Molecular Determinants of Latency-Associated Nuclear Antigen Function in Gammaherpesvirus Pathogenesis Open Access
Paden, Clinton (2012)
Published
Abstract
Gammaherpesviruses establish a lifelong latent infection in the host and are associated with a myriad of lymphoprolilferative disorders. Kaposi's sarcoma-associated herpesvirus (KSHV) is tightly associated with the AIDS-defining illness Kaposi's Sarcoma, as well as other diseases. The Latency-Associated Nuclear Antigen (LANA) is the only viral gene expressed in every KSHV-associated malignancy, so it is of great interest in the gammaherpesvirus field as a candidate viral oncogene. However, due to the tight species-specificity of the herpesviruses, little is known about its role in infection, before and during transition to disease.
Murine gammaherpesvirus 68 (MHV68) is a now well-described rodent pathogen we can use to do controlled pathogenesis experiments. In this dissertation, I describe my work on the MHV68 LANA (mLANA) during natural infection.
We found that mLANA is expressed throughout lytic infection and that it regulates the cascade of lytic gene expression required for efficient virus replication. In the absence of mLANA, there is a 90-99% reduction in output virus.
Previously, our lab determined that mLANA was necessary to establish latency after intranasal infection, supporting hypotheses based on observations from KSHV. To extend this work, we infected mice intraperitoneally with an mLANA-null virus to expose the natural latency reservoir to the virus. We found the mLANA-null virus actually establishes a chronic infection, but it is incapable of reactivating from those cells. Further, we demonstrated that the mLANA-null genome is not episomal, but the virus apparently integrates into the host chromosome. Despite that unusual arrangement, the mLANA-null virus expressed latency genes similar to wild type, causing us to rethink traditional descriptions and molecular definitions of latency
Of the many described functions of LANA, we chose to
examine its role as a transcriptional modulator. We generated a
library of random point mutants and designed an assay to screen
them for loss of function. Many of these mutations occurred in a
conserved region of LANA genes that is predicted to be a
DNA-binding domain. Notably, each of these mutations, when cloned
into the virus, results in mLANA-null phenotypes, leading us to
hypothesize that this LANA function is absolutely
essential.
Table of Contents
Table of Contents
Abstract.......................................................................................................
iii
Acknowledgements.......................................................................................
v
Table of
Contents..........................................................................................vi
List of Figures and
Tables.............................................................................viii
Chapter 1. Introduction
..................................................................................1
A. Herpesviruses
i. Background
a. Basic information
................................................................................2
b. Taxonomy
.........................................................................................3
c. Replication and life cycle
.....................................................................4
ii. Human Gammaherpesviruses: Pathogenesis and Disease
Associations
a. Epstein-Barr Virus
(EBV/HHV-4).............................................................5
b. Kaposi's Sarcoma-Associated Herpesvirus (KSHV/HHV-8)
...........................7
iii. Non-human Gammaherpesviruses
a. Herpesvirus saimiri
(HVS)......................................................................9
b. Murine Gammaherpesvirus 68 (MHV68/MuHV-4)
......................................10
B. Latency-Associated Nuclear Antigen (LANA)
i. Identification and Background
..................................................................14
ii. LANA in disease and Pathogenesis
a. Introduction
.....................................................................................15
b. Protein-Protein interactions
.................................................................17
c. Transcriptional regulation
....................................................................20
d. Latent replication and episome maintenance
...........................................22
C. MHV68 LANA
i. Introduction
.........................................................................................24
ii. Transcription and cell-type expression of
mLANA..........................................25
iii. Pathogenesis studies of MHV68 mLANA-null mutants
....................................27
D. Figures 1.1-1.4
.......................................................................................29
Chapter 2. Roles for mLANA and p53 in MHV68 Replication
...............................33
A. Introduction
...........................................................................................34
B. Materials and Methods
..............................................................................38
C. Results
..................................................................................................46
D. Discussion
..............................................................................................59
E. Figures 2.1-2.9
........................................................................................68
Chapter 3. MHV68 LANA is essential for virus reactivation
from splenocytes, but
not long-term carriage of viral genome
...........................................................81
A. Introduction
............................................................................................82
B. Materials and methods
...............................................................................85
C. Results
...................................................................................................90
D. Discussion
...............................................................................................99
E. Figures 3.1-3.8
.......................................................................................106
Chapter 4. Unbiased mutagenesis of MHV68 LANA reveals a
DNA-binding
domain required for LANA function in vitro and
in vivo
....................................116
A. Introduction
...........................................................................................117
B. Materials and methods
.............................................................................119
C. Results
.................................................................................................129
D. Discussion
.............................................................................................144
E. Figures 4.1-4.8
.......................................................................................152
F. Table 4.1
...............................................................................................167
Chapter 5. General Discussion and Future Possibilities
....................................168
References
..................................................................................................182
List of Figures and Tables
Figure 1.1.
