1. Analysis of HIV Evolution Bobak Seddighzadeh and Kristoffer Chin Department of Biology Loyola Marymount University Bio 398-01 February 23, 2010

2. Outline Background Question Methods Results Discussion References

3. Function of the HIV Virus  HIV is a retrovirus that affects immune cells, specifically T cells.  Retroviruses contain reverse transcriptase also known as RNA-dependent DNA polymerase.  Reverse transcriptase synthesizes single stranded RNA into a c-DNA molecule.  Since viruses cannot synthesize their own proteins, they require a host cell to carry out its replication process.  HIV infects the host cell. It then injects its genome from its capsid and uses reverse transcriptase to create a c-DNA template that it then incorperates into the host cell ’ s DNA. The host cell transcribes and translates proteins that assemble into more copies of the virus.  Virus can have either the lytic or lysogenic life cycle

4. Markham Study of HIV Virus Evolution is the change in the allele frequency in a population over a period of time More specific to HIV, the population refers to all the HIV viruses in an affected individual, and the allele refers to each variant of the HIV env gene. Different individuals in the population vary in heritable traits that can be passed onto offspring that may or may not give them a selective advantage. Markham et al. (1998) Study Nucleotide sequences of the env region were used to study evolution The viruses were classified into groups according to rate of CD4 T cell decline: progressors and non-progressors Progressors had increased rates of diversity and divergence in comparison to non-progressors and ultimately higher rates of CD4 T cell decline. We wanted to know why a disparity exists in the rate of evolution amongst variants

5. Differences in Nucletide Sequences? More specifically our question was: what is or are the nucleotide sequences in the env region of the V3 domain that are responsible for the increased genetic diversity, divergence, and rate of CD4 T cell decline amongst rapid progressors and non-progressors My partner and I predict that the increased rate of CD4 decline, genetic divergence and diversity are directly related to two possible mechanism: A) HIV-1 variants termed rapid progressors selected for a structural mechanism that does not protect the genetic material against mutations as effectively B) or HIV-1 variants termed rapid progressors selected for a mechanism that induces mutations on its own DNA. We also predict that both these mechanism are related to nucleotide differences found in the env region of the V3 domain.

6. Methods: Selection of Subjects, ClustalW, ClustDist, Counting Nucleotide Differences Selection Subjects and visits – highest rate of CD4 T Cell decline Clones – Largest Max distance, for more variation ClustalW Analysis and comparison of S Analysis and comparison of Unrooted trees of subjects ClustDist Analysis of Max Distance of Nucleotide sequence differences Counting nucleotide differences Analysis of specific nucleotide differences from Non and Rapid Progressors using Excel

7. Results:Non and Rapid Progressors Subjects, Visits, and Clone Selection Non Progressors SubjectVisitClonesMax Distance 21, 3, 4(1,5) (4,5) (3,8)(3) (11) (14) 123, 4, 5(1,3) (1,2) (5,6)(3) (6) (9) 132, 3, 5(1,2) (3,6) (3,6)(1) (3) (4) Rapid Progressors SubjectVisitClonesMax Distance 42, 3, 4(5, 13) (2,16) (1,4)(16) (14) (15) 104, 5, 6(5,8) (3,10) (4,7)(13) (12) (20) 112, 3, 4(1,6) (2,5) (6,8)(3) (7) (7)

