Following human immunodeficiency virus (HIV-1) entry into the host cell, the viral capsid gradually disassembles in a process called uncoating. A proper rate of uncoating is important for reverse transcription of the HIV-1 genome. Host restriction factors such as TRIM5alpha; and TRIMCyp bind retroviral capsids and cause premature disassembly, leading to blocks in reverse transcription. Other host factors, such as cyclophilin A, stabilize the HIV-1 capsid and are required for efficient infection in some cell types. To identify additional cellular factors that alter retroviral core uncoating, we developed a novel in vitro assay of HIV-1 capsid stability. Using this assay, we have shown that a factor in the cytoplasm of cells from multiple vertebrate species slows the spontaneous disassembly of HIV-1 capsid-nucleocapsid (CA-NC) complexes in vitro. We identified the PDZ-Domain-containing protein 8 (PDZD8) as a critical component of the capsid-stabilizing activity in the cytoplasmic extracts. PDZD8 has been previously reported to bind the HIV-1 Gag polyprotein and to make a positive contribution to the efficiency of HIV-1 infection. PDZD8 knockdown accelerated the disassembly of HIV-1 capsids in infected cells, resulting in decreased reverse transcription. The PDZD8 coiled-coil domain is sufficient for HIV-1 capsid binding, but other parts of the protein, including the PDZ domain, are apparently required for stabilizing the capsid and supporting HIV-1 infection. In summary, PDZD8 interacts with and stabilizes the HIV-1 capsid and thus represents a potentially targetable host cofactor for HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) infection causes general loss of immune response in humans. Presently, an estimated 34 million (31.4-35.9 million) people worldwide are HIV-1 positive and many more are being newly infected. In the absence of a definitive cure, anti-HIV-1 drug therapy helps to manage the infection by suppressing virus replication. However, extensive drug resistance against most of existing drugs demands alternative anti-HIV-1 strategies. The proper knowledge about HIV-1 replication is essential to guide the development of new anti-HIV-1 strategies. The research presented in this thesis aims to understand the role of HIV-1 Integrase (IN) and cellular co-factors interactions in the early stage virus replication. In the cytoplasm, HIV-1 cDNA exists as a high molecular weight nucleoprotein complex called pre-integration complex (PIC). The cDNA enters the nucleus as a part of PIC by active nuclear import and integrates into the host genome. HIV-1 Integrase (IN) protein has been recognized as a primary viral factor for HIV-1 nuclear import, but the key contributing cellular factor(s) is unknown. We have examined the requirement of different Importinα (Impα) isoforms for HIV-1 replication and identified the requirement of Impα3 for HIV-1 replication in HeLa cells, C8166T cells, and human macrophages. Further investigations showed the specific requirement of Impα3 for HIV-1 nuclear import. By analyzing the Impα3 interaction with HIV-1 proteins, we detected the IN interaction with Impα3 and C-terminal domain (CTD) of IN was essential for Impα3 interaction. These data led to the conclusion that Impα3 is required for HIV-1 nuclear import and interacts with IN. The IN-CTD consists of conserved basic amino acid rich motifs (211KELQKQITK, 236KGPAKLLWK, and 262RRKAK) that closely resemble the consensus classical nuclear localization signal (NLS) for Impα interaction. By substitution mutation and interaction analysis, 211KELQKQITK and 262RRKAK motifs in IN were identified as required for Impα3 interaction, IN nuclear localization, and HIV-1 nuclear import. Together, these data were useful in explaining the molecular mechanism of IN and Impα3 interaction and its requirement for HIV-1 nuclear import. Retrograde transportation of macromolecules in the cytoplasm is one of the prerequisites for their nuclear import. Although an earlier study implicated the dynein complex in retrograde transport of HIV-1, cellular and viral factors that are involved in this process are unknown. In this study, we have elucidated the HIV-1 IN interaction with the dynein light chain 1 (DYNLL1) in 293T cells, in vitro, and in HIV-1 infected cells. DYNLL1 is one of the adapter proteins that mediate the cargo recruitment to dynein complex. However, our data suggested that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not for nuclear import. Surprisingly, DYNLL1 interaction of IN was dispensable for HIV-1 recruitment to dynein complex. These data led to the conclusion that the IN and DYNLL1 interaction is essential for proper HIV-1 uncoating and cDNA synthesis but not required for HIV-1 recruitment to the dynein complex or for retrograde transport. In summary, this study advances our knowledge on the role of IN and cellular factors interactions in different early steps of HIV-1 replication and offers potential contributions in the development of future anti-HIV-1 strategies.

