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Postdoc talk

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by

Jaturong Sewatanon

on 28 October 2013

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Transcript of Postdoc talk

Of Mice and Men:
Gammaherpesvirus Evasion of Intrinsic Antiviral Defenses
Gammaherpesvirus

Evasion of

Intrinsic Antiviral Defenses
Epstein-Barr virus (EBV)
Establish latency in lymphocytes & macrophages
Kaposi's Sarcoma-associated herpesvirus (KSHV)
Problematic in immunocompromised hosts
Highly associated with
cancers
Promyelocytic leukemia (PML) nuclear bodies
Latency & Reactivation
Kaposi's Sarcoma
Burkitt's Lymphoma
Nasopharyngeal Carcinoma
-inducible
Targeted for by many viruses
Interferon
disruption
MHV68
PML
Murine gammaherpesvirus 68
Closely-related genome organization
Naturally infects laboratory mice
Establish latency & lymphoproliferative diseases
Previous Study
ORF75c
FGARAT homolog (23% identity)
Viral tegument protein
All gammaherpesviruses
130 KDa
WHY?
HOW?
What is the role of during infection
in vivo
?
How does induce PML degradation?
What is the role of
PML-degradation function 0f ORF75c
during infection?
PML
ORF75c
Aims of the study
Significance
Acknowledgements
plaque-forming efficiency in
Higher
PML0 cells
acute infection
(day 7-22)
latent infection
(day 42)
MHV68
PML-/-
MHV68 Life Cycle
Ex vivo reacivation assay
Limiting-dilution PCR assay
PML represses MHV68 reactivation
in peritoneal cells
ORF75c degrades PML through
proteasome-dependent
pathway
ORF75c is a viral ubiquitin E3 ligase?
ORF75c interacts with PML?
ORF75c increases PML poly-ubiquitination?
ORF75c contains E3 ligase activity?
ubiquitin E3 ligase ?
ORF75c

is a viral
Colocalization
Physical association
ORF75c has self-ubiquitination activity
ORF75c increases PML poly-ubiquitination
acute infection
(day 7-22)
latent infection
(day 42)
ORF75c-mutant MHV68
wild-type
ORF61 disrupts PML NBs
to track-like structures
ORF61 is sufficient to induce
track-like structure of PML NBs
ORF61 colocalizes with PML NBs
ORF61 knockdown blocks
track-like structures formation
Summary
Mechanism?
Mechanism?
MHV68 double-mutant
New insights
into the role of
PML
and
ORF75c & ORF61
in regulating
latency & reactivation
Advisory Committee
Paul D. Ling, Ph.D.
Mary K. Estes, Ph.D.
Ronald T. Javier, Ph.D.
B.V.V. Prasad, Ph.D.
Andrew P. Rice, Ph.D.

Ling Lab
RongSheng Peng
Jie Tan

Financial Supports
Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand

Baylor College of Medicine Comprehensive Cancer Training Program (RP101499) by Cancer Prevention and Research Institute of Texas (CPRIT)

Speck Lab
Kathleen S. Gray, Ph.D.
Dan L. Duncan Cancer Center
(Division of Biostatistics)

Hao Liu, Ph.D.
Prasad Lab
Sreejesh Shanker, Ph.D.
Antiviral defenses
(antibody- and cell-mediated)
(interferon-regulated and viral pattern-recognition)
Adaptive immunity
Innate immunity
Intrinsic immunity
pre-existing
cellular factors
repress viral replication during
initial stages of infection

(Formylglycinamide ribonucleotide amidotransferase)
Roles of PML and PML NBs on herpesvirus infection in vivo
Precise mechanism by which ORF75c induce PML degradation
is an ubiquitin E3 ligase itself?
recruits cellular E3 ligase?
ORF75c

PML ubiquitination in vitro?
?
?
Increases PML poly-ubiquitination in vivo
Interacts weakly with PML
Contains self-ubiquitination activity
New insights into the function of viral FGARAT
Represses reactivation of MHV68
Limits establishment of latency in PECs
Represses persistent infection in vivo
New insights into the role of PML in controlling herpesvirus latency & reactivation in vivo
ORF61
Track-like structures
of PML NBs
during infection of
ORF75c-mu MHV68
Homolog of
ribonucleotide reductase (RNR) large subunit
80 KDa
Early gene
All herpesvirus subfamilies
Synthesize dNTPs (important for DNA synthesis)
Replication in non-dividing cells & reactivation from latency
(some lack RNR activity)
Important for establishment of MHV68 infection in respiratory tract
ORF75c-mu MHV68 has similar phenotype as wild-type MHV68 in vivo
ORF61 disrupts PML NBs to track-like structures
(similar to adenovirus type 5 E4 ORF3)
New insights into a novel function of viral RNR large subunit and new PML NB-modifying protein
PML-NB formation
(co-expression in 293T cells)
(infection in 3T12 cells)
(co-expression in 293T cells)
ORF75c interacts weakly with mouse PML
(in vitro ubiquitination assay)
No increase in lytic replication in PML-/- mice
Higher reactivation frequency in
peritoneal cells of PML-/- mice
Higher latently infected cells & reactivation efficiency in PML-/- mice
ORF75c-mu MHV68 has similar phenotype as wild-type MHV68 in vivo
ORF61 disrupts PML NBs to track-like structures
(similar to adenovirus type 5 E4 ORF3)

Proximity to
PML nuclear bodies
regulates
HIV-1 latency
in
CD4+ T Cells
Full transcript