8. Results: Unrooted tree of selected Rapid Progressors # of ClonesSThetaMin DistanceMax Distance 1810029451

9. Results: Unrooted tree of selected Non Progressors # of ClonesSThetaMin DistanceMax Distance 187120147

10. Results: Unrooted tree of selected Rapid and Non Progressors # of ClonesSThetaMin DistanceMax Distance 3614234152

11. Results: Samples of differences of Nucleotide Sequence in Non and Rapid Progressors S4V4-4 GAGGTAGTAATTA GATCTGAAAATTT CACGAACAATGCT AAAATTATAATAGT ACAGCTG S4V4-4 AATAAATCTGTAGA AATTAATTGTACAA GACCCAACAACAAT ACAATAAGAAGGAT A--- S4V4-4 CCTATAGGACCCG GCAGAGCATTTTAT ACAACAGG-------- CAGAATAGGCAATA TAAGGCAA S4V4-4 GCTCATTGTAACA TTATTGAAACAAAA TGGAATAACACTTT AAAACTGATAGTT GACAAA S4V4-4 TTAAGAGAACA GTTTGG----- GAATAAAACAA TAATCTTTAATC AATCCTCA S10V4-5 GAGGTAGTAATTA GATCTGAAAATTT CACGGACAATGCT AAAACCATAATAG TACAGCTG S10V4-5 AATGAATCTGTAGA AATTAATTGCACAA GACCCAACAACAAT ACAAGAAGAAGGAT A--- S10V4-5 CATATGGGACCAG GCAGAGCATTTTAT GCAACAGGAGAAA TAATAGGAGATATA AGGCAA S10V4-5 GCACATTGTAACC TTAGTAGAACAAAA TGGAATGACACTTT AAAACAGGTAGTT GACAAA S10V4-5 TTAAGAGAACA ATTTAG----- GAATAAAACAA TAATCTTTAAG CAATCCTCA S2V1-1 GAGGTAGTAATTA GATCCGAAAATTT CACGAACAATGCT AAAATCATAATAGT ACAGCTG S2V1-1 AATGAATCTGTAGA AATTAATTGTACAA GACCCAACAACAAT ACAAGAAAAAGTAT A--- S2V1-1 CATATAGGACCAG GTAGAGCATTTTAT ACAACAGGAGACAT AATAGGAGATATAA GACAA S2V1-1 GCATATTGTAACAT TAGTAGAGCAGAA TGGAATAACACTTT AAAACAGATAGTTA TAAAA S2V1-1 TTGAGAGAACA CTTTGG----- GAATAAAACAA TAGTCTTTAATC ACTCCTCA S13V3-3 GAGATAGTAATTAG ATCCGAAAATTTCA CAAACAATGCTAAA ATCATAATAGTACA GCTG S13V3-3 AAGGAATCTGTAGA AATTAATTGTACAAG ACCTGGCAACAATA CAAGAAGAAGTATA- -- S13V3-3 AATATAGGACCAGG TAGAGCATTTTATG CATCAAGAGGAATA ATAGGAGATATAAG ACAA S13V3-3 GCATATTGTAACAT CAGTAAAGCAAAAT GGGATAACACTTTA GGACAGGTAGCTA CAAAA S13V3-3 TTAAGAGAACA ATTTAG---------- GAATGCTACAA TAGTCTTTAATC ACTCATCA

12. Past Studies in the Nucleotide Sequences in HIV Hill and Hernandez found a certain loop in the gene sequence in Rapid progressors that in the beginning of infection and showed up again in the end. They also found that lack of immunity shows diverse forms, but there is a significant domination once there is a fit form of the disease Found significant diversification in the beginning of the infection and slowly lowered. Specific Changes in the V3 region alter phenotype that correlated to disease progression

13. Past Studies in the Nucleotide Sequences in HIV Usage of CCR4 is related to high sequence conservation in contrast theCXCR4 tropic viruses that spread sequence space over larger regions. The location of the sequences in the space can be used to improve the accuracy of the prediciton of the coreceptror usage. Bozek found that patients with high CD4 T cell counts had highly conservessequences in comparison to patients that had low CD4 T cell counts.

14. Interpretation of Differences found in Nucleotide Sequences 10 nucleotide sequence difference were found in the comparison of the non and rapid progressor subjects that were chosen These 10 differences shows that there were some common sequences that may be necessary to the rapid decline of T Cells that is caused by the HIV virus The 10 differences may not seem a lot, but the unrooted tree of both Non and Rpaid progressors shows a strong diversity in visits 13 and 11, 12 and 11. The other branches of the trees shows close relation among the other visitors which can be interpreted that there is no significant changes in the nucelotide sequences in non and rapid progressors Markham found a lot more diversity in the rapid progressors, it is possible that the clones that we chose was not as effective We were more concerned with nucleotide differences, and we did not even consider the sequence space. This may provide insight as to why there are not significant nucleotide differences amongst progressors and non-progressors yet they functionalally different.

15. Translation of Sequences and Nucleotide Spaces In future studies, we believe that a translation of the nucleotide sequences will show light to the results we found There may be no specific change that is found in the nucleotide sequence, but it is quite possible with the amino acids Hill an Hernandez found differences in the amino acids as finding more Basic than acidic Analyzing the Nucleotide spacing found in the V3 region as analyzed by Bozek

16. References Borrego P, Marcelino JM, Rocha C, Doroana M, Antunes F, Maltez F, Gomes P, Novo C, Barroso H, and Taveira N. The role of the humoral immune response in the molecular evolution of the envelope C2, V3 and C3 regions in chronically HIV-2 infected patients. Retrovirology 2008 Sep 8; 5 78. Hill MD and Hernandez W. Nucleotide and amino acid mutations in human immunodeficiency virus corresponding to CD4+ decline. Arch Virol 2006 Jun; 151(6) 1149-58. Templeton AR, Reichert RA, Weisstein AE, Yu XF, and Markham RB. Selection in context: patterns of natural selection in the glycoprotein 120 region of human immunodeficiency virus 1 within infected individuals. Genetics 2004 Aug; 167(4) 1547-61. Bozek K, Thielen A, Sierra S, Kaiser R, and Lengauer T. V3 loop sequence space analysis suggests different evolutionary patterns of CCR5- and CXCR4-tropic HIV. PLoS One 2009 Oct 9; 4(10) e7387.