• MSA Progressing Groups at Initial Visits
• MSA Progressing Groups at Final Visits

Diagnosed Group Multiple Sequence Alignments

Figure 1. Multiple Sequence alignment comparing all AIDS diagnosed sequences at the first visit. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus.

Figure 2: Tree for the AIDS diagnosed patients at the first visit. Each clone is most closely related to clones from its own species, as expected.

Figure 3. Multiple Sequence alignment comparing all AIDS diagnosed sequences at the final visit. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus.

Figure 4. Tree for the AIDS diagnosed patients at the first visit. Each clone is most closely related to clones from its own species, as expected.

Figure 5. Multiple Sequence alignment comparing all AIDS diagnosed sequences. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus.

Figure 6. Tree for the AIDS diagnosed patients at the first visit. Each clone is most closely related to clones from its own species, as expected.

Progressing Group Multiple Sequence Alignments

Figure 7. Multiple Sequence alignment comparing all progressing amino acid sequences at the first visit. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus. For all first visit progressing sequences, 9 residues had no consensus, 17 strong groups were conserved and 9 weak groups were conserved.

Figure 8. Phylogenetic tree generated for all AIDS progressing sequences at the first visit. Clones from the same virus were more genetically similar to each other, than to clones from other subjects.

Figure 9. Multiple Sequence alignment comparing all progressing amino acid sequences at the final visit. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus. For all final visit progressing sequences, 12 residues had no consensus, 24 strong groups were conserved and 3 weak groups were conserved.

Figure 10. Phylogenetic tree generated for all AIDS progressing sequences at the final visit. Clones from the same virus were more genetically similar to each other, than to clones from other subjects.

Figure 11. Multiple Sequence alignment comparing all progressing amino sequences. Blue asterisks (*) denote single, fully conserved residues. Green colons (:) denote conservation of strong groups, and Dark Blue periods (.) denote conservation of weak groups. Black (no symbol) indicates no consensus. For all progressing sequences, 14 residues had no consensus, 23 strong groups were conserved and 7 weak groups were conserved.

Figure 12. Phylogenetic tree generated for all AIDS progressing sequences at the first and last visits. Clones from the same virus were still more genetically similar to each other, than to clones from other subjects.

Non-trending Group Multiple Sequence Alignments

Figure 13. Multiple Sequence alignment comparing all progressing sequences at the first visit.

Figure 14. Phylogenetic tree comparing all progressing sequences at the first visit.

Figure 15. Multiple Sequence alignment comparing all progressing sequences at the final visit.

Figure 16. Phylogenetic tree comparing all progressing sequences at the final visit.

Figure 17. Multiple Sequence alignment comparing all progressing sequences.

Figure 18. Phylogenetic tree comparing all progressing sequences.

DNA Sequence Multiple Sequence Alignment

AIDS Diagnosed Groups

Figure 19. Multiple Sequence alignment comparing all AIDS Diagnosed DNA sequences at the first visit. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 69 total differences were observed between the DNA sequences.

Figure 20. Multiple Sequence alignment comparing all AIDS Diagnosed DNA sequences at the final visit. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 79 total differences were observed between the DNA sequences.

File:AIDSDiagnosedSA DNA.png

Figure 21. Multiple Sequence alignment comparing all AIDS Diagnosed DNA sequences. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 94 total differences were observed between the DNA sequences.

Visit	Type	Number of Differences	Percentage Different
1	Amino Acids	37	37/95 = 38.9%
1	DNA	69	69/285 = 24.2%
Final	Amino Acids	45	45/95 = 47.3%
Final	DNA	79	79/285 = 27.7%
Both	Amino Acids	49	49/95 = 51.6%
Both	DNA	94	94/285 = 33%

Table 2. Table illustrating the number of differences between the amino acid sequences and the DNA sequences at both visits.

AIDS Progressing Groups

Figure 22. Multiple Sequence alignment comparing all progressing DNA sequences at the initial visit. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 52 differences were observed between the DNA sequences.

Figure 23. Multiple Sequence alignment comparing all progressing DNA sequences at the final visit. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 66 differences were observed between the DNA sequences.

Figure 24a. Part 1 of the multiple Sequence alignment comparing all progressing DNA sequences. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 80 total differences were observed between the DNA sequences.

Figure 24b. Part 2 of the multiple Sequence alignment comparing all progressing DNA sequences. Blue asterisks (*) denote single, fully conserved residues. Black indicates no consensus. 80 total differences were observed between the DNA sequences.

