Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtil 4 uppsatser.

HIV and TB are among top causes of mortality and morbidity globally. Incident TB cases in 2015 were approximately 10.5 million, with 11% having HIV. Having HIV is also the biggest risk factor for developing TB disease. Clinical studies show HIV-2/TB coinfection cases progress to disease slower than HIV-1/TB. However, the underlying cellular, immunological, and molecular host-pathogen interactions accounting for these observations remain unclear. The host immune response to both viral and mycobacterial infection of macrophages is partly dependent on type I interferon, which in turn induces expression of various interferon-stimulated genes (ISGs) to control the pathogens. GBP5, a GTPase with a role in activating the inflammasome; and Sp110, a nuclear body protein, are among various ISGs whose expression levels are elevated during individual viral and bacterial infections in macrophages. GBP5 seems to restrict HIV-1 replication in immune cells, while Sp110 is known to restrict MTB proliferation in macrophages, but is now thought to also promote HIV-1 replication. We hypothesized differences in the effect of these proteins on the infectivity of the two HIV subtypes in macrophages. We also hypothesized that the viral protein Vpr is associated with the host expression levels of GBP5 and Sp110. We used small hairpin RNA to knock out GBP5 and Sp110 from THP-1 human monocytoidcell line, differentiated using PMA into a macrophage phenotype;and infected these cells with GFP-expressing HIV-2 ROD 9ΔenvΔnefstrain pseudotyped with VSV-G envelope for single cycle infection. Percentages of macrophages infected with GFP-expressing viruses were measured by Flow cytometry. To determine effect of GBP5 and Sp110 onreplication of the two HIV subtypes, HIV-1 and HIV-2, we measured virus production in supernatants of productively infected THP-1 macrophages by titering cell-free supernatants on TZMBl-reporter cells at day 3 and 6 post infection. There was a marginal increase, and a dramatic decrease, of HIV-2ΔenvΔnef GFP+ virus infectivity inall THP-1 macrophage conditions in the absence of viral Vpr, and Vpx, respectively. Knocking downmacrophage Sp110 gene expression reduced single cycle (VSV-G pseudotyped) HIV-2 ROD 9ΔenvΔnefGFP+wild type and Vpr mutant virus infectivity but promoted Vpx mutant.There was a reduction in number of infectious HIV-1 particles released from Sp110 knockdown compared to normal macrophages over 6 days post infection; no difference in HIV-1 virus production between GBP5 knock down and normal macrophages in one experiment; and a decrease in HIV-1 virus production from GBP5 knockout compared to normal macrophages 3 and 6 days post infection in a follow up experiment.Interestingly, reduced Sp110 expression corresponded to increased HIV-2 production from macrophages in one experiment but no difference in viral production between normal and Sp110 knock down macrophages by day 3 post infection in the follow up experiment. There was no HIV-2 virus production from the Sp110 knock down cells on day 6 in the follow up experiment and virus spread was reduced in GBP5 knock down, relative to normal macrophages. Interestingly, reduced Sp110 expression corresponded to increased HIV-2 production from macrophages in one experiment, but there was no difference in viral production between normal and Sp110 knock down macrophages by day 3 post infection in the follow up experiment. There was no virus production from the Sp110 knock down cells on day 6 in the follow up experiment. HIV-2 virus production was also reduced in GBP5 knock down, relative to normal, productively infected THP1/PMA macrophages. Our results support the argument that HIV-2, just like HIV-1, possibly utilizes Vpr to prevent sensing of virus infection in macrophages, thus allowing viral replication without inducing interferon stimulation, and subsequent viral restriction. Our results also suggest that the host macrophage restriction factor Sp110 may indeed enhance VSV-G pseudotyped GFP+ HIV-2 and HIV-2delVpr infectivity but restrict HIV-2 Vpx mutant. This could imply presence of a yet unknown association between another HIV-2 viral accessory protein, Vpx, and the macrophage restriction factor, Sp110 known to counter invading intracellular bacteria. We report much lower productive infectious virus release of HIV-2, compared to HIV-1, from macrophages. Overall, our results suggest that Sp110 may transiently restrict HIV-2 infection of macrophages, but is eventually manipulated by the virus to promote viral replication.