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MENDES, Marta Maria Lavouras (2011) Analysis of changes in the
host cell proteome during hepatits D vírus infection, Dissertação
de Doutoramento no ramo de Ciências Biomédicas, especialidade
de Biologia Celular e Molecular. IHMT. 2011.
Hepatitis delta virus is a very simple virus with a 1.7 kb, circular, ssRNA
genome which codes for only one protein – the delta antigen (HDAg/ Ag).
Replication occurs in the nucleus of host cells by a rolling circle mechanism and
using host RNA polymerase II giving rise to genomic and antigenomic RNA and
a 5‟-capped, polyadenylated RNA that acts as mRNA. During replication an
editing mechanism occurs in the antigenomic RNA extending the reading frame
and a second form of the delta antigen is produced. Despite its simplicity, little is
known about which host factors and/or mechanism are involved in, or used by
HDV during replication and pathogenesis, mainly due to the lack of an
appropriate cell culture system to study it. In a previous work, two proteins,
HSP105 and hnRNP H were found to be differentially expressed during HDV
replication. In order to determine if and how these proteins may be involved in
HDV replication, several assays were performed. Furthermore, using a new
model for HDV replication based on a tetracycline inducible cell line, alterations
in the proteome of those cells resultant from HDV replication were also
determined. This model devises a tetracycline inducible cell line, 293- Ag, in
which Ag expression is under the control of tetracycline and cells 293-HDV, in
which 293- Ag cell line is transiently transfected with HDV RNAs allowing HDV
replication. Using an MS-based quantitative proteomics approach consisting in
16O/18O enzymatic labeling together with systems biology, an attempt was
made to clarify HDV replication and pathogenesis mechanisms by determining
changes in the host cell proteome during hepatitis D infection.
Results showed that HSP105 and hnRNP H affect HDV replication. HSP105
knockdown induced a decrease in LHDAg expression levels and an increase of
HDV mRNA levels. Furthermore, HSP105 was also shown to interact with both
delta antigens. HSP105 seems to be involved in the ribonucleoprotein (RNP)
transport or anchoring in the ER, or even in viral particle assembly. As for
hnRNP H, a knockdown of the proteins led to an increase of HDV mRNA levels
and a decrease in the expression of the delta antigens. It was also shown that
hnRNP H binds to SHDAg. hnRNP H may be recruited by HDV in order to
induce alternative splicing of pre-mRNAs that will originate key proteins
essential to viral replication.
In order to determine alterations in the host cell proteome during HDV
replication, five proteome comparisons, between controls and experiments,
were performed. Approximately, 1000 proteins per experiment were identified
and from those about 600 proteins were quantified. Finally, 88 proteins were
found differentially expressed during HDV replication. From those, most
proteins were found to be involved in protein metabolism and energy pathways.
It was also possible, using GOTM and IPA, to perform a deeper analysis of the
results0, by determining interactions between differentially expressed proteins
and determine which canonical pathways were most affected by HDV
replication. Results showed that several metabolic pathways and key proteins
were shown to be associated with HDV replication. Anaerobic glycolysis, HIF1
signaling and G2/M checkpoint regulation were three of the most affected