Thesis advisor: Welkin E. Johnson / For lentiviruses such as HIV-1, the viral capsid protein (CA) plays a crucial role in replication by facilitating active transport across nuclear pore complexes (NPCs). Nucleoporin Nup358/RanBP2 – a large, multidomain protein that comprises the main component of cytoplasmic NPC filaments – was previously identified as a potential cofactor for HIV-1 nuclear entry, and its C-terminal cyclophilin-like domain (Nup358Cyp) is able to interact with the CA of both HIV-1 and HIV-2. The importance of this interaction to viral replication is unclear though as certain cell-culture experiments suggest CA interaction with Nup358Cyp is dispensable for viral replication, and the CA of several other lentiviruses like SIVmac do not appear to interact with the Nup358Cyp domain. However, we have found that CA interaction with Nup358 is widely conserved among primate lentiviruses and is maintained by natural selection. The exception, SIVmac, likely reflects an evolutionary trade-off allowing escape from rhesus macaque TRIM5Cyp. Together, our observations are strong evidence that the interaction between viral CA and the Nup358Cyp domain must be biologically relevant in vivo. Specifically, by comparing interactions between multiple SIVsm/HIV-2 lineage CAs and several primate orthologs of Nup358, we identified interspecies differences in the Nup358Cyp domain that affect the CA interaction, but only when assayed in conjunction with the preceding Ran-binding domain 4 (Nup358R4). We next found that selection preserves the interaction during cross-species transmission, resulting in adaptation to differences between the Nup358Cyp homologs of the reservoir and spillover hosts. For example, SIVsm CA does not interact with human Nup358R4-Cyp, while HIV-2 CA interacts with both the human and sooty mangabey orthologs. We confirmed these distinct interaction phenotypes in an extended set of SIVsm/HIV-2 CAs, and mapped the difference to a single position – residue 3173 – in the Nup358Cyp domain. The differing ability to interact with human Nup358R4-Cyp is due to residue 85 in the CA 4-5 loops; most SIVsm strains encode a glutamine at position 85, whereas most HIV-2 strains encode an isoleucine. Reciprocal swaps reverse the interaction phenotypes, such that the SIVsm Q85I CA mutant strongly interacts with human Nup358R4-Cyp, while HIV-2 I85Q CA mutant does not. This difference also correlates with differences in single- and multi-cycle infectivity on human cell lines and levels of nuclear import in HeLa cells. Together, these results indicate that HIV-2 adapted to human Nup358 during emergence in humans. We also examined the ability of our CA panel to interact with Cyclophilin A. While all HIV-2 CA interact with CypA, the ability to interact varied among the other SIVsm CA tested, and was absent for SIVpbj. Thus, conservation of CA interaction with Nup358Cyp does not correlate to the ability to interact with CypA, and is not simply a consequence of maintaining the CA-CypA interaction. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Les adénovirus (AdV), comme d'autres virus à réplication nucléaire, ont besoin d’arriver jusqu’aunoyau cellulaire afin de libérer leur génome. Pour ce faire, les particules des AdV contenant l’ADNviral sont transportées jusqu’au complexe du pore nucléaire (NPC), via le centre d’organisation desmicrotubules, par un mécanisme encore mal compris qui implique l’exportine cellulaire CRM1. Lacapside des AdV dépasse la taille limite d’entrée dans le noyau, et par conséquent, elle doit êtredésassemblée au niveau du NPC. Le mécanisme d’import de molécules d’ADN n’est pas un processusphysiologique. Pour cela, les AdV doivent détourner la machinerie cellulaire afin d’importer leurgénome dans le noyau. Le NPC est un complexe de protéines appelées nucléoporines. LaNup358/RanBP2, principal composant des filaments cytoplasmiques, sert de plateforme de liaison àdes karyopherines (e.g Importin-β, CRM1) et à la protéine GTPase Ran. Les karyopherinesreconnaissent des signaux spécifiques présents dans les cargos et facilitent leur transport d’unemanière très régulée dépendante de RanGTP. Nous avons constaté que l’import du génome AdV estmoins efficace en l’absence de Nup358. Dans ces conditions, nous avons observé que certaineskaryopherines deviennent limitantes pour l’import du génome viral, et identifié la région minimale deNup358 requise pour compenser ce défaut. D’autre part, nous avons confirmé l’implication de CRM1dans l’arrivé des particules virales au noyau et identifié un nouveau rôle de CRM1 dans ledésassemblage de la capside des AdV. Ces travaux contribuent à mieux connaître le mécanismed’entrée du génome AdV dans le noyau et donnent une idée de la façon dont les virus peuventcontourner la machinerie de transport cellulaire pour leur propre bénéfice. / Nuclear delivery of viral genomes is an essential step for nuclear replicating DNA viruses such asAdenovirus (AdV). AdV particles reach the nuclear pore complex (NPC) in the form of genomecontaining, partially disassembled capsids, through a poorly understood CRM1-dependent mechanism.These capsids exceed the NPC size limit and therefore, they must disassemble at the NPC to releasethe viral genome. Nuclear import of DNA cargos is not a physiological process. Consequently, AdVneed to divert the cellular transport machinery for nuclear genome delivery. The NPC is a multiproteincomplex consisting of nucleoporins (Nups). The Nup358/RanBP2 is the major component ofthe cytoplasmic filaments of the NPC and serves as binding platform for factors includingkaryopherins (i.e Importin-β, CRM1) and the small GTPase Ran. Selective transport of cargo throughthe NPC is mediated by karyopherins, which recognize specific signals within the cargos and facilitatetheir transport in a RanGTP-dependent regulated manner. We identified that Nup358-depleted cellsreduce nuclear import efficiency of the AdV genome. Indeed, we observed that karyopherins are ratelimitingfor AdV genome import under these conditions and we mapped the minimal region ofNup358 necessary to compensate the import defect. On the other hand, we could confirm therequirement of CRM1 in nuclear targeting of AdV capsids and identified and additional role inmediating AdV capsid disassembly. This work helps to understand the strategy used by AdV todeliver their genome and gives insight about how viruses hijack the cellular transport machinery fortheir own benefit.

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