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Made available in DSpace on 2012-09-06T01:12:29Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 840.pdf: 810675 bytes, checksum: d3525f79141d4c4b7c949d4be32026a0 (MD5) Previous issue date: 2005 / Cabo Verde é um arquipélago de 10 ilhas, situado a 445 Km da Costa Ocidental da África, politicamente estável e com uma população residente de 436.412 habitantes. A realização dos testes sorológicos para pesquisa de anticorpos anti-HIV-1 e 2 teve início em 1987, sendo diagnosticados no país os dois tipos virais circulantes (HIV-1 e HIV-2). Na expectativa de contribuir para o conhecimento da epidemia no país e na estruturação da rede de diagnóstico laboratorial do HIV, efetuamos um estudo descritivo, de natureza retrospectiva, com base nos dados disponíveis no laboratório de referência para diagnóstico de HIV em Cabo Verde. Foram preenchidas 1149 (Fichas de Investigação Individual Soro-Epidemiológica), sendo 993 pacientes com infecção pelo HIV e 156 pacientes com amostra indeterminada/inconclusiva. Observamos que, na população estudada, a epidemia vem crescendo em progressão linear, com padrão de transmissão predominantemente heterossexual, sendo as idades mais afetadas as que vão de 30 a 49 anos. Praia, o interior de Santiago e S. Vicente são as regiões em que se concentra o maior número de amostras positivas, sendo a maioria com Aids eselecida. Entre 1987 a 1991, os pacientes eram em sua maioria devido ao HIV-2 e com uma maior participação proporcional do sexo masculino. De 1992 a 1998, observa-se uma nítida inversão do predomínio do tipo viral, com participação proporcional similar em ambos sexos. De 1999 a 2002, predominam os pacientes com infecção pelo HIV-1, com uma contribuição progressivamente maior de mulheres. Quanto às amostras indeterminadas/inconclusivas, a reanálise efetuada concluiu que a maioria deve ser considerada simplesmente (inconclusiva). O número expressivo de amostras inconclusivas para a infecção para o HIV-1 e/ou com positividade para apenas uma das glicoproteínas de membrana específica do HIV-1 exige estudos mais aprofundados, capazes de esclarecer o quanto às evidências laboratoriais disponíveis correspondem, de fato, à distribuição dos tipos virais na dinâmica da epidemia em Cabo Verde.

Pathogenic mechanisms that account for the dramatic differences between the HIV-1 and HIV-2 epidemics remain unknown. Myeloid dendritic cells (DCs) are sentinels of the immune system, which sense invading pathogens and initiate immune responses. I hypothesize that failure of HIV-2 to overcome DC-intrinsic defense mechanisms results in diminished virus replication and reduced pathogenesis in vivo. Recent studies from our laboratory have identified capture of HIV-1 by CD169 (Siglec1), which results in preservation of virus infectivity in peripheral non-lysosomal compartments and transfer to CD4+ T cells, a mechanism of DC-mediated trans infection. HIV-1 interaction with CD169 was dependent on incorporation of a ganglioside, GM3, in the virus particle membrane. We hypothesized that reduced interaction of HIV-2 with CD169 is crucial for its attenuated pathogenic phenotype in vivo. Interestingly, HIV-2 virion assembly sites were divergent from HIV-1, which correlated with reduced incorporation of GM3 in HIV-2 virions, and a significant decrease in capture of HIV-2 compared to HIV-1 by mature DCs. Furthermore, reduced CD169-dependent HIV-2 capture by DCs attenuated access of HIV-2 to DC-mediated trans infection. In contrast to the trans infection pathway, HIV-2 could establish productive infection in DCs, though productive infection of DCs by HIV-2 resulted in innate immune activation, induction of IFN-α production and attenuated spread of virus in DC – CD4+ T cell co-cultures. As opposed to HIV-2, productive infection of DCs by HIV-1 was attenuated and failed to trigger type I IFN responses, thus allowing for efficient spread of HIV-1 in DC – CD4+ T cell co-cultures. These results suggest that immune sensing of HIV-2 in productively infected DCs limits viral spread. Finally, we investigated GM3-expressing nanoparticles (GM3-NPs) for delivery of therapeutics that trigger innate immune responses in CD169+ myeloid cells as a novel strategy to mimic myeloid cell-intrinsic virus control observed in HIV-2 infection. We tested the ability of GM3-coated nanoparticles that incorporated a TLR2 ligand, Pam3CSK4, to activate CD169+ cells. Interestingly, Pam3CSK4 containing GM3-NPs robustly activated CD169+ cells. These results suggest that induction of dendritic cell-intrinsic type I IFN responses might be a fruitful therapeutic strategy to restrict HIV-1 replication in vivo.