In the infection process, native poliovirus (160S) first converts to a cell-entry intermediate (135S) particle, which causes the externalization of capsid proteins VP4 and the N-terminus of VP1 (residues 1-53). The externalization of these entities is followed by release of the RNA genome, leaving an empty (80S) particle. Three antibodies were utilized to track the location of VP1 residues in different states of poliovirus by cryogenic electron microscopy (cryo-EM). "P1" antibody binds to N-terminal residues 24-40 of VP1. Three-dimensional reconstruction of 135S-P1 showed that P1 binds to a prominent capsid peak known as the "propeller tip". In contrast, our initial 80S-P1 reconstruction showed P1 Fabs also binding to a second site, ~60 Å distant, at the icosahedral twofold axes. Analysis of 80S-P1 reconstructions showed that the overall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at the propeller tips; only at the twofold axes; or simultaneously at both positions. Our results indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry. Similar deviations from icosahedral symmetry may be biologically significant during other viral transitions. "C3" antibody binds to 93-103 residues (BC loop) of VP1. The C3 epitope shifts outwards in radius by 4.5% and twists through 15° in the 160S-to-135S transition, but appears unchanged in the 135S-to-80S transition. In addition, binding of C3 to either 160S or 135S particles causes residues of the BC loop to move an estimated 5 (±2) Å, indicating flexibility. The flexibility of BC loop may play a role in cell-entry interactions. At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and the N-terminus of VP1 externalize reversibly. An antibody, binding to the residues 39-55 of VP1, was utilized to track the location of the N-terminus of VP1 in 160S particle in the "breathing" state. The resulting reconstruction showed the capsid expands similarly to the irreversibly altered 135S particle, but the N-terminus of VP1 is located near the twofold axes, instead of the propeller tip as in 135S particles.

No description available.

Les vecteurs viraux dérivés du virus adéno-associé (AAV) apparaissent depuis deux décennies, comme des outils efficaces pour le transfert de gène in vivo. Cependant, malgré une faible immunogénicité et une absence de toxicité in vivo, leur optimisation requiert encore un effort important vers une meilleure compréhension de leur biologie et, en particulier, de leur interaction avec le système immunitaire. Au cours de ce travail de thèse, nous avons utilisé une méthode de sélection dirigée in vitro dans le but d’obtenir un variant de capside capable de transduire efficacement un type cellulaire non-permissif aux vecteurs AAV : les cellules dendritiques (DC). En effet, ces cellules jouent un rôle primordial dans l’établissement de la réponse immunitaire et, par conséquent, dans la persistance de l’expression du transgène in vivo. Cette technologie, très répandue dans la communauté AAV, a permis de sélectionner un variant de capside aux propriétés très intéressantes. La mutation sélectionnée, caractérisée in vitro comme induisant une instabilité de la capside, a permis d’identifier et de surmonter un point de blocage majeur dans le processus de transduction des DC par les vecteurs AAV consistant dans l’étape de décapsidation du génome du vecteur dans le noyau cellulaire. De manière intéressante, le variant obtenu exhibe un avantage en terme de transduction non seulement dans les DC mais aussi dans différents modèles de cellules primaires humaines (e.g. HUVEC) ou animales (OBC), peu ou pas permissive à l’AAV. De plus, des expériences de transfert de gène in vivo réalisées dans un modèle murin, indiquent que le variant sélectionné conduit à une meilleure expression du transgène, possiblement due à la mise en place d’un processus de tolérisation. Les propriétés remarquables de ce variant de capside, font de lui un candidat intéressant pour des applications médicales. / Vectors derived from the Adeno-associated virus (AAV) have emerged as an efficient system for in vivo gene transfer. However, despite their low immunogenicity and good tolerance in vivo, a better characterization of the host-AAV interaction is required to be able to fully exploit AAV’s potential fora gene therapy or gene vaccination. In this PhD project, we have used an in vitro directed evolution strategy to select an AAV capsid variant able to transduce human dendritic cell (DC), a non-permissive cell type which plays a critical role in the initiation of immune responses and, consequently, on the persistence of the expression of transgene in vivo. This procedure allowed us to identify an AAV variant characterized by a decreased stability of the capsid in vitro. The use of this mutant as a vector to transduce human DC resulted in an improved uncoating of the vector genome in the cell nucleus, thus identifying this step as major barrier toward DC transduction. Interestingly, the selected variant also displayed an increased transduction efficiency not only in DC but also in different primary human and animal cell types, poorly or non-permissive to AAV. Finally, when injected in mice, this AAV variant resulted in a higher expression of the transgene, associated to a low level of immune responses, suggesting the induction of tolerant state. The remarkable features suggest that our selected variant capsid is a promising candidate for medical applications.