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Evolution in fast motion – Resistance to HIV (Exercise)

Resistance to HIV In the early 1990s, a number of studies revealed that some people – although they had repeatedly contact with HIV – did not become carriers of HIV or, in the case of a confirmed HIV-infection, showed a delayed onset of the disease (AIDS) (a delay of several years was reported). First attempts to explain these phenomena were observed a few years later, when scientists identified important co-receptor molecules on the surface of the host cells, which are essential for HIV to infect the host cell. Scientists assumed that resistant persons may carry an aberrant version of the co-receptor molecule, which makes it impossible for the virus to enter the host cell. Such a co-receptor is the chemokine co-receptor CCR5, which is normally involved in the host’s immune answer (Dean & O’Brien, 1998). In order to test their hypothesis, the scientists sequenced the which code for the co- receptor CCR5. They investigated more than 700 samples from HIV-infected patients and compared them with the CCR5-sequences from more than 700 healthy persons. The results of the DNA-sequencing revealed mutations in the CCR5- in HIV-infected persons with a delayed onset of AIDS, as well as in some samples of healthy persons (but not in HIV- patients with typical onset of AIDS) (Samson et al., 1996).

Exercises

1. In material 1, you can find two CCR5-gene-sequences selected from the data set of the scientists. Compare the sequences and a. find out where the mutation is (identify the position and label it in both! sequences). b. define the kind of mutation.

Advice: In order to facilitate your investigation, the section of the sequence containing the mutation is marked red. Start with the comparison of the in a forward direction, label the beginning of the mutation exactly and then proceed with the comparison in a backward direction, starting at the end of the red section! Label any difference you find!

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Evolution in fast motion – Resistance to HIV (Exercise)

2. Formulate hypotheses about the consequences such a mutation may have for the subsequent translation of the gene in protein biosynthesis, where the sequence of nucleotides is translated into a sequence of amino acids. ______

3. Test your hypotheses by translating the underlined triplets within the red sections of both DNA-sequences (material 2) into amino acid sequences using the RNA-Code (tool 1). What is the difference between the resultant proteins? Consider that the code (tool 1) applies to RNA-nucleotides, whereas the present sequences consist of DNA-nucleotides!

Advice: Using the RNA-code for the translation of DNA The double-stranded DNA is composed of the coding strand and the ‘antisense-strand’. The strand serving as the template for the into mRNA is the ‘antisense- strand’. It is read in 3’5’ direction, while the synthesis of the mRNA is carried out in 5’3’ direction. The resulting RNA molecule is called ‘sense-strand’ and its - sequence corresponds to the coding strand of the DNA [except for the substitution of thymidin (T) and uridin / (U)].

______

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Evolution in fast motion – Resistance to HIV (Exercise)

Additional information: The gene for the co-receptor CCR5 is located on the third human pair of chromosomes. There are two alleles for this locus. If the two alleles are identical, the person is homozygous. If the alleles are different, the person is heterozygous. If a person is homozygous for the “standard”-allele CCR5 (CCR5/CCR5), he or she is not resistant to HIV. If the person is heterozygous, which means he or she bears both a standard allele CCR5 and a mutant allele CCR5Δ32 (CCR5/CCR5Δ32), the onset of the disease will be delayed. In fact, only persons who are homozygous for the mutant allele (CCR5Δ32/ CCR5Δ32) are resistant to an infection with HIV. 4. Explain why only persons who are homozygous for the mutant allele are resistant to a HIV-infection, while being heterozygous just means that the onset of the disease is delayed! ______

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Evolution in fast motion – Resistance to HIV (Exercise)

Tool 1: The (RNA-) Code

Onie, 2007 (www.wickipedia.org) Licenced by: Creative Commons Attribution ShareAlike 3.0 (http://creativecommons.org/licenses/by-sa/3.0/legalcode)

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Evolution in fast motion – Resistance to HIV (Exercise)

Material 1

) www.ncbi.nlm.nih.gov (

bank Accessionnr.: AY463215.1 Gen

) www.ncbi.nlm.nih.gov ( Genbank Accessionnr.: U66285.1

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Evolution in fast motion – Resistance to HIV (Exercise) Material 2

