Cell-host interaction : assessing the role of helper factors in HIV-1 replication

• Author(s): Rato, Sylvie Ferreira, 1982-
• Date: 2010
• Persistent ID: http://hdl.handle.net/10451/2242
• Origin: Repositório da Universidade de Lisboa
• Subject(s): Microbiologia; Fosfatases; Teses de doutoramento - 2010
• Document Type: Doctoral thesis
• Language: English
• Advisor(s): Gonçalves, João, 1967-
• Contributor(s): Repositório da Universidade de Lisboa

Description

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2010.

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.

Sylvie Ferreira Rato was financially supported by a PhD fellowship (SFRH/BD/24129/2005) from Fundação para a Ciência e Tecnologia (FCT), Lisboa, Portugal. This work was supported by the grant PTDC/SAU‐MII/65346/2006 from FCT, Lisboa, Portugal.