Vpr is a 14 kDa accessory protein conserved amongst extant primate lentiviruses that is required for virus replication in vivo. Although many functions have been attributed to Vpr, its primary role, and the function under selective pressure in vivo, remains elusive. The minimal importance of Vpr in infection of activated CD4+ T cells in vitro suggests that its major importance lies in overcoming restriction to virus replication in quiescent CD4+ T cells and non-cycling myeloid cell populations, such as macrophages and dendritic cells. Previous studies from our laboratory demonstrated that HIV-1 replication is attenuated in the absence of Vpr in monocyte-derived dendritic cells (MDDCs) and macrophages, which is correlated with the ability of HIV-1 Vpr to overcome a post-integration transcriptional defect in these cells. In contrast to HIV-1 Vpr-mediated transcriptional enhancement of the viral LTR, here I describe a role for HIV-2 and SIVmac Vpr homologs in the suppression of innate immune sensing of primate lentiviral infection in monocyte-derived dendritic cells (MDDCs). Specifically, the Vpr proteins of HIV-2 and SIVmac, but not that of HIV-1, suppress innate immune detection and induction of type I and type III IFN at two distinct stages of the viral life cycle: prior to and during integration. We posit that HIV-2/SIVmac-lineage Vpr homologs gained this function upon the acquisition of Vpx, a Vpr paralog in the lentiviral genome, that targets the retroviral restriction factor SAMHD1 for proteasomal degradation. Mutational analysis shows that suppression of pre-integration innate immune sensing by HIV-2/SIVmac Vpr homologs is tied to their interaction with the DNA damage response protein human Uracil DNA glycosylase hUNG. Interestingly, the HIV-1 Vpr degrades hUNG, whilst the HIV-2/SIVmac Vpr homologs do not. This difference correlates with the inability of HIV-1 Vpr to suppress type I and III IFN responses in SIVmac Vpx supplemented infections of MDDCs. These results highlight how divergent lentiviruses have tailored interactions of their Vpr proteins with members of the DNA damage response to promote replication in diverse cellular contexts. This work also describes the conserved role of primate lentiviral Vpr homologs in the transcription of extrachromosomal or unintegrated viral DNA. This function is dependent on Vpr engagement with the host E3-ubiquitin ligase complex Cul4-DDB1-DCAF1 (DCAFCRL4) and ability to activate the DNA damage response. These findings give insight into the mechanisms driving transcription from an underappreciated and long-lived source of viral antigen, and further the field of non-integrating lentiviral vectors, a frequently used tool for the genetic modification of non-dividing cells. Together, both studies shed light on the way Vpr proteins from diverse primate lentiviruses converge in their manipulation of the DNA damage response to facilitate multiple stages of the virus lifecycle. / 2024-01-24T00:00:00Z

Depuis la découverte du virus de l'Immunodéficience humaine, un lentivirus, comme agent pathogène responsable de l'épidémie du SIDA en 1983, beaucoup de progrès sur le sujet ont été réalisés. Il existe deux types de virus différents pouvant infecter l'Homme, le HIV-1 et le HIV-2. Ces deux virus se regroupent en différents groupes et sous-types qui témoignent d'une grande diversité inter et intra individus (notions de quasi-espèces). La découverte de lentivirus infectant naturellement au moins quarante-cinq espèces de primates en Afrique sub-saharienne, a permis un enrichissement des connaissances sur les origines des épidémies lentivirales humaines. Aujourd'hui , il est clairement admis que l'origine des épidémies d'HIV-1 et HIV-2 sont le résultat de transmissions zoonotiques de virus de chimpanzés/gorilles et de mangabeys enfumées, respectivement. La mise en évidence de nombreux SIV circulant chez ces primates non-humains indique bien le risque potentiel de nouvelles zoonoses dans la population humaine exposée, cependant, il peut paraître surprenant que jusqu'à maintenant, deux lignées lentivirales seulement ont été capables de franchir cette barrière d'espèce. Pour pouvoir se répliquer dans les cellules d'un nouvel hôte, un lentivirus doit pouvoir contrecarrer les différents facteurs de restriction exprimés par les cellules cibles tout en exploitant au maximum la machinerie cellulaire. La famille de protéines TRIM5, APOBEC3 et les protéines Tetherin/Bst2 et SAMHD1 sont capables de bloquer une infection rétrovirale. Dans ce travail, le rôle des protéines TRIM5 a été étudié ainsi que celui d'autres protéines interagissant avec des capsides rétrovirales, dans un contexte de transmission inter-espèces de lentivirus de primates. L'étude de TRIM5α humain a montré que cette protéine n'était capable de bloquer aucune des infections par les lentivirus primates testés dans cette étude, ni par les autres SIV. Nous avons pu mettre en évidence que la dépendance de la liaison à la Cyclophiline A pour l'infection des différents SIV était variable en fonction de la capside testée. Ainsi, si