Characterization of the Novel CCR5 Lysine

to Arginine Mutation Using U937 Cells

A. Clarke, A. Lilly, K. Jones, S.

Mares, B. Long, A Calvitti, E.

McCallister

The Family

Figure 1. A pedigree showing HIV infection in an African American family.

IB gave birth to all of her

children naturally, with no

anti-retrovirals.

IIB, the focus of our

research, was exposed

but not infected with

HIV.

HIV Isolate Similarity

Figure 2: Using MacVector, a comparison of genetic distance within the family was modeled.

HIV isolates from infected family

members were taken and compared to

confirm the mother, IB, as the source of

infection.

Child IIB

Figure 3. Amino acid map of the CCR5 receptor showing location of TG5 mutation.

IIB’s CCR5 gene was

sequenced and the lysine to

arginine mutation was

found.

CCR5 is a gene that codes for a cell

receptor whose secondary function is

to act as an HIV co-receptor

alongside primary receptor CD4.

Location ofLys to Arg

(TG5)mutation.

Research Questions/Hypothesis

What is the function of the TG5 mutated

gene?

Does the mutation confer HIV resistance?

Hypothesis: The CCR5 mutation TG5 does not

protect a cell from HIV infection.

Overview

To characterize the TG5 mutation, we have decided

upon transfection and transduction techniques to

attempt to express the mutation in U937 promonocytic

human stem cell cancer cells.

U937 cells (pictured in slide backgrounds) were chosen because

the cells possess the CCR5 gene but do not express the

extracellular receptor.

Transfection

Figure 4. Map of constructed transfection plasmid vector pcDNATG5.

The CCR5 allele containing the Lys to Arg

missense mutation observed in subject IIB

was cloned into pcDNA3.1 (Invitrogen).

The map was confirmed by restriction

endonuclease digestion and analysis.

U937 cells were grown in RPMI with 10%

FBS and 1% Pen-Strep (Invitrogen).

Using Lipofectamine 2000

(Invitrogen), U937 cells were transfected

with pcDNATG5.

TransfectionA drug study was done to find the

optimal amount of G418 to use in

selection.

Cells were treated with increasing

amounts of G418, from 0μg/μL to

1000μg/μL for a week and counted

three times.

Before counting, cells were treated with

Trypan Blue Stain (Gibco) and dead

cells took in dye.

After cells were transfected they were

treated with 200μg/μL for five weeks.

Ultimately it the transfection was

determined to be unsuccessful.

Figure 5. Graph of G418 drug study U937 cell counts.

TransductionAfter transfection proved to be unsuccessful,

a transduction using pLNCX2 (Clontech)

retroviral vector was started.

First, the vector had to be constructed by

cutting TG5 out of pcDNA with the use of

restriction enzymes XbaI and BamHI.

Oligonucleotide ‘linkers’ were ligated onto

TG5 which converted the BamHI end into a

HindIII site, and the XbaI end into a NotI

site.

This conversion made TG5 compatible for

ligation into the Multiple Cloning Site

(MCS) of pLNCX2.

Figures 6&7. Map of

original pLNCX2

retroviral vector and

MCS sequence.

TransductionNow strands of pLNCX2 containing the

TG5 mutation are being transformed using

competent E.Coli cells to produce sufficient

amounts of plasmid for transduction.

Next, plasmid will be transfected into the

associated packaging cell line PT67 which

will assemble the virus particles.

These particles will be introduced into U937

cultures, and cells will be allowed to grow.

Then successfully transduced cells will be

selected for using the same G418 antibiotics

used in the transfection.

Figure 8. Map of pLNCX2 adapted to show

CCR5 TG5 gene.

Future Plans

If the TG5 mutation can be characterized successfully, HIV

infectability may be tested using viral envelope or further testing

if necessary.

We also plan to test any effects of the TG5 mutated gene on both

the CCR5 and CXCR4 (another HIV co-receptor secondary to CD4) receptors.

Provided the mutation is characterized successfully, we will find

another facility to send our cells to in order to test infectability

with actual HIV.

If it is found that the lysine to arginine (TG5) mutation confers

resistance, there are many possibilities for gene therapies, other

treatments, or even a cure.

Acknowledgements

Harry Kestler PhD, Rosa Hainaj PhD, John Crooks

PhD, Margaret Gorensek MD, X. Z. Ma MD, Jalpa

Nagisetty, Jessica Jenkins, Anthony George, Katelyn

Witte, Diana Shuman, Kelly Harrison, and Support of

the Lorain County Community College Foundation

Picture Credits

CCR5 map slide - Figure 3:

www.microbytes.com/blog/2010/07/

pLNCX2 map and MCS - Figure 6&7:

http://www.clontech.com/US/Products/Viral_Transduction/Retroviral_Vector_Systems/C

onstitutive_Promoter?sitex=10020:22372:US