Sequence Differences Between Progressor Groups

Visit	Type	Number of Differences	Percentage Different
1	Amino Acids	31	31/95 = 32.6%
1	DNA	52	52/285 = 18.2%
Final	Amino Acids	39	39/95 = 41%
Final	DNA	66	66/285 = 23.2%
Both	Amino Acids	44	44/95 = 46%
Both	DNA	80	80/285 = 28.1%

Table 3. The calculated percentage differences of amino acid and DNA residues for Progressor groups at the initial visits, final visits, and combined groups. The data suggests a trend towards increasing diversity over time, within the progressor group. Amino acid sequences also show less consensus than DNA sequences.

Non Trending Groups

Visit	Type	Number of Differences	Percentage Different
1	Amino Acids	36	36/95 = 37.9%
1	DNA	67	67/285 = 23.5%
Final	Amino Acids	38	38/95 = 40%
Final	DNA	67	67/285 = 23.5%
Both	Amino Acids	43	43/95 = 45.3%
Both	DNA	79	79/ 285=27.7%

Table 4. Showing the percent difference in the Protein Sequence and DNA Sequence for Subject 5,6,13 for the first visit, final visit, and all the visits.

Secondary Structure Prediction in V3 Fragment

PSIPRED Protein Sequence Analysis Workbench was accessed. The multiple sequence alignments created through ClustalW were uploaded to the page for analysis for each group at the initial visit, final visit, and then for each subject. The following results were generated by the program. Yellow indicates beta sheets, pink cylinders indicate helices, and the black indicates the coil. The varying levels of confidence by the program is indicated by the height of size and darkness of the blue bar above each sequence.

AIDS Diagnosed

Figure 25. PsiPred result for the first visit of all the AIDS diagnosed patients. The V3 region is located at 29-53. Like subject 15, it does not have a beta sheet at residues 47-49, likely due to the influence of subject 15.

Figure 26. PsiPred result for the final visit of all the AIDS diagnosed patients. The V3 region is located at 29-53. Like subject 15, it does not have a beta sheet at residues 47-49, likely due to the influence of subject 15.

Figure 27. PsiPred result for both the first and last visits of subject 3. The V3 region is located at 29-53.

Figure 28. PsiPred result for both the first and last visits of subject 10. The V3 region is located at 29-53.

Figure 29. PsiPred result for both the first and last visits of subject 15. The V3 region is located at 29-53. This is the only subject in which there is not a beta sheet at residues 47-49.

• With the exception of subject 15, each of the sequences matched the consensus sequence. Perhaps due to the influence of subject 15, the first and final visits results for all the sequences also are missing the beta sheet that subject 15 is missing.

AIDS Progressing

Figure 30. PSIPred secondary structure prediction for the Progressing groups at their initial visit. Beta sheets and alpha helices are predicted throughout the protein. The V3 region (residue 29-63) contains a 3 residue beta sheet (47-49), and a 6 residue alpha helix (56-61).

Figure 31. PSIPred secondary structure prediction for the Progressing groups at their final visit. Beta sheets and alpha helices are predicted throughout the protein. The V3 region (residue 29-63) contains a 3 residue beta sheet (47-49), and a 6 residue alpha helix (56-61). The final visit prediction is the same as the initial visit prediction.

Figure 32. PSIPred secondary structure prediction for subject 7 at their first and final visit. Beta sheets and alpha helices are predicted throughout the protein. The V3 region (residue 29-63) contains a 3 residue beta sheet (47-49), and a 6 residue alpha helix (56-61). The predicted structure is the same as all other progressing group predictions.

Figure 33. PSIPred secondary structure prediction for subject 8 at their first and final visit. Beta sheets and alpha helices are predicted throughout the protein. The V3 region (residue 29-63) contains a 3 residue beta sheet (47-49), and a 6 residue alpha helix (56-61). The predicted structure is the same as all other progressing group predictions.

Figure 34. PSIPred secondary structure prediction for subject 14 at their first and final visit. Beta sheets and alpha helices are predicted throughout the protein. The V3 region (residue 29-63) contains a 3 residue beta sheet (47-49), and a 6 residue alpha helix (56-61). The predicted structure is the same as all other progressing group predictions.

Non Trending

Figure 35. The results from PsiPred using the amino acids from all Subjects 5,6, and 13 for Visit 1.

Figure 36. The results from PsiPred using the amino acids from all Subjects 5,6, and 13 for last visit.

Figure 37. The results from PsiPred using the amino acids from Subject 5, for first and last visit

Figure 38. The results from PsiPred using the amino acids from Subject 6, for first and last visit)

Figure 39. The results from PsiPred using the amino acids from Subject 13, for first and last visit

Figure 40. The results from PsiPred using the amino acids from Subjects 5,6, and 13, for first and last visit

Analysis of V3 Structure

Huang et al. (2005) Structure 2B4C was uploaded onto StarBiochem. Images were developed by selecting various structural levels and adjusting size of atoms and groups. Using StarBiochem, the V3 region was isolated and the sequence was found.

