1. The Other Coreceptor: CXCR4 Cloning and Sequencing in CD4+ T cell Normal and Depleted Sooty Mangabeys Irene Bukh Post-rotational Talk Guido Silvestri Lab September 21, 2009
2. CXCR4 background GPCR, 7-TM CXCR4/fusin receptor for CXCL12/SDF-1 ...variety of biological roles Wide cellular distribution in immature and mature hematopoietic cells (neutrophils, monocytes, T + B cells, dendritic cells, Langerhans cells, macrophages) Also found on vascular endothelial cells and neuronal/nerve cells HIV co-receptor (Moyle et al. 2006) (Alkhatib, 2009)
6. Hypothesis Could it be that the phenylalanine in the V3 extracellular loop region in SM CXCR4, rather than the tyrosine in humans, makes it more difficult to use the CXCR4 co-receptor due to a tertiary structural change as a result of the amino acid difference? Furthermore, in CD4+ T cell depleted SMs, is there an amino acid mutation away from phenylalanine in the same V3 region that would change the conformation of CXCR4 such that there is less resistance to its use?
7. Let’s get this project started… Select CXCR4 primers based on previous sequences Obtain SM blood samples from Yerkes – including both normal and depleted CD4+ T cell animals Purify RNA and run RT-PCR Initially tested 2 pairs of primers optimized to 1 pair Run agarose gel to cut out CXCR4 bands, extract more pure sample and send for sequencing Analyze sequence
8. SM Blood Samples CD4+ T cell normal FYm Fav FFv FBq FTr FQg FFq FKn CD4+ T cell depleted FFr FBr FAl
10. SM CXCR4 sequence results Human Chimpanzee Rhesus macaque Crab-eating macaque Rhesus macaque Sooty mangabey SM FQg SM FKn SM FFq SM FBq SM FFv SM FYm SM FQg SM FKn SM FFq SM FBq SM FFr SM FBr SM FAl
11. Conclusions Due to small sample size, one cannot exclude the possibility that some SMs with low CD4+ T cell levels may have a mutation in the V3 region lending to changes in tertiary structure
12. Thank you Silvestri Lab! Guido Silvestri MirkoPaiardini VandyVanderford Steve Bosinger Barbara Cervasi Katrina Nolan Jill Adamski Jessica Engram Paul Carnathan Alex Ortiz Jessica Taaffe Beth Cramer Nick Francella Elane Reyes And thank you Sootys….
13. References Alkhatib G. The biology of CCR5 and CXCR4. Curr Opinion in HIV and AIDS 2009. 4:96-103. De Clercq E. The bicyclam AMD3100 story. Nature Rev Drug Discov 2003. 2(7): 581-7. Marx PA, Chen Z. The function of simian chemokine receptors in the replication of SIV. Seminars in Immunology 1998. 10: 215-223. Milush JM, Reeves JD, et al. Journal of Immunology 2007. 179: 3047-3056. Waters LJ, Mandalia S, Wildfire A, Gazzard B, Moyle G. CXCR4/mixed-tropic HIV-1 is associated with more rapid CD4 cell decline compared with CCR5-tropic virus in antiretroviral-naive individuals. In: Program and abstracts of the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 27-30, 2006; San Francisco, Calif. Abstract H-1667. Vodros D, Fenyo EM. Primate models for human immunodeficiency virus infection: evolution of receptor use during pathogenesis. ActaMicrobiol et ImmunoHungarica 2003. 51:1-29.
14. While I was working on this talk, apparently Mila thought sleep was more interesting… And on that note, if anyone wants to or knows someone who wants to adopt a spay dog, let me know!
Editor's Notes
So today I am going to give you all a relatively brief rundown of my project this summer, which had significant assistance from Vandy.
CXCR4 is a chemokine receptor that belongs to the family of G protein coupled receptors. Its only known endogenous ligand is stromal cell-derived factor 1/CXCL12. This receptor-ligand pair play important roles in hematopoiesis, cardiogenesis, vasculogenesis, neuronal development, immune cell trafficking, malignant cell growth, and metastasis due to its wide cellular distribution. These various biological functions are why it has been less attractive to develop a CXCR4 blocking agent for HIV. So moving on to that, as everyone here already knows, CXCR4 is used by HIV as a co-receptor to gain entry into CD4+ T-cells. X4-tropic viruses, as compared to R5-tropic ones, are found with increased frequency in late disease, are associated with CD4 decline, HIV RNA increase and clinical progression.
