[PDF] Analyzing Contributions Of Host Cellular Proteins And Signaling Pathways To Hiv 1 Replication And Cell Cell Spread eBook

Author : Eunju Park
Publisher :
Page : 185 pages
File Size : 20,90 MB
Release : 2018
Category :
ISBN :

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Human immunodeficiency virus type 1 (HIV-1) is a highly successful human pathogen responsible for the global AIDS epidemic, leading to 35 million deaths since its discovery in 1983. Current antiretroviral therapies reduce viral load, but are problematic due to long-term toxicity, the development of viral resistance, and the inability to target persistent viral reservoirs. Thus, there is a need for new and improved anti-HIV-1 therapeutics, whose rational development requires better understanding of HIV-1 replication. HIV-1 infection depends on interactions with host membranes to facilitate virtually every step of virus replication. Reticulon homology domain proteins (RHPs) are a family of membrane-shaping proteins with functions in membrane rearrangement and vesicle trafficking. Previously, our laboratory found that plasmid-expressed short hairpin RNA (shRNA) against RHPs significantly stimulated HIV-1 virion release, suggesting that RHPs might normally function to restrict HIV-1 virion production. However, further work reported here showed that the RHP-targeted shRNAs nonspecifically stimulated not only HIV-1 release but secretion of multiple HIV-1-independent proteins, implicating off-target effects of plasmid-expressed shRNAs, while more efficient, specific knockdown of RHPs by small interfering RNAs and microRNAs did not modulate HIV-1 release. Therefore, we conclude that RHPs are not essential for HIV-1 virion release. HIV-1 primarily spreads by direct contacts between infected and uninfected cells. To map cell contact-induced dynamic signaling pathways and responses during viral spread, we used mass spectrometry with cell-specific protein labeling combining stable isotope labeling of amino acids in cell culture with tandem mass tagging of peptide pools after isolation. These approaches revealed phosphoproteomic and proteomic changes induced in HIV-1-infected T cells by contact with uninfected T cells, including, from 5 through 60 min of co-culture, significant alterations in cell division-related signaling pathways mediated by the LCK, WEE1 and Aurora B kinases. We further validated that specifically inhibiting LCK, WEE1 or Aurora B kinase activity modulates infected cell and uninfected cell interaction, virion assembly and infection spread. Our results reveal that on contacting uninfected cells, HIV-1-infected cells undergo rapid and expanding changes including reprograming the cell cycle network to facilitate virus spread, adding new dimensions to prior results on HIV-1-induced cell cycle changes.

Author : Nadine Laguette
Publisher : Frontiers Media SA
Page : 119 pages
File Size : 11,58 MB
Release : 2015-05-07
Category : Microbiology
ISBN : 2889194841

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Productive HIV infection requires completion of all the steps of the replication cycle, the success of which largely relying on the multiple interactions established by viral proteins with cellular partners. Indeed, cellular and viral fates are intertwined and this interplay may involve rerouting of cellular factors/pathways to the benefit of the viral life cycle. To gain a foothold into host cells, HIV has to take advantage of available cellular factories and overcome the numerous potential blocks opposed to its replication while ensuring cellular survival. Viral auxiliary proteins are a perfect paradigm to illustrate the complexity of the relationship between HIV and its host. Although these accessory proteins are mostly unnecessary for viral replication in permissive cells in vitro, they play a crucial role in regulating viral spread ex vivo in non-permissive cells and in vivo in hosts. Most accessory proteins are pleiotropic and instrumental in the counteraction of restriction factors and proteins involved in innate immune response. Several proteins of the “intrinsic” immune system that detect the presence of the assailant and initiate a subsequent immune response, as well as restriction factors that are directly devoted to arresting the replication cycle at precise steps have been characterized. Despite the numerous cellular mechanisms dedicated to preventing viral replication, HIV is able to efficiently replicate in humans. Indeed, as a master regulator of cellular machineries and processes, not only has HIV evolved strategies to avoid triggering of pattern recognition receptors, but HIV has also elaborated ways to counteract host restriction factors, thereby overcoming the hurdles that oppose efficient replication. This review collection is dedicated to the manipulation of host cells by HIV-1 and HIV-2, with a particular focus on viral accessory proteins.