Four polypeptide subunits were defined using the Quatenary structure methods:

• Chain G (Yellow)
  • Residues: 84:G - 492:G
  • Amino end: Valine (position 84)
  • Carboxyl end: Glutamate (position 492)
• Chain C (Light Pink)
  • Residues: 1:C - 175:C
  • Amino end: Lysine (position 1)
  • Carboxyl end: Valine (position 175)
• Chain L (Green)
  • Residues: 1:L - 214:L
  • Amino end: Glutamate (position 1)
  • Carboxyl end: Cysteine (position 214)
• Chain H (Dark Red)
  • Residues: 2:H - 216:H
  • Amino end: Glutamine (position 2)
  • Carboxyl end: Cysteine (position 216)

Figure 41. Amino and carboxyl termini of the gp120 protein polypeptide subunits depicted using StarBiochem.

Figure 42. The four polypeptide quatenary structures of the gp120 protein depicted using StarBiochem.

Secondary Structure Elements:

• Beta Sheets: 22
• Alpha Helices: 17
• Random Coil: 84

Figure 43. Alpha helices (pink) and beta sheets (blue) as depicted as secondary structures within the gp120 protein using StarBiochem.

V3 Region

• Located between 296:G and 331:G
• Sequence as follows:
  • C T R P N Q N T R K S I H I G P G R A F Y T T G E I I G D I R Q A H C
• V3 Amino Acid Properties
  • Nonpolar: 12
  • Polar: 11
  • Positively charged: 6
  • Negatively charged: 2
  • Aromatic: 2

Figure 44. Image of the isolated V3 region from the gp120 protein as imaged on StarBiochem. The region was determined between 296:G and 331:G. This region contains a beta sheet and alpha helix, similar to the previous PSIPreds for the other

Figure 45. Image of the V3 region in the context of the entire gp120 protein structure as depicted on StarBiochem.

Figure 46. V3 region sequence from 296:G to 331:G in the amino acid sequence viewer of StarBiochem.

The gp120 protein from Huang et al. was ran on PSIpred in order to predict secondary structures. The V3 region sequence was identified at residues 293 through 326. This was compared to the PSIPred's for the Markham et al. sequences. The amino acid for the Markham et al. sequences were identified from residues 29-63. In each of the PSIPred runs, a corresponding beta sheet and alpha helix exists, as expected. This shows that the proper portion of the Markham et al. sequences were identified as the V3 regions.

Figure 47. PSIPred of the Huang et al. gp120 protein. The V3 sequence region was identified from residues 293 to 326

Amino Acid Sequence Effects on V3

The amino acids of the Markham et al. sequences with non conserved residues within the V3 region (residues 29-63 of the 95 amino acid sequence) were analyzed for the progressing group

Visit	Position (from start of V3 sequence)	Conservation	Residues
1	10	strongly conserved	K, R
1	13	no conservation	N, S, P, L
1	14	no conservation	T, I
1	20	strong	F
1	22	strong	A, T
1	25	strong	D, E
1	29	strong	D, N
1	10	no conservation	N, S, P, L
F	5	strong	N, H
F	10	strong	K, E
F	11	no conservation	R, S
F	13	no conservation	S, H, N
F	14	strong	L, I
F	19	weak	V, A
F	20	none	Y, F, L
F	22	strong	T, A
F	25	none	Q, E, K, A
F	29	strong	D, N
F	32	strong	K, Q
F	34	strong	Y, H
All	5	strong	N, H
All	10	weak	K, E
All	11	no conservation	R, S
All	13	no conservation	S, H, N, L, P
All	14	none	L, I
All	19	weak	V, A
All	20	none	Y, F, L
All	22	strong	T, A
All	25	none	Q, E, K, A, D
All	29	strong	D, N
All	32	strong	K, Q
All	34	strong	Y, H

Table 5: Sequences analyzed for progress or group changes at the V3 region.

Presentation

HIV Structure Project

• Isabel Gonzaga
• BIOL 368 Fall 2014

Weekly Assignments

• Week 1 Assignment
• Week 2 Assignment
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• Week 4 Assignment
• Week 5 Assignment
• Week 6 Assignment
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• Week 10 Assignment
• Week 11 Assignment
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• Week 13 Assignment
• Week 15 Assignment

Class Journals

• Class Journal Week 1
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Electronic Lab Notebook

• Isabel Gonzaga Week 2
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• Isabel Gonzaga Week 8
• Isabel Gonzaga Week 9
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• Isabel Gonzaga Week 13
• Isabel Gonzaga Week 15