1. A Mutational Investigation of an HIV Patient’s GP120 Glycoprotein and it’s Implications on CD4 Binding Salita Kaistha Usrinus College, Collegeville PA 19426 November 28, 2003 Background HIV is a deadly virus killing more than 3 million people a year. This fact alone motivates researchers to learn as much as possible about the virus, its structure, and it’s entry into cells, in hopes of eventually developing a cure. Viewed under a microscope an HIV particle would look like this: An HIV particle is approximately 0.0001 mm. An HIV particle consists of two main parts, the inner core and the viral membrane. This viral membrane contains two main glycoprotein's, GP120 and GP41. GP120 allows the HIV particle to bind to CDR+ T cells of our immune system. GP41 facilitates membrane fusion. Due to GP120’s involvement in cell recognition and binding, it is of great interest to researchers. GP120 is encoded for by 500 amino acids, but only a small portion of these are involved in binding to CD4. In fact, GP120 interactions come from 6 fragments located on the V1/V2 stem, Loop D, B15-alpha15 excursion, B20-B21 hairpin, B23, and the B24-alpha5 connection. This coincides with the general principle that much of a protein’s secondary structure is simply needed to provide a scaffold, and it is the loops that allow the protein to function. The progression of HIV is monitored by an individual’s CD4 T cell count. Prior Research This study was conducted using research and data collected by Markham et al. Markham et al. followed 15 patients for anywhere from 1 ½ - 4 ½ years. They monitored the number of clones of HIV within the individual and the individuals CD4 T cell count. The number of clones per visit ranged anywhere between 2 and 18. The researchers gathered over 666 DNA sequences. Methods and Materials This study was conducted using the data gathered by Markham et al. The study focused on patient # 11, a rapid progressor for HIV. Data was collected for the last two years of the patient’s life. Resources used include those found at PDB, NCBI and ExPASy. DNA Results DNA Results Continued One advantage that HIV has over our immune system is its rapid rate of mutation. There are both synonymous and non-synonymous mutations. Synonymous mutations are those which involve changes in the nucleotide sequence, but still produce the same amino acid sequence. Non-synonymous mutations are those which change the nucleotide sequence and the amino acid sequence. Clone 3 from Visit 1 is what I consider to be the original strain. All other clones are variations of this strain. Clone 4 from Visit 4 is an example of a synonymous mutation, for there is a change in the DNA sequence (1 nucleotide), but the resulting amino acid sequence is the same (Fig. 1). Visit 1 clone 1 is an example of a non-synonymous mutation. There was a change in one nucleotide of the DNA sequence, however this time the DNA sequence produces a different amino acid sequence (Fig. 1). Figure 2 portrays only the amino acid positions that encountered changes over the 2 years. There are 3 predominant changes taking place. These changes include converting the charge (+  -), converting to a smaller amino acid (a.a.  G), or a conservative change (+  +). As the disease progresses there is a simultaneous drop in CD4+ T cell counts and an increase in the number of mutations. Over two years there are 32 strains and only 17 amino acid positions altered over the sequence of 96 amino acids (less than 18%). The percentage of non-synonymous mutations increases as the disease progresses. There are never more than 3 amino acid positions altered per strain of HIV, regardless of the visit number. The number of strains with an increasing number of mutations per strain increases as the disease progresses. Amino Acid Results Figure 2. The amino acids found at selected positions for all of the clones found at all visits for patient 11. The selected positions are those which experienced a mutation at any time during the two years. The mutated amino acids are highlighted according to the following color scheme: yellow for Glycine’s, green for hydrophobic, pink for negatively charged, blue for positively charged and purple for polar amino acids. Figure 3. This chart highlights the information found in the amino acid sequences listed above and also gives the CD4+ T cell counts for the patient at each visit. Figure 1. Codons encoding amino acid positions that were mutated over the 2 years are highlighted in blue. The codons containing the point mutations are highlighted in pink. These sequences demonstrate the fact that these mutations are not occurring throughout the entire sequence, but rather at specific portions of the amino acid sequence over the 2 ½ years (Fig. 4). In other words certain portions are subject to mutations, where as other portions are conserved and experience no mutation over the two year period. As mentioned earlier, GP120 interacts with CD4 through 6 different fragments or portions on GP120. The DNA sequences obtained for patient 11 encode for one of these 6 fragments, that is the V1/V2 stem (Fig. 5). I have located the 6 amino acids that are critical for this interaction. They are AHCNVS. Due to the fact the HIV must bind to CD4 in order to infect an individual’s cells, these 6 amino acids are critical to the virus’s infection and survival. Thus, logically, these 6 amino acids would be conserved, and this is precisely what was discovered. This crucial conserved sequence, AHCNVS, is highlighted in green in Figure 4. Structural Results Figure 4. This chart presents the 96 amino acid sequences for the envelope gene of the GP120 glycoprotein of HIV in the various clones of patient 11 at visits 1 and 4. Amino acids at positions were subject to mutation at any time during the two years are highlighted in blue. Amino acids there were mutated are highlighted in pink. The amino acids highlighted in green represent a region on the V1/V2 steam that is critical for GP120 to bind to CD4. Figure 5. This is a schematic representation of a portion of GP120 that interacts with CD4. It shows the V1/V2 stem on HIV’s GP120, which is encoded for by patient 11’s DNA sequence. The green box highlights 6 amino acids on the V1/V2 stem that are necessary for binding to CD4. The pink box highlights the fragment of CD4 that the V1/V2 stem interacts with. References Kwong, P., Wyatt, R., and J. Robinson. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-659. Markham, R., Wnag, W. and A. Weisstein. (1998) Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc. Natl. Acad. Sci. 95, 12568-12573. The Protein Data Bank. (2003) http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics&pdbId=1G9M&page=&pid=151221070341180. The Molecules of HIV. (2003) http://www.mcld.co.uk/hiv/?q=HIV%20virus%20particle. Aids in the World. (2003) http://www.yale.edu/yaw/world.html. Acknowledgements I would like to thank Dr. Roberts, Tom Seegar, Derese Getnet and Drew Foy for all their help in completing this poster.