The gp120 viral envelope has an affinity for the CD4 receptor which is expressed on the cell surface of the CD4+ cell. Binding of gp120 to the CD4 receptor causes a conformational change in the gp120 protein. This change exposes a binding site for a chemokine co-receptor, such as CCR5 or CXCR4. Once gp120 binds the coreceptor, this causes another conformational change, this time in the gp41 protein, allowing insertion of a fusion peptide from the gp41 protein into the cell membrane. This insertion draws the virus and CD4+ cell close enough together to allow the virus envelope and cell membrane to fuse. Now the viral core can enter the CD4+ cell and infection begins.
These figures are from Don Sodora’s 2007 JI paper which showed that CD4+ T cell depletion by SIV is not an adequate stimulus, by itself, to induce AIDS in a natural host – here, the sooty mangabey. They identified two SMs that had a multitropic SIV infection emerge after 43 or 71 weeks post-infection. This was associated with an extreme loss of CD4+ T cells, but without clinical signs of AIDS. In the top figure, they looked at coreceptor usage at three time points – acute stage, a second CD4 decline, and a chronic stage. At 195 weeks post infection, CD4-healthy mangabeys predominantly use the CCR5 coreceptor. This is typical at all time points. But SM1 and SM2, which are the CD4+ T cell depleted SMs, start using mostly CCR5 in the acute stage, but then when the second CD4 decline begins, they expand their coreceptor usage to include CXCR4, among some others. Sodora’s group concluded that there must be some sort of adaptation that the envelope protein acquired to use these other co-receptors.Also, I don’t show it here, but they found an increase in basic amino acid residues in the V3 region of the envelope gene after CD4 decline in only the CD4 depleted SMs, which has previously been shown to be associated with an expanded co-receptor usage.The figure at the bottom is showing a SIVnegativemangabey, where more than 90% of CD4+ T cells expressed CXCR4 on their surface, while less than 20% expressed both CCR5 and CXCR4. This shows how a broadened coreceptor usage to include CXCR4 could more drastically deplete T cells.
Marx and Chen came out with a paper in 1998 that described the function of simianchemokine receptors in SIV. They took human, chimpanzee, rhesus, and sooty mangabey CCR5 and CXCR4, amplified and cloned it, and then determined the sequences from cDNA. They found a high percent homology between humans and the non-human primates, but stated that the possibility exists that co-receptor amino acid differences between humans and monkeys play a role in resistance. They also said that thus far, no naturally occurring SIV strains tested use CXCR4. So looking at the sequences of CXCR4 in this paper, and specifically where the yellow arrow is pointing in the V3 extracellular loop region, you can see there is a switch from tyrosine (Y) that humans have in their sequence, to a phenylalanine (F) that SMs have. Tyrosine can be post-translationally modified in GPCRs to undergo sulfation, which plays a role in strengthening protein-protein interactions. Could something about this amino acid difference be the reason why CXCR4 is not normally used in the sooty mangabey?
CXCR4 use is very rare, overall, in SIV isolates. Tyrosine, as in humans, undergoes sulfation, which may play a role in strengthening protein-protein interactions.
Peripheral Blood Mononuclear Cell is any cell having a round nucleus such as a lymphocyte or a monocyte. With a CpT tube, you can separate PBMCs from Whole blood and thus obtain a sample that is just lymphocytes and monocytes, which then includes CD4+ T cells.
I had never made primers before, so I learned that there are several factors that go into the creation of primers, including an optimal length of 18-22 bp, specific melting and annealing temperatures, the G-C content, and avoiding cross homology. Initially, I tested 2 pairs of CXCR4 primers on the 8 CD4+ T cell normal SMs. Based on the results of the gel, it appeared that the third combination was optimal. These were the primers we had named F2 and R1. I extracted these bands to send for sequencing and when they returned, Vandy and I analyzed the results using the Sequencher program. Four of the samples returned fairly poor results, so when three more blood samples with CD4+ depleted animals came a couple of weeks later, I re-processed the CD4+ T cell normal animals again (seen in the gel on the right as #4-7), together with the CD4+ T cell depleted animals (seen as #1-3 on the right), and again sent them to be sequenced. Numbers 2 and 3 are the same animals that were SM1 and SM2 in Don Sodora’s study, which were the persistently low CD4+ T cell SMs I discussed a few slides back.
