Towards Inhibition of Vif-APOBEC3G Interaction: Which Protein to Target?

APOBEC proteins appeared in the cellular battle against HIV-1 as part of intrinsic cellular immunity. The antiretroviral activity of some of these proteins is overtaken by the action of HIV-1 Viral Infectivity Factor (Vif) protein. Since the discovery of APOBEC3G (A3G) as an antiviral factor, many advances have been made to understand its mechanism of action in the cell and how Vif acts in order to counteract its activity. The mainstream concept is that Vif overcomes the innate antiviral activity of A3G by direct protein binding and promoting its degradation via the cellular ubiquitin/proteasomal pathway. Vif may also inhibit A3G through mechanisms independent of proteasomal degradation. Binding of Vif to A3G is essential for its degradation since disruption of this interaction is predicted to stimulate intracellular antiviral immunity. In this paper we will discuss the different binding partners between both proteins as one of the major challenges for the development of new antiviral drugs

An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1β Secretion

The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1β secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1β secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1β secretion due to elevation of IL-1β and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases

The structure-function relationship of HIV-1 Vif protein and its regulation

Tese de doutoramento em Farmácia (Microbiologia), apresentada à Universidade de Lisboa através da Faculdade de Farmácia, 2008One of the critical determinants of HIV-1 infectivity is the capacity of the virus to infect new cells. The viral Vif protein is one of the critical factors involved in the increase infectivity of the virus. Vif protein is required for HIV to replicate in some human cell types (termed non-permissive' cells), but not others ( permissive' cells). The reason for this is the expression, in non-permissive cells, of a potent antiretroviral enzyme: APOBEC3G (A3G) and its paralog APOBEC3F (A3F). Each of these enzymes contains two RNA-binding motifs and incorporates into assembling HIV-1 capsids where they cause lethal dC-to-dU hypermutations in the single-stranded viral DNA that transiently forms during reverse transcription. One of the mechanisms that Vif uses to overcome this viral block is by reducing A3G/A3F intracellular expression levels recruiting it for polyubiquitination and proteasomal degradation in the 26S proteasome. In order to understand how A3G half-life can influence its antiviral activity, both at the level of viral encapsidation and catalytic activity in the cell, we decided to model the intracellular half-life of A3G by targeting it to the N-end rule pathway. This system allowed us to show that A3G variants with different stabilities are capable to be targeted for proteasomal degradation in a Vif-independent manner. Our results confirmed the importance of A3G steady-state expression for the maintenance of its antiretroviral activity indicating that in order to counteract A3G activity, Vif has to act at the very early stages of A3G life-time, probably within the first 13 minutes after its synthesis, in order to impede its incorporation into virions. In addition, we show that active deamination in the cytoplasm does not seem to be important for antiviral activity of A3G and deamination activity does not increase with the half-life of A3G. Identification of protein biding partners between Vif and A3G proteins is one of the major challenges for the development of new antiviral drugs, as disruption of A3G-Vif interaction is predicted to stimulate natural antiviral infectivity. In this work, we used two different approaches to study the interaction of Vif with A3G. One strategy was based on an oligomerization-assisted interaction whereby fragments of Vif and A3G were fused to independently folding and interacting domains of homodimerizing GCN4 leucine zipper-forming sequences. This allowed us to show that the C-terminal region of Vif alone is not sufficient to induce A3G degradation and that binding of Vif to A3G must involve an additional co-factor that is probably linked to the N-terminus of Vif and may be essential for the interaction and consequent degradation of A3G. The other strategy involved a Protein Complementation Assay (PCA). We produced several mutants of both Vif and A3G bearing mutations on key residues that are thought to be crucial for the interaction between these two proteins. By using this strategy we were able to show that some mutations in A3G protein that had been described as being involved in the interaction with HIV-1 Gag, were also important for the interaction with Vif. As a result, we were able to achieve a quantitative assessment of HIV-1 Vif-A3G interactions, which allowed us to do a fine dissection of the regions involved in this interaction. Finally, we found that a region in Vif (DRMR) may be important for the selection of the APOBEC protein to inactivate. By studying the interaction of Vif with A2 protein we reached the conclusion that an additional co-factor may be required in order to A2 to be able to exert antiviral action. Therefore, we provided supporting information for a new direction of the study of Vif-A3 problematic regarding the involvement of anadditional co-factor.Fundação para a Ciência e Tecnologia (FCT), (SFRH/BD/18805/2004

