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Gene-targeted strategies to eliminate HIV latent cells through synthetic activators and suicide lentivectors

Publication . Perdigão, PRL; Gonçalves, João, 1967-; Marta, Mariana Santa, 1978-

Despite the success of antiretroviral therapy, a cure for HIV-1 infection remains elusive. The persistence of cellular reservoirs harboring transcriptionally silent (latent) HIV provirus is responsible for the viremia rebound observed following treatment withdrawal. Stimulation of latent viral expression is considered critical to target HIV reservoirs for elimination through a “shock and kill” approach. Pharmacological drugs have systematically proven ineffective to drastically reduce the reservoir size and may cause severe side effects owing to their indiscriminate mode of action. In the present thesis, gene-targeted strategies were explored to stimulate and eliminate HIV latent cells. To stimulate latent virus expression, we designed synthetic activators based on transcription activator-like effector (TALE) proteins that recognize conserved regions on HIV 5’LTR promoter. Four TALE activators strongly induced HIV transcription, acting in cooperation to specifically enhance viral expression from cell line models of HIV-1 latency. Moreover, we show that histone deacetylase inhibitors can further enhance the effect of TALE-mediated activation in highly repressed latent cells. To further potentiate the elimination of stimulated latent cells, we conjugated an HIV-responsive suicide lentivector to our TALE activator technology. For this purpose, we incorporated a modified 5’LTR promoter into the suicidal lentivector as a safety mechanism to dissociate TALE-driven activation, restricting the responsiveness of this plasmid to the HIV regulatory proteins. The therapeutic plasmid was capable of specifically eliminate latently infected cells stimulated by TALE activators through a Tat/Rev-dependent expression of the diphtheria toxin. Finally, we presented a “gene-free” approach to specifically activate latent HIV expression through protein delivery of cell-penetrating zinc-finger activators (CPP-ZFA). A single activator based on Cys2His2 zinc-finger domains proved effective at inducing viral expression from the primer binding site downstream of 5’LTR promoter. When conjugated with positively charged nuclear localization signal repeats, this synthetic activator efficiently translocated across cell membrane without the need of carriers. Short-term presence of CPP-ZFA following protein delivery was sufficient to stimulate gene expression in HIV-1 latent cells, offering a safer alternative to avoid off-target effects from prolonged exposure to these synthetic activators. In resume, this work provides proof-of-concept that synthetic activators and suicide lentivectors constitute promising candidates for the eradication of HIV-1 reservoirs through gene-targeted strategies.

2017Doctoral thesis

Open access

Enfuvirtide-protoporphyrin IX dual-loaded liposomes: in vitro evidence of synergy against HIV-1 entry into cells

Publication . Figueira, Tiago Nascimento; Domingues, Marco; Illien, Françoise; Cadima Couto, Carla Iris; Todorovski, Toni; Andreu, David; Sagan, Sandrine; Castanho, Miguel A. R. B.; Walrant, Astrid; Veiga, Ana Salomé

We have developed a nanocarrier consisting of large unilamellar vesicles (LUVs) for combined delivery of two human immunodeficiency virus type 1 (HIV-1) entry inhibitors, enfuvirtide (ENF) and protoporphyrin IX (PPIX). The intrinsic lipophilicity of ENF and PPIX, a fusion inhibitor and an attachment inhibitor, respectively, leads to their spontaneous incorporation into the lipid bilayer of the LUVs nanocarrier. Both entry inhibitors partition significantly toward LUVs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a 9:1 mixture of POPC:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (DPPE-PEG2000), representative of conventional and immune-evasive drug delivery formulations, respectively. These colocalize in the core of lipid membranes. Dual-loaded nanocarriers are monodispersed and retain the size distribution, thermotropic behavior, and surface charge of the unloaded form. Combination of the two entry inhibitors in the nanocarrier resulted in improved synergy against HIV-1 entry compared to combination in free form, strongly when immuneevasive formulations are used. We propose that the improved action of the entry inhibitors when loaded into the nanocarriers results from their slow release at the site of viral entry. Overall, liposomes remain largely unexplored platforms for combination of viral entry inhibitors, with potential for improvement of current antiretroviral therapy drug safety and application. Our work calls for a reappraisal of the potential of entry inhibitor combinations and delivery for clinical use in antiretroviral therapy.

2020Journal article

Restricted access

Mechanisms of vesicular stomatitis virus inactivation by protoporphyrin ix, zinc- protoporphyrin ix, and mesoporphyrin ix

Publication . Oliveira, Christine Cruz; Almeida, Andreza F.; Freire, João M.; Caruso, Marjolly B.; Morando, Maria A.; Ferreira, Vivian N. S.; Miranda, Iranaia Assunção; Gomes, Andre M. O.; Castanho, Miguel A. R. B.; Poian, Andrea T. da

Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.

2017Journal article

Open access

Mining viral proteins for antimicrobial and cell-penetrating drug delivery peptides

Publication . Freire, João Miguel; Dias, Susana Almeida; Flores, Luís; Veiga, Ana Salomé; Castanho, Miguel A. R. B.

Motivation: The need for more effective and safer pharmaceuticals is a persistent quest. Microbial adaptations create the need to permanently develop new antimicrobials, for instance. Similarly, intracellular delivery of drugs is still a challenge and translocation of membranes for drug delivery is an area of intense research. Peptides can be used both as antimicrobial drug leads and drug carrier systems for intracellular delivery. Multifunctional proteins are abundant in viruses but, surprisingly, have never been thoroughly screened for bioactive peptide sequences. Results: Using the AMPA and CellPPD online tools we have evaluated the propensity of viral proteins to comprise antimicrobial (AMP) or cell-penetrating peptides (CPP). Capsid proteins from both enveloped and non-enveloped viruses, and membrane and envelope proteins from enveloped viruses, in a total of 272 proteins from 133 viruses, were screened to detect the presence of potential AMP and CPP sequences. A pool of 2444 and 426 CPP and AMP sequences, respectively, were discovered. The capsids of flaviviruses are the best sources of these peptides reaching more than 80% of CPP sequence coverage per protein. Selected sequences were tested experimentally and validated the results. Overall, this study reveals that viruses form a natural multivalent biotechnological platform still underexplored in drug discovery and the heterogeneous abundance of CPP/AMP sequences among viral families opens new avenues in viral biology research.

2015Journal article

Restricted access

The rigid amphipathic fusion Inhibitor dUY11 acts through photosensitization of viruses

Publication . Vigant, Frederic; Hollmann, Axel; Lee, Jihye; Santos, Nuno C.; Jung, Michael E.; Lee, Benhur

Rigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen (1O2), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE.

2014Journal article

Open access