cette interaction est largement répandue parmi les différentes lignées de SIV, elle n'est toutefois pas universelle. La sensibilité des SIV à la déplétion de nucléoporines qui sont connues pour affecter l'infection par HIV-1, était également variable pour différents SIV, et la même diversité a été observée concernant les déplétions de RanBP2 et Nup153. De plus, nous avons découvert une capside de SIV soumise à une forte restriction de son infection dans les cellules humaines, ce phénotype a été nommé Ref2.Il a été suggéré qu'il existait une possible corrélation entre des variations de la capside de HIV-2 et la progression vers le SIDA, nous avons donc élaboré une étude afin de déterminer si les protéines TRIM5 étaient impliquées dans ce phénotype. La conclusion est que TRIM5α humaine ne restreint fortement aucune des capsides de HIV-2 testées provenant d'une cohorte d'individus à “progression rapide“ ou “lente“ vers le SIDA. Cependant nous avons observé une capacité d'infection qui corrélait avec la pathogénicité. Il est intéressant de noter que toutes les capsides d'HIV-2 testées étaient dépendantes de la présence de Cyclophiline A pour leur infection. Toutes ces capsides étaient sensibles à la déplétion de RanBP2, et l'interaction est très probablement médiée par le motif C-ter de RanBP2 qui a une forte homologie avec la Cyclophiline A. En conclusion, il est très probable que des SIV infectant naturellement des singes puissent utiliser les mêmes protéines que HIV-1, pour un éventuel passage inter-espèces. TRIM5α ne semble pas être une barrière efficace aux différents SIV, et l'interaction avec la Cyclophiline A est probablement très conservée par les lentivirus primates / Ever since HIV has been discovered to be the pathogenic agent that causes AIDS in 1983, much progress has been made in the field. Two different viruses are now known to infect humans, HIV-1 and HIV-2. These two distinct viruses have many sub-types and clades representing a high diversity inter and intra-individuals (quasi-species). The finding of HIV simian counterparts, the Simian Immunodeficiency Viruses (SIVs), has broadened the knowledge of primate lentiviruses and to date forty-five species of non-human primates are known to be infected with SIVs in sub-saharan Africa. It is now clear that HIV-1 and HIV-2 epidemics are the result of zoonosis from chimpanzees/gorillas and sooty mangabeys, respectively. With such a big diversity of SIVs in the wild and a frequent contact of SIV infected monkey species with humans, it is interesting that so far, only two lineages breached the species barrier and infected human populations. To be able to correctly infect a cell, a lentivirus has to overcome the installed cellular barriers known as restriction factors while at the same time correctly exploiting the established host cellular machinery. Proteins such as TRIM5, APOBEC3, Tetherin/Bst2, SAMHD1 are able to restrict retroviral infections in certain conditions. In this thesis, it has been evaluated the role of TRIM5 proteins and other capsid interacting proteins with a scope to the eventuality of a cross-species transmission infection. The results showed that human TRIM5alpha does not restrict any of the primate lentiviruses tested, and so far, no primate lentivirus is known to be restricted by it. Cyclophilin A binding and dependence is variable depending on the SIV capsid; this interaction is widespread among the primate lentiviruses phylogenetic tree but not a universal phenotype. Different capsids from SIVs have been tested for the sensitivity to the depletion of nucleoporins that are known to be used by HIV-1 in its infection; it has been concluded that the same diversity applies to the interaction with RanBP2 and Nup153. Additionally, we identified a SIV capsid that is highly restricted in human cells; this phenotype was called Ref2. With the report of a possible correlation between HIV-2 capsid variations and different levels of progression to AIDS, we devised a study aiming to identify if TRIM5 proteins were involved in this phenotype. We concluded that human TRIM5alpha does not restrict any HIV-2 capsid obtained from a HIV-2 cohort, in which individuals were presenting different levels of progression to AIDS. However, we observed a different viral fitness that correlated with pathogenicity. Moreover, Cyclophilin A dependence seems ubiquitous among all of the tested HIV-2 capsids. All of these capsids are sensitive to RanBP2 depletion and the interaction is much likely mediated by RanBP2's C-terminal motif that shares a high homology with Cyclophilin A. Summing up, it is much likely that some SIVs that still circulate in the wild can hijack the same specific cellular co-factors as HIV-1 to produce a new epidemic in humans. TRIM5α does not seem to be a potent barrier to an eventual cross-species transmission from lower primates to humans, and Cyclophilin A interaction seems to play a major role to the infection of some SIVs.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 71-78).

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.