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Cell-host interaction : assessing the role of helper factors in HIV-1 replication
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Much progress has been made in the past twenty‐seven years to understand the complexity
of human immunodeficiency virus type 1 (HIV‐1) infection and to develop an efficient strategy
that can eliminate the virus from its host. Despite all efforts this strategy has not been
achieved mainly due to the emergence of resistant viruses and to the persistence of latently
infected viral reservoirs. Hence, it is crucial to identify novel drug targets and new therapeutic
strategies to combat the acquired immunodeficiency disease syndrome (AIDS). It is
conceivable that the use of cellular proteins as antiviral targets instead of viral proteins could
be a good alternative strategy, once cellular proteins are less variable than those from viral
origin, avoiding effectiveness fluctuations of drug efficacy. Therefore it is important to
understand HIV‐1 and host interaction, by studying the exploitation of the cellular machinery
by HIV‐1.
The work described in this thesis was focused on the interaction between HIV‐1 and its host
cell and on the discovery of new co‐factors for HIV‐1 infection. We have developed an
iterative shRNA screen in Jurkat T CD4+ cells to identify co‐factors for HIV‐1 infection, focusing
on kinases and phosphatases, a very druggable class of proteins. With this innovative screen
we were able to identify 14 new cellular proteins essential for HIV‐1 replication in
T lymphocytes but that do not interfere with cell survival. We have identified two
phosphatases, PTPN9 and PTPRE; five kinases, PRKD1, MAP3K2, MAPK9, SGK and STK24; one
hypothetical kinase‐binding‐protein, CIB2; two phosphatase‐binding‐proteins, PPFIA2 and PPFIBP1; and four other proteins with diverse functions, RAD23B, EZH2, WT1 and ELA1. The
role of these proteins in HIV‐1 replication was validated through replication assays with T cell
lines‐expressing‐shRNA for each gene in study and the role of the identified co‐factors in
different steps of the HIV‐1 life cycle was then evaluated. It was verified that none of the
studied proteins have a relevant role in HIV‐1 proviral integration. Instead, all proteins
seemed to play an important role before viral integration, in an early step of HIV‐1 life cycle.
Moreover, our results indicate that PRKD1, MAP3K2, MAPK9, RAD23B, EZH2, PPFIA2, PPFIBP1,
WT1 and STK24 have an additional effect on HIV‐1 LTR transcription.
The identification of CIB2, a hypothetical DNA‐PKcs binding protein, in the initial screen led us
to study the cellular protein DNA‐PKcs and its importance in HIV‐1 replication. We assessed its
role in different steps of HIV‐1 life cycle and verified that DNA‐PKcs is essential for HIV‐1
replication. Our results indicated that this co‐factor does not have a role in the early steps of
HIV‐1 life cycle until viral cDNA integration but it is crucial to HIV‐1‐LTR driven transcription,
having dramatic effects in the expression of Tat viral protein levels.
This study brings new insights for the complex interplay of HIV‐1/host cell, showing additional
knowledge on cellular proteins and pathways that are essential for HIV‐1 replication but
non‐important for cell viability and opens new possibilities for antiviral strategies. HIV‐1; shRNA; kinases; phosphatases; DNA‐PKcs.
Descrição
Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2010.
Palavras-chave
Microbiologia Fosfatases Teses de doutoramento - 2010