Author : Paul Spearman
Publisher : Springer Science & Business Media
Page : 210 pages
File Size : 11,30 MB
Release : 2009-12-11
Category : Medical
ISBN : 3642021751

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The study of viruses necessarily involves dissecting the intimate details of cellular pathways. Viruses have often been employed as tools in studying cellular pathways, as was done by early retrovirologists such as Peyton Rous in attempting to understand the mechanism of cellular transformation and oncogenesis. On the other side of the coin, virologists seek to de?ne those cellular elements interacting intimatelywiththeir virus ofinterestinorder to better understand viral replication itself, and in some cases to develop antiviral strategies. It is in the intersection of virology and cell biology that many of us ?nd the most rewarding aspects of our research. When a new discovery yields insights into basic cellular mechanisms and presents new targets for int- vention to ?ght a serious pathogen, the impact can be high and the excitement intense. HIV has been no exception to the rule that viruses reveal many basic aspects of cellular biology. In recent years, in part because of the importance of HIV as a major cause of human suffering, numerous cellular processes have been elucidated through work on processes or proteins of this human retrovirus. The excitement in this ?eld is especially well illustrated by the discovery of new innate means of resisting viral replication, such as the work on APOBEC3G, TRIM5a, and BST-2/ tetherin presented in this volume.

Author : Yingfeng Zheng
Publisher :
Page : 0 pages
File Size : 42,92 MB
Release : 2008
Category :
ISBN :

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Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

Author : Emily Elizabeth Whitaker
Publisher :
Page : 406 pages
File Size : 37,45 MB
Release : 2021
Category : HIV infections
ISBN :

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Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and the causative agent of Acquired Immunodeficiency Syndrome (AIDS). HIV-1 can spread through multiple modes of transmission including cell-to-cell transmission between CD4+ T cells at a transient junction known as the virological synapse (VS). The VS forms upon HIV-1 Envelope (Env) on the surface of an infected (producer) cell binding CD4 on an uninfected (target) cell. While the VS typically resolves with complete cell separation and transfer of virus particles, Env can occasionally facilitate cell-cell fusion at this site, forming a multinucleated infected cell (syncytium). Excessive syncytium formation is prevented by viral and host factors, though this subpopulation of infected cells can still comprise ~20% of all infected cells in vivo. T cell-based syncytia detected in vivo are unique from mononucleated infected cells as they contain 2-4 nuclei, can have an elongated morphology, and appear highly motile. Despite such significant presence of syncytia, little is known about how these multinucleated infected entities contribute to HIV-1 spread and pathogenesis. During cell-to-cell transmission at the VS, viral and host factors are enriched at this site to support virus spread (reviewed in Chapter 2). This thesis focused on fusion inhibitory factors HIV-1 Gag and several host proteins, including tetraspanins, ezrin, and EWI-2. We determined that EWI-2 is recruited specifically to the producer cell side of the VS (the presynapse) where it inhibits HIV-1-induced cell-cell fusion in a dose-dependent manner (Chapter 3). Although both EWI-2 and tetraspanins are typically downregulated upon infection, both tetraspanin CD81 and EWI-2 surface levels are partially restored on HIV-1-induced CD4+ primary T cell-based syncytia compared to mononucleated infected cells. We sought to determine whether target cells influence the surface profile upon fusion and whether the altered protein levels are maintained for the lifetime of a syncytium (Chapter 4). We demonstrated that EWI-2 surface levels on syncytia correlate with levels of the target cell population, suggesting that EWI-2 brought along by target cells at least partially restores surface expression in syncytia. Further, we determined that newly formed, “young” syncytia, have higher levels of EWI-2 than older ones, suggesting that downregulation of EWI-2 continues in syncytia. We expect that higher levels of EWI-2 on young syncytia will render them less susceptible to continued cell-cell fusion than mononucleated infected cells and may also reduce virus particle infectivity. This will be tested by analysis of a purified syncytia population to measure fusogenicity and particle infectivity relative to fusogenicity and particle infectivity of mononucleated infected cells. Those data will be included in a future manuscript. Collectively, the work presented in this dissertation has furthered our understanding of HIV-1-induced cell-cell fusion regulation and allowed us to characterize distinct differences in protein expression between syncytia and mononucleated infected cells. These findings open the door to future investigations aimed at understanding how syncytia contribute to virus transmission and pathogenesis.