A3G antiviral activity revisited

The human APOBEC3G (A3G) is a potent inhibitor of HIV-1 replication and its activity is suppressed by HIV-1 virion infectivity factor (Vif). Vif neutralizes A3G mainly by inducing its degradation in the proteasome and blocking its incorporation into HIV-1. - FCT [POCI/BIAMIC/60038/2004]. - We greatly appreciate the gift of pcDNA3.1-APOBEC3G-HA (A3G) by Dr. Strebel K., and pEGFP*IRES-Ub-X vector, kindly supplied by Dr. Mark A. Muesing. The work was supported by FCT grant POCI/BIAMIC/60038/2004

Repurposing anti-cancer porphyrin derivative drugs to target SARS-CoV-2 envelope

Antiviral medicines to treat COVID-19 are still scarce. Porphyrins and porphyrin derivatives (PDs) usually present broad-spectrum antiviral activity with low risk of resistance development. In fact, some PDs are clinically approved to be used in anti-cancer photodynamic therapy and repurposing clinically approved PDs might be an alternative to treat COVID-19. Here, we characterize the ability of temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles. PDs light-dependent and –independent effect on SARS-CoV-2 infectivity were evaluated. PDs photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light dose. However, only temoporfin and verteporfin inactivated SARS-CoV-2 in the dark, being verteporfin the most effective. PDs treatment reduced viral load in infected Caco-2 cells, while not inducing cytotoxicity. Furthermore, light-independent treatment with temoporfin and verteporfin act on early stages of viral infection. Using lipid vehicles as membrane models, we characterized PDs interaction to the viral envelope. Verteporfin presented the lowest IC50 for viral inactivation and the highest partition coefficients (Kp) towards lipid bilayers. Curiously, although temoporfin and redaporfin presented similar Kps, redaporfin did not present light-independent antiviral activity, and only temoporfin and verteporfin caused lipid membrane disorder. In fact, redaporfin is located closer to the bilayer surface, while temoporfin and verteporfin are located closer to the centre. Our results suggest that viral envelope affinity, with penetration and destabilization of the lipid bilayer, seems critical to mediate PDs antiviral activity. Altogether, these findings open new avenues for the off-label application of temoporfin and verteporfin in the systemic treatment of COVID-19

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

© 2019 American Chemical SocietyWe 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.This work was supported by European Research Area Networks (ERA-Net) project HIVERA/0002/2013 and Fundação para a Ciência e a Tecnologia (FCT-MCTES) project VIH/SAU/0029/2011. This work has also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 828774. T.N.F., M.M.D., and I.C. acknowledge individual fellowships SFRH/BD/5283/2013, SFRH/BPD/122779/2016, and SFRH/BPD/65531/2009 funded by FCT-MCTES, respectively. A.S.V. acknowledges funding under the Investigator Programme (IF/00803/2012) from FCT-MCTES.info:eu-repo/semantics/publishedVersio

Penetrating the blood-brain barrier with new peptide-porphyrin conjugates having anti-HIV activity

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/52150011), by the European Union (H2020-FETOPEN-2018-2019-2020-01 grant no 828774), and by the Spanish Ministry of Science and Innovation (AEI/FEDER grant CTQ2017-84371-P). NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. Additional funding from Fundação para a Ciência e Tecnologia (FCT-MCTES) is also acknowledged for DA Mendonça (PD/BD/136752/2018) and for C. Cruz-Oliveira and I. Cadima-Couto (PTDC/BIA-VIR/29495/2017)

Anti-HIV-1 Activity of pepRF1, a Proteolysis-Resistant CXCR4 Antagonist Derived from Dengue Virus Capsid Protein

International audienceThere is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53 000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases

Anti-HIV-1 Activity of pepRF1, a Proteolysis-Resistant CXCR4 Antagonist Derived from Dengue Virus Capsid Protein

There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53 000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.This work was supported by Fundação para a Ciência e a Tecnologia (FCT-MCTES, Portugal) project PTDC/QEQMED/4412/2014, and by the Institut National de la Santé et de la Recherche Medicale (Inserm). I.C.C., J.M.F., T.N.F., and I.B. acknowledge fellowships SFRH/BPD/65531/2009, SFRH/BD/70423/2010, SFRH/BD/5283/2013, and SFRH/BPD/76225/2011, respectively, funded by FCT-MCTES. A.T. is the recipient of a fellowship from Agence Nationale de Recherche sur le Sida et les Hepatites Virales (ANRS). A.S.V. acknowledges funding under the Investigator Programme (IF/00803/2012) from FCT-MCTES. Work at Pompeu Fabra University was supported by grants AGL2014-52395-C2 and AGL2017-84097-C2-2-R and by the “María de Maeztu” Program for Units of Excellence in R&D from the Spanish Ministry of Innovation and Competitiveness (MINECO).info:eu-repo/semantics/publishedVersio