Comparison of genome organization among
gammaherpesviruses................................29
Figure 1.2.
Schematic diagram of LANA proteins from different rhadinoviruses.
............................30
Figure 1.3.
Structure of transcripts encoding ORF73.
.............................................................31
Figure 1.4.
Inefficient establishment of latency after intranasal inoculation
of
MHV68.73.Stop.
..............................................................................................32
Figure 2.1.
mLANA promotes efficient viral replication in primary
murine fibroblasts.
.......................................................................................68
Figure 2.2.
mLANA is a nuclear/cytoplasmic protein expressed during
lytic replication.
..........................................................................................69
Figure 2.3.
mLANA-deficient virus exhibits increased kinetics of cell death
and
PARP
cleavage.............................................................................................70
Figure 2.4.
73.Stop virus exhibits increased replication, viral antigen
expression,
and p53 induction.
.......................................................................................71
Figure 2.5.
73.Stop-induced cell death, p53 phosphorylation, and
dysregulated
early gene expression occur independent of viral replication.
...............................73
Figure 2.6.
mLANA inhibits p53 induction and reduces etoposide-induced
cell death.
.................................................................................................75
Figure 2.7.
p53 regulates MHV68 replication
.....................................................................77
Figure 2.8.
Overexpression of p53 and etoposide treatment induviral gene
expression.
................................................................................................78
Figure 2.9.
Model of predicted mLANA functions to regulate host-cell
stress
and promote efficient viral replication
..............................................................80
Figure 3.1.
Intranasal infection of IFNαβR-/- mice with 73.Stop
allows for
greater lytic replication and mLANA-independent seeding of
latency
in the spleen, but not reactivation of virus
.....................................................106
Figure 3.2.
mLANA is required for efficient reactivation, but not
establishment
of latency, in PECs and splenocytes following intraperitoneal
inoculation of immunocompetent C57Bl/6 mice
.................................................108
Figure 3.3.
B cells account for the majority of mLANA null virus
infected
splenocytes.
.............................................................................................109
Figure 3.4.
Viral genomes are maintained long-term in mice in the
absence of mLANA.
....................................................................................110
Figure 3.5.
Latency-associated MHV68 transcripts can be detected in
mLANA-
null virus infected splenocytes.
....................................................................111
Figure 3.6.
mLANA-deficient virus is not impaired in the induction of a
strong
germinal center
response.............................................................................112
Figure 3.7.
A functional mLANA gene is required for efficient replication
of
MHV68 following transfection of viral DNA
in permissive fibroblasts.
.............................................................................113
Figure 3.8.
Digestion-Circularization PCR reveals mLANA-dependent
episome
formation during early latency in the
spleen.....................................................114
Figure 4.1
mLANA represses a promoter that initiates transcription within
the
MHV68 terminal repeat (TR).
........................................................................152
Figure 4.2.
Deletion analysis of TR reveals DNA sequence required for
mLANA-mediated repression..
.......................................................................155
Figure 4.3.
DNaseI footprinting of the terminal repeat with recombinant
mLANA shows sequence-specific DNA binding.
................................................157
Figure 4.4
Random mutagenesis of mLANA reveals residues important for
transcriptional repression.
...........................................................................159
Figure 4.5.
mLANA mutants lose transcription repression function, but not
the
capacity to dimerize
..................................................................................161
Figure 4.6.
MHV68-73TRN mutants display similar growth kinetics and
virus
output compared to 73.Stop virus
................................................................163
Figure 4.7.
Intraperitoneal infection of C57Bl/6 mice with MHV68-73TRN
mutants reveals mLANA DNA-binding/transcriptional repression
function is essential for reactivation from splenocytes
......................................165
Table 4.1
Repression activity of mLANA mutants in the MHV68 TR-driven
luciferase reporter assay
.............................................................................167
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