) www.ncbi.nlm.nih.gov ( bank Accessionn r.: AY463215.1 Gen

) www.ncbi.nlm.nih.gov ( Genbank Accessionnr.: U66285.1 www.evolution-of-life.com Janina Jördens (corresponding author) 7

Evolution in fast motion – Resistance to HIV (Exercise)

Expected answers to the problems 1a. Standard allele CCR5 (red section, material 1): CATTACACCTGCAGCTCTCATTTTCCATACAGTCAGTATCAATTCTGGAAGAATTT CCAGACATTAAAGATAGTCATCTTGGGGCTGGTCCT GCCGCTGCTTGTCATGGT CATCTGCTACTCGGGAATCCTAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAG AGGCACAGGGCTGTGAGGCT TATCTT

Mutant allele CCR5 (red section, material 1): CATTACACCTGCAGCTCTCATTTTCCATACA – lack of 32 bases– TTAAAGATAGTC ATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGCTACTCGGGAATCC TAAAAACTCTGCTTCGGTGTCGAAATGAGAAGAAGAGGCACAGGGCTGTGAGGC TTATCTT

1b. Deletion

2. a. Frameshift mutation and thus a different sequence of amino acids (primary structure) and, as a further consequence, different secondary and tertiary structure of the protein. b. Frameshift mutation and thus a different sequence of amino acids and early generation of a stop-codon, which determines a shorter protein. c. no frameshift mutation, number of deleted bases is a multiple of 3.

3. Frameshift mutation and thus a different sequence of amino acids and early generation of a stop-codon, which determines a shorter protein. This leads to a lack of three transmembrane segments in the CCR5 receptor, which are essential for binding to HIV (see: “illustration of the modification”). standard allele CCR5 (red section underlined, material 2): … His, Tyr, Thr, Cys, Ser, Ser, His, Phe, Pro, Tyr, Ser, Gln, Tyr, Gln, Phe, Trp, Lys, Asn, Phe, Gln, Thr, Leu, Lys, Ile, Val, Ile, Leu, Gly, Leu, Val, Leu, Pro, Leu, Leu, Val, Met, Val, Ile, Cys, Tyr, Ser, Gly, Ile, Leu, Lys, Thr, Leu, Leu, Arg, Cys, Arg, Asn, Glu, Lys, Lys, Arg, His, Arg, Ala, Val, … mutant allele CCR5 (red section underlined, material 2): … His, Tyr, Thr, Cys, Ser, Ser, His, Phe, Pro, Tyr, Ile, Lys, Asp, Ser, His, Leu, Gly, Ala, Gly, Pro, Ala, Ala, Ala, Cys, His, Gly, His, Leu, Leu, Leu, Gly, Asn, Pro, Lys, Asn, Ser, Ala, Ser, Val, Ser, Lys, STOP

4. In persons, who are homozygous for the mutant allele, the co-receptor CCR5 is exclusively produced in the modified, nonfunctional version, thus HIV is not able to infect the host cell. If the person is heterozygous for the mutant allele, the functional co-receptor is expressed to a minor degree. HIV is able to infect the host-cells, however to a lesser degree, so that the onset of the disease is delayed.

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Evolution in fast motion – Resistance to HIV (Exercise)

Illustration of the modification:

Intact co-receptor CCR5 and the binding of HIV on the cell-surface

Mutant co-receptor CCR5Δ32

Because of the early interruption of the protein biosynthesis, the mutant protein lacks the last three transmembrane segments. Thereby HIV cannot bind to the host cell and the fusion of the membranes is not initiated.

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Evolution in fast motion – Resistance to HIV (Exercise)

Literature & links: Dean, M. and S. O'Brien (1997). "In Search of AIDS-Resistance Genes." Scientific American 9: 28-35. Samson, M., F. Libert, et al. (1996). "Resistance to HIV-1 infections in caucasian individuals bearing mutant allels of the CCR-5 chemokine receptor gene." Nature 382: 722-725.

HIV: the ultimate evolver (www.evolution.berkeley.edu/evolibrary/article/_0_0/medicine_04) (last access: July 2009) Ghost of epidemic past (www.evolution.berkeley.edu/evolibrary/news/081001_hivmalaria) (last access: July 2009) HIV immunity (www.pbs.org/wgbh/evolution/library/10/4/l_104_06.html) (last access: July 2009)

www.evolution-of-life.com Janina Jördens (corresponding author)