KSHV-encoded MicroRNAs Promote Angiogenesis Through Inhibition of TGF-2 During Latent Viral Infection

Sreetha Sidharthan1, Terri DiMaio2, Kimberley Gutierrez2, Michael Lagunoff2

1Department

of Biochemistry, 2Department of Microbiology, University of Washington, Seattle 98195

KSHV down-regulates TGF-2 to promote capillary stability

Figure 1
Mock

Abstract

Angiogenesis is the development of new blood vessels from pre-exis=ng vasculature and a hallmark of Kaposis sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals, especially AIDS pa=ents. The e=ological agent of KS, Kaposis sarcoma- associated herpesvirus (KSHV), induces angiogenesis of endothelial cells during latent infec=on. Interes=ngly, KSHV infec=on leads to the down-regula=on of transforming growth factor-beta 2 (TGF-2), an an=-angiogenic cytokine. By down-regula=ng TGF-2, KSHV is able to promote and stabilize capillary morphogenesis, a measure of angiogenesis. We have iden=ed two virally encoded microRNAs, miR-K3 and miR-K8, that are involved in the downregula=on of TGF-2. By iden=fying specic targets of KSHVs miRNAs such as TGF-2, we can improve our understanding of how KSHV is able to maintain latent infec=on, disrupt the normal regula=on of cell prolifera=on, and induce angiogenic phenotypes. These studies will further our understanding of KSHV pathogenesis and the development of the KS tumor.

KSHV targets TGF-2 3UTR

Luciferase
Mock Control Mock TGF-b2 KSHV Control KSHV TGF-b2

Figure 3 A
Mock

B
10

TGF-2 3 UTR Target

Number of Connec=ons per Field

KSHV-infected

INTERACTION

NO INTERACTION

KSHV-infected (-) control

miRNA RISC complex

4 hours

24 hours

KSHV-infected TGF-B2

Figure 1. KSHV-infec2on stabilizes capillary forma2on. At 24 hours post-pla=ng and 48 hours post-infec=on, KSHV-infected cells maintained capillary stability as compared to mock cells. Capillary stability is a measure of angiogenesis.

Low level luciferase transla=on

24hrs

Luciferase expression

-2

Figure 7 Figure 7
TGF-2 3'UTR Luciferase
TGFB2_Mock

Figure 2
Fold decrease over Mock
8 6 4 2 0

6 hours

24 hours

TGF-2 Transcript Levels

Background

Kaposis Sarcoma: The KS tumor exhibits extensive angiogenesis, the development of new blood vessels. The e=ological agent of KS is Kaposis Sarcoma-associated herpesvirus (KSHV).

Figure 2. KSHV down-regulates TGF-2 transcript levels. Transcript levels were measured 48 hpi using qRT-PCR.

Mock

KSHV

Figure 3. KSHV-induced capillary stability is inhibited by exogenous TGF-2 . (A) 48 hpi, cells are plated on Matrigel and monitored for 24 hours. The addi=on of exogenous TGF-2 to KSHV-infected cells destabilized capillary maintenance at 24 hours while having no eect on forma=on at 6 hours. (B) Quan=ta=ve analysis of tube forma=on indicates that at twenty-four hours, a signicant decrease in the number of capillaries was evident in KSHV-infected cells with exogenous TGF-2.

TGFB2_KSHV

20

40

60

80

100

120

% Luciferase Expression

KS Tumor

KSHV is a double-stranded DNA virus that encodes more than 80 open reading frames (ORFs) and, as with all herpesviruses, exhibits both latent and ly=c gene expression. In the KS tumor and in infected cells in culture, KSHV primarily exists in the latent state where only six viral genes and 17 recently discovered microRNAs (miRNAs) are expressed. miRNA: Small RNAs that downregulate gene expression by binding to the 3 untranslated region (3 UTR) of messenger RNA (mRNA) and either target it for degrada=on or inhibit transla=on.
3UTR Protein-coding region of target mRNA

MicroRNAs downregulate TGF- 2 levels

miRNA TGF-2 transcripts

Antagomirs partially restore TGF- 2 levels

Antagomirs: Chemically engineered oligonucleo=des that inhibit miRNA ac=vity through an=sense binding. Antagomir miRNA TGF-2 transcripts

Figure 7. KSHV down-regulates TGF-2 by directly targe2ng the 3UTR. HEK293 cells expressing the TGF-2 3UTR were infected with KSHV and harvested for luciferase 48 hpi. Further studies tes=ng the specicity of the miRNAs and antagomirs are ongoing.

Conclusions

I. KSHV promotes angiogenesis by stabilizing capillary morphogenesis. II. KSHV-induced capillary stability requires TGF-2 downregula=on during latent infec=on. III. KSHV-encoded miRNAs miR-K3 and miR-K8 inhibit TGF- 2 expression. IV. KSHV down-regulates TGF-2 by directly targe=ng the 3UTR.

Figure 4
5

TGF-2 Transcript Levels

Fold Decrease

4 3 2 1 0 M K pSIN miR-K3 miR-K8 miR-K12

Figure 6
TGF-2 Transcript Levels
16
14
12
10
8
6
4
2
0
Mock KSHV KSHV ant-K3/8

miRNA RISC complex

Figure 4. miR-K3 and miR-K8 downregulate TGF-2 transcript levels. Len=viral vectors expressing the individual KSHV miRNAs were used to infect endothelial cells. TGF-2 transcript levels were then measured 48 hpi by qRT-PCR and normalized to gapdh.

Fold Decrease

These studies demonstrate the signicant role KSHV miRNAs play in altering host cell gene expression to promote KSHV pathogenesis.

Acknowledgements

I would like to thank Dr. Michael Laguno, Ph.D. for giving me the amazing opportunity to experience science and research by allowing me to work in his lab this year. Thank you to Kim Gu=errez and the other members of the Laguno lab for guiding me with this project, teaching me numerous laboratory techniques, and taking the =me to answer all my ques=ons. This research was supported in part by a grant to the University of Washington from the Howard Hughes Medical Ins=tute through the Undergraduate Science Educa=on Program. Thanks also to the Mary Gates Research Endowment for funding my research.

Hypothesis

KSHV-encoded miRNAs regulate host cell gene expression to induce angiogenesis.

Figure 5 miR-K3: 5 TCACATTCTGAGGACGGCAGCGA 3

miR-K8: 5 TAGGCGCGACTGAGAGAGCACG 3

Figure 5. Seed sequence determines target specicity. miR-K3 and miR-K8 seed matches in TGF-2 3UTR were iden=ed.

Figure 6. Antagomirs to miR-K3 and miR-K8 par2ally restore TGF-2 levels. Cells were transfected with antagomirs specic to miR-K3 and miR-K8. 24 hpt, the cell were infected with KSHV. Transcript levels were measured by quan=ta=ve RT-PCR 48 hpi. (n=1) Further experiments are underway.