silvestri post rotational talk
TRANSCRIPT
The Other Coreceptor: CXCR4 Cloning and Sequencing
in CD4+ T cell Normal and Depleted Sooty Mangabeys
Irene BukhPost-rotational TalkGuido Silvestri Lab September 21, 2009
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, Langerhanscells, macrophages)
• Also found on vascular endothelial cells and neuronal/nerve cells
• HIV co-receptor
(Moyle et al. 2006) (Alkhatib, 2009)
HIV binding
(De Clercq, 2003)
Impetus for a project
Severe CD4+ T cell loss is associated with expanded co-receptor usage
(Sodora et al., 2007)
CXCR4 sequence
(Marx and Chen, 1998)
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?
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
SM Blood Samples
CD4+ T cell normalFYmFavFFvFBqFTrFQgFFqFKn
CD4+ T cell depletedFFrFBrFAl
CXCR4 Optimized PrimersCXCR4-F2 5'-GCCTGAGTGCTCCAGTAGCC-3’
CXCR4-R1 5'-TTTTACATCTGTGTTAGCTGGAGTG-3’
CXCR4-seqF1 5'-CCAAGGAAGCTGTTGGCTG-3’
CXCR4-seqR1 5'-TGGAACACAACCACCCACAAGTC-3'
CD4+ depleted CD4+ normal
SM CXCR4 sequence resultsHuman
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
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
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….
ReferencesAlkhatib 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 InterscienceConference 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 ImmunoHungarica2003. 51:1-29.
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 aspay dog, let me know!