INDUCING TELOMERE LOSS IN B CELLS INFECTED WITH KAPOSI'S SARCOMA ASSOCIATED...
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INDUCING TELOMERE LOSS IN B CELLS INFECTED WITH KAPOSI'S SARCOMA ASSOCIATED HERPESVIRUS. Elena M. Cortizas a , Santas A. Rosario b , Enrique A. Mesri b and Ramiro E. Verdun a . (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 KSHV KSHV KSHV -infected B cell (see Figure 3) - Telomere stability Determine: - Proliferation capacity UNG inhibition B cell KSHV KSHV KSHV -infected B cell - AID expression levels - Class switching capacity (see Figure 2) Determine: Figure 2 Figure 3 B 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- KSHV Control % IgG1 cells B cells B cells Class switching to IgG1 20- 10- 0- KSHV Control Arbitrary Units 100- 10- 1- P<0.001 P<0.001 A A B B Cell proliferation GFP days post-infection Ugi GFP KSHV Ugi KSHV B Cells 0.6- 0.4- 0.2- 1.0- 0.8- I 1 I 3 I 5 I 7 I 9 0- 10 (6) cells / ml Fig 3. UNG inhibition decreases telomere stability (A) and pro- liferation capacity of B cells infected with KSHV (B). Telomeres lost per metaphase 5- 4- 3- 2- 1- 0- C-rich G-rich telomere strand CO-FISH (telomeres) CO-FISH (telomeres) CO-FISH KSHV -infected B cells KSHV -infected B cells Normal Telomere loss in leading strand G-rich lagging strand C-rich leading strand telomeric probes GFP Ugi Ugi + shAID GFP Ugi Ugi shAID 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 Normal B cell UNG-deficient B cell MMR Stop cell proliferation AID AID Telomere Sub-Tel Repair of telomere UNG-BER Continue cell proliferation deaminated telomere Telomere loss KSHV UNG inhibitor 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.
Transcript of INDUCING TELOMERE LOSS IN B CELLS INFECTED WITH KAPOSI'S SARCOMA ASSOCIATED...
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.
