1. Herpesviral reactivation:
A key step for disease development.
Olga D. Gonzalez-Lopez, Ph.D candidate
University of Medicine and Dentistry of New Jersey
Dept. Microbiology and Molecular Genetics

2. • Introduce myself
• Overview of the Human Herpesvirus family
• Molecular mechanism of reactivation in Kaposi’s
sarcoma-associated herpesvirus (KSHV).
– Promoter specification by Rta and RBP-Jk
• GSBS Summer Research Program @ UMDNJ

4. Activation of
lytic program
Establish
Latency
Maintain
Latency
Reactivation

5. Latency:
 life-long persistent infection.
 poor target of host immune response.
 enhances cell survival and promotes proliferation.
 proper segregation of the viral episomes into
daughter cells during mitosis.

6. Reactivation

7. α β
γ
HSV-1
VZV
CMV
HHV-6
HHV-7
EBV
KSHV
HSV-2

8. Kimberlin, DW. 2005. Semin Pediatr Infect Dis. 16(4): 271–281.
 HSV-1: infections above the waist
 HSV-2: infections below the waist
 Diseases:
o Oral herpes or cold sore
o Genital herpes
Treatment: Acyclovir

9. Treatment: Live attenuated vaccine

10.  Congenital disease:
o Virus can spread via the placenta to
the fetus and congenital abnormalities
can occur
o Different route of transmission: breast
milk, during birth
Centers for Disease Control and Prevention (CDC): h"p://www.cdc.gov/features/dscytomegalovirus/
Treatment: Ganciclovir

11. Epstein Barr Virus
Kaposi’s sarcoma-
associated herpesvirus

12. 95% of people in the U.S. have been infected with EBV.
Up to 80% of students entering college in the US are seropositive.
Infectious mononucleosis

13. Burkitt lymphoma Nasopharyngeal carcinoma
NO TREATMENT

15. J. Bras. Pneumol. vol.31 no.6 São Paulo Nov./Dec. 2005
h"p://www.dermis.net/dermisroot/en/17372/image.htm

17. TPA
HDAC
inhibitors
IE genes DE genes Late genesvDNA-R
TFs
IE

19. Rta
vector!
ORF50/Rta! TPA!
untreated!

20. TranscripIonal AcIvaIon Domain
DNA binding
NLS=Nuclear localiza/on signal
TetramerizaIon

21. Rta

22.  Notch signaling pathway  is evolutionarily conserved pathway
regulate cell fate decision in various cell types during and after development
 Aberrant Notch signaling  tumor formation and progression.

24. 1
(45 kb.)
(90 kb.)
(140 kb.)
3x
2x
3x
2x
2x

25. Jk
x
 Only Rta, but not the RBP-Jk-dependent activators
NICD-1 nor EBV EBNA2, productively reactivate KSHV
from latency
 RBP‐Jk is not consItuIvely and broadly
bound to KSHV DNA.
CANT CANT CANT CANT
RtaRtaRta
RBP-Jk Rta sImulates DNA binding of RBP‐Jk.

26. Rta

27. Rta transacIvaIon
308.0x (19.0)
TATA
‐30
‐957
Luciferase
Jk
‐136 ‐106 ‐54
7.6x (0.6)
TATA
‐30
‐957
Luciferase
Jk
‐136
X
‐106 ‐54

28. HDAC
XX X X Ac Ac AcAc Ac
RBP-Jk
VP16
AD
X RBP-Jk
HDAC
RBP-Jk
RBP- Jk
VP16
AD

29. 1 530 RtaΔSTAD
Rta WT

30. TATA
‐30
‐957
Luciferase
Jk
‐136 ‐106 ‐54
Mta Promoter: ‐957 ‐136

31. [A/T]3 - N7 - [A/T]3 N7 - [A/T]3
[A/T]3 - N17 - [A/T]3
Liao, et al.,
2003
Ziegelbauer, et
al., 2006
CCCACTTC
Palmeri, et al.,
2011
ANTGTAACANTA/T
A/TT
Guito,J and Lukac DM, 2012.

32. G + A ladder
0 0
0 +
+ +
++ +
3’AGTGCCATTGTGAATACTCAGTCACAAAACGGTCGTTCACATTGTTATTACAAGGGTGCCGGGTAAAAAGCAAAC 5’
RBP‐Jk
‐136 ‐62
Mta
3’AGTGCCATTGTGAATACTCAGTCACAAAACGGTCGTTCACATTGTTATTACAAGGGTGCCGGGTAAAAAGCAAAC 5’
5’TCACGGTAACACTTATGAGTCAGTGTTTTGCCAGCAAGTGTAACAATAATGTTCCCACGGCCCATTTTTCGTTTG3’
‐62
‐136
RBP‐Jk
Palmeri, et al. 2011

33. 3’AGTGCCATTGTGAATACTCAGTCACAAAACGGTCGTTCACATTGTTATTACAAGGGTGCCGGGTAAAAAGCAAAC 5’
5’TCACGGTAACACTTATGAGTCAGTGTTTTGCCAGCAAGTGTAACAATAATGTTCCCACGGCCCATTTTTCGTTTG 3’
1R 2R 3R
1F 2F 3F 4F
‐62 ‐136
RBP‐Jk
Palmeri, et al. 2011
1F
2F
3F
4F
1R
2R
3R
CONS
A
T
A
C
G
A
G
A
C
A
G
A
C
T
T
N
G
T
T
C
C
T
G
T
G
G
G
G
A
G
G
G
T
A
T
G
A
T
G
T
A
G
A
C
A
T
A
A
A
T
A
C
A
A
A
A
C
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
C
G
A
T
C
C
T
N
T
T
T
T
T
T
T
T
T
G
A
T
G
T
A
A
T
A
T
A
G
T
T
T
T
T
C
C
T
T
A/T
A/T

34. ‐136
0 5 10 15 20 25 30
3R
2F
1R 2R
1F 4F 3F
Jk
4F
3R
3F
Jk
4F
Jk
Jk
4F
3R
3F
Jk
3R
2F
1R 2R
3F
Jk
3R
2F
1R
3F
Jk
E
F
G
H
I
J
Palmeri, et al. 2011

35. RBP‐Jk
Rta
RBP-Jk
RtaRta
RtaRtaRta
RtaRta Rta
3’AGTGCCATTGTGAATACTCAGTCACAAAACGGTCGTTCACATTGTTATTACAAGGGTGCCGGGTAAAAAGCAAAC 5’
5’TCACGGTAACACTTATGAGTCAGTGTTTTGCCAGCAAGTGTAACAATAATGTTCCCACGGCCCATTTTTCGTTTG3’
‐62 ‐136
Mta

36. • Understanding of molecular mechanism for disease
development.
• Manipulation of host signaling pathways. “Molecular
piracy”
• Understanding of cancer development and
progression.

37. • Many cis-elements contribute to Rta/
RBP-Jk transactivation.
• The number and positions of CANT and
[A/T]3 repeats and their relative
positions to the RBP-Jk binding site,
determines Rta transactivation.