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!