INDUCING TELOMERE LOSS IN B CELLS INFECTED WITH KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS.

Elena M. Cortizasa, Santas A. Rosariob, Enrique A. Mesrib and Ramiro E. Verduna.(a) University of Miami, Miller School of Medicine, Dep. of Medicine. Sylvester Comprehensive Cancer Center (SCCC). Miami, Florida.(b) University of Miami, Miller School of Medicine, Dep. of Microbiology and Immunology . SCCC. Miami, Florida.

Methods

KSHVKSHV

KSHV-infected B cell

(see Figure 3)

- Telomere stabilityDetermine:

- Proliferation capacity

UNGinhibitionB cell

KSHVKSHV

KSHV-infected B cell

- AID expression levels- Class switching capacity

(see Figure 2)

Determine:

Figure 2 Figure 3B cell

Conclusions-KSHV induces expression of mutagenic protein AID and increases class switching efficiency in B cells.

-Inhibition of UNG induces telomere loss and decreases the proliferation capacity of KSHV-infected B cells.

References

Acknowledgments

- A role for host activation-induced cytidine deaminase in innate immune defense against KSHV.Bekerman E, Jeon D, Ardolino M, Coscoy L.PLoS Pathogology. (2013). 9(11):e1003748. doi: 10.1371/jour- nal.ppat.1003748.

- UNG protects B cells from AID-induced telomere loss.Cortizas EM, Zahn A, Safavi S, Reed JA, Vega F, Di Noia JM, Verdun RE.Journal of Experimental Medicine. (2016). Oct 3. doi: 10.1084/jem.20160635.

We are indebted to Dr. T. Honjo (Kyoto Univ.) for mouse B cells. This work was supported by grants from NIAID R56-AI106894-01A1 and Miami CFAR developmental grant 1P30AI073961 to RV.

Results

Fig 2. KSHV induces expression of AID (A) and increases class switching efficiency in B cells (B).

IgM

IgG1

AID expression (RT-PCR)

0-KSHVControl

% Ig

G1

cells

B cellsB cells

Class switching to IgG1

20-

10-

0-KSHVControl

Arb

itrar

y U

nits

100-

10-

1-

P<0.001 P<0.001

A

A

B

BCell proliferation

GFP

days post-infection

UgiGFP KSHVUgi KSHV

B Cells

0.6-0.4-0.2-

1.0-0.8-

I1

I3

I5

I7

I9

0-

10(6

) cel

ls /

ml

Fig 3. UNG inhibition decreases telomere stability (A) and pro-liferation capacity of B cells infected with KSHV (B).

Telo

mer

es lo

st p

er m

etap

hase

5-4-3-2-1-

0-

C-richG-rich

telomerestrand

CO-FISH (telomeres)

CO-FISH (telomeres)

CO-FISH

KSHV-infected B cells

KSHV-infected B cells

Normal

Telomere loss inleading strand

G-rich lagging strandC-rich leading strand

telomeric probes GFP Ugi Ugi + shAID

GFP Ugi UgishAID

V

V

(white arrows indicate missing telomere staining from single sister chromatids)

Failure to faithfully repair off-target DNA damage initi-ated by the antibody diversification enzyme activa-tion-induced deaminase (AID) can lead to B cell lym-phoma or cell death. AID is targeted to the immuno-globulin genes through specialized structural fea-tures that are shared with telomeres. We found that AID localizes to and damages telomeres in activated B cells. This damage is repaired by the uracil-DNA glycosylase UNG. In the absence of UNG activity, processing of AID-induced telomere lesions leads to telomere loss and cell proliferation defects. Interest-ingly, B cells infected with Kaposi’s Sarcoma Associ-ated Herpesvirus (KSHV) have a rapid induction of AID expression. Here we show that inhibition of UNG affects the telomere stability in KSHV-infected B cells. Considering the relevance of telomere mainte-nance on cell viability, the proposed experiments could represent a proof of principle template for a new therapeutic regimen against KSHV, the most common malignancy in AIDS patients.

Abstract

Background

Fig 1. Schematics showing the mechanism for AID-dependent telomere loss in UNG-de-ficient B cells.

IgM

IgA

NormalB cell

UNG-deficientB cell

MMR

Stop cellproliferation

AID

AID

TelomereSub-Tel

Repair of telomere

UNG-BER

Continue cellproliferation

deaminated telomere

Telomere loss

KSHV UNGinhibitor

Cell dead due to telomere loss

KSHV induces expression of mutagenic protein

AID in B cell

B cell

KSHV

Hypothesis

UNG inhibition will induce telomere loss and limit the proliferation capacity of KSHV-infect-ed B cells.