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

•  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

Activation of lytic program

Establish Latency

Maintain Latency

Reactivation

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.

Reactivation

α β

γ

HSV-1

VZV

CMV HHV-6

HHV-7

EBV

KSHV

HSV-2

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

Treatment: Live attenuated vaccine

 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

Epstein Barr Virus Kaposi’s sarcoma-associated herpesvirus

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

Burkitt lymphoma Nasopharyngeal carcinoma

NO TREATMENT

J. Bras. Pneumol. vol.31 no.6 São Paulo Nov./Dec. 2005 

h"p://www.dermis.net/dermisroot/en/17372/image.htm 

TPA

HDAC inhibitors

IE genes DE genes Late genes vDNA-R

TFs IE

Rta

vector!

ORF50/Rta! TPA!

untreated!

TranscripIonal AcIvaIon Domain 

DNA binding 

NLS=Nuclear localiza/on signal 

TetramerizaIon 

Rta

  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.

1

(45 kb.)

(90 kb.)

(140 kb.)

3x 2x 3x 2x 2x

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 

Rta Rta Rta

RBP-Jk  Rta sImulates DNA binding of RBP‐Jk.

Rta

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 

HDAC

X X X X Ac Ac Ac Ac Ac

RBP- Jk VP16 AD

X RBP-Jk

HDAC

RBP-Jk

RBP- Jk VP16 AD

1 530 RtaΔSTAD

Rta WT

TATA ‐30 

‐957  Luciferase Jk ‐136  ‐106   ‐54 

Mta Promoter:         ‐957           ‐136 

[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/TA/TT

Guito,J and Lukac DM, 2012. 

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 

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 CCTG

T

GGGGAGG

G

TATGATG

T

AGACATA

A

ATACAAA

A

CCCCCCC

C

AAAAAAA

A

CGATCCT

N

TTTTTTT

T

TGATGTA

ATATAGT

TTTTCCT

T A/TA/T 

 ‐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 

RBP‐Jk 

Rta

RBP-Jk

Rta Rta

Rta Rta Rta

Rta Rta Rta

3’AGTGCCATTGTGAATACTCAGTCACAAAACGGTCGTTCACATTGTTATTACAAGGGTGCCGGGTAAAAAGCAAAC 5’ 5’TCACGGTAACACTTATGAGTCAGTGTTTTGCCAGCAAGTGTAACAATAATGTTCCCACGGCCCATTTTTCGTTTG3’

‐62 ‐136 

Mta 

•  Understanding of molecular mechanism for disease development.

•  Manipulation of host signaling pathways. “Molecular piracy”

•  Understanding of cancer development and progression.

•  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.