Author : Ann Arvin
Publisher : Cambridge University Press
Page : 1325 pages
File Size : 35,19 MB
Release : 2007-08-16
Category : Medical
ISBN : 1139461648

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This comprehensive account of the human herpesviruses provides an encyclopedic overview of their basic virology and clinical manifestations. This group of viruses includes human simplex type 1 and 2, Epstein–Barr virus, Kaposi's Sarcoma-associated herpesvirus, cytomegalovirus, HHV6A, 6B and 7, and varicella-zoster virus. The viral diseases and cancers they cause are significant and often recurrent. Their prevalence in the developed world accounts for a major burden of disease, and as a result there is a great deal of research into the pathophysiology of infection and immunobiology. Another important area covered within this volume concerns antiviral therapy and the development of vaccines. All these aspects are covered in depth, both scientifically and in terms of clinical guidelines for patient care. The text is illustrated generously throughout and is fully referenced to the latest research and developments.

Author : Guido Silvestri
Publisher : Springer
Page : 253 pages
File Size : 25,97 MB
Release : 2018-10-11
Category : Medical
ISBN : 303002816X

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This volume summarizes recent advances in understanding the mechanisms of HIV-1 latency, in characterizing residual viral reservoirs, and in developing targeted interventions to reduce HIV-1 persistence during antiretroviral therapy. Specific chapters address the molecular mechanisms that govern and regulate HIV-1 transcription and latency; assays and technical approaches to quantify viral reservoirs in humans and animal models; the complex interchange between viral reservoirs and the host immune system; computational strategies to model viral reservoir dynamics; and the development of therapeutic approaches that target viral reservoir cells. With contributions from an interdisciplinary group of investigators that cover a broad spectrum of subjects, from molecular virology to proof-of-principle clinical trials, this book is a valuable resource for basic scientists, translational investigators, infectious-disease physicians, individuals living with HIV/AIDS and the general public.

Author : Justin T. Massey
Publisher :
Page : 364 pages
File Size : 20,27 MB
Release : 2016
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ISBN :

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Introduction: HIV-1 is a retrovirus that requires membranes for multiple stages of viral replication. Recently, global screens identified host-membrane shaping reticulon homology proteins (RHPs) as having possible roles in un-determined early (König et al., 2008) stages of HIV-1 replication as well as interacting with HIV-1 accessory protein VPU (Jager et al., 2012). RHPs are ER-resident proteins and contribute to stabilization of ER-tubules and have can affect the kinetics of nuclear envelope formation during mitosis (Anderson and Hetzer, 2008). Therefore, we investigated roles of RHPs in early replication as well as potential functional consequences of RHP interactions with VPU. Results: We did not detect HIV-1 entry defects with RTN3 knockdown in Jurkat T-cells, or THP-1 monocytes, despite efficient RTN3 depletion. We determined that effects in HEK293T cells were due to off target effects of the siRNA used under conditions of cell crowding, independent of RTN3 knockdown. Further, we confirmed an interaction between HIV-1 VPU protein and RHPs, but did not observe a consequence of RHP depletion on VPU-mediated antagonism of CD4 or tetherin. In contrast to our IP results, using super-resolution microscopy we determined that VPU does not co-localize with RTN3 or RTN4 in cells. Conclusion: We were able to reproduce the early defect in HIV-1 early replication reported in the König screen. Further analysis revealed that this defect was not specific to knockdown of RTN3, and instead was a non-specific effect of siRNA on HIV in over-crowded, adherent cells. Taken together, these results showed that RTN3 depletion alone did not affect HIV entry. Additionally, while we confirmed that VPU interacted with the RHP family, we were unable to assign a function to this interaction. Furthermore, VPU did not appear to co-localize with RHPs under super-resolution microscopy, suggesting the IP results were an artifact of cell lysis. However, it remains formally possible that single gene knockdown of RHPs may not be enough to see a phenotype, and that depletion of several compensating RHPs by a multi-gene knockout approach, such as creating multiple gene deletions, might be required to fully test and to reveal a potential RHP contribution to HIV replication.