RESUMO
Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.
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Pesquisadores , Relatório de Pesquisa , Humanos , Poder PsicológicoRESUMO
OBJECTIVES: The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. METHODS: Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. RESULTS: R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. CONCLUSIONS: Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.
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Infecções por HIV , Inibidores de Integrase de HIV , Integrase de HIV , HIV-1 , Substituição de Aminoácidos , DNA , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Mutação , Oxazinas/farmacologia , Piperazinas/farmacologia , Provírus/genética , Piridonas/farmacologiaRESUMO
The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 µM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 µM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.
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Antivirais/farmacologia , Quelantes/farmacologia , Vírus da Dengue/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Picolinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Aedes , Substituição de Aminoácidos , Animais , Antivirais/síntese química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Quelantes/síntese química , Cricetinae , Vírus da Dengue/enzimologia , Vírus da Dengue/genética , Relação Dose-Resposta a Droga , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Histidina/genética , Histidina/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Cinética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Picolinas/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Sulfonas/síntese química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
UNLABELLED: Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE: Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3' processing indicates the importance of the 3' processing step and highlights a therapeutic angle that needs to be better exploited.
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Substituição de Aminoácidos , Farmacorresistência Viral , Infecções por HIV/virologia , Integrase de HIV/genética , HIV-1/enzimologia , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Integrase de HIV/química , Integrase de HIV/metabolismo , HIV-1/classificação , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Alinhamento de SequênciaRESUMO
UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.
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Farmacorresistência Viral/genética , Inibidores de Integrase/farmacologia , Seleção Genética , Vírus da Imunodeficiência Símia/genética , Animais , Clonagem Molecular , Primers do DNA/genética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Leucócitos Mononucleares/virologia , Macaca mulatta , Mutagênese , Mutação de Sentido Incorreto/genética , Oxazinas , Piperazinas , Piridonas , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Especificidade da EspécieRESUMO
Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype.
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HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , CinéticaRESUMO
OBJECTIVES: Of the currently approved HIV integrase strand transfer inhibitors (INSTIs), dolutegravir has shown greater efficacy than raltegravir at suppressing HIV-1 replication in treatment-experienced individuals. Biochemical experiments have also shown that dolutegravir has a longer dissociative half-life when bound to HIV integrase than does raltegravir. In order to study the intracellular efficacy of various INSTIs, we asked whether drug removal from INSTI-treated HIV-1-infected cells would result in different times to viral rebound. In addition, we assessed the role of the R263K substitution within the integrase ORF that is associated with low-level resistance to dolutegravir. METHODS: HIV-infected MT-2 cells were treated with dolutegravir, raltegravir or a third experimental INSTI (MK-2048) and the drugs were washed out after varying times. Viral replication was monitored by measuring reverse transcriptase (RT) activity in the culture fluids. RESULTS: We observed a significantly slower increase in RT activity after the removal of dolutegravir compared with raltegravir or MK-2048. The incubation time before the drug was removed also had an impact on the level of RT activity independently of the drug and virus used. The R263K substitution did not significantly impact on levels of RT activity after drug washout, suggesting that dolutegravir remained tightly bound to the integrase enzyme despite the presence of this mutation. CONCLUSIONS: These results suggest that the residency time of INSTIs on integrase is a key factor in the activity of these drugs and that the anti-HIV activity of dolutegravir persists more effectively than that of other INSTIs after drug washout.
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Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Infecções por HIV/virologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Oxazinas , Piperazinas , Piridonas , Linfócitos T/virologiaRESUMO
UNLABELLED: Studies on the in vitro susceptibility of SIV to integrase strand transfer inhibitors (INSTIs) have been rare. In order to determine the susceptibility of SIVmac239 to INSTIs and characterize the genetic pathways that might lead to drug resistance, we inserted various integrase (IN) mutations that had been selected with HIV under drug pressure with raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) into the IN gene of SIV. We evaluated the effects of these mutations on SIV susceptibility to INSTIs and on viral infectivity. Sequence alignments of SIVmac239 IN with various HIV-1 isolates showed a high degree of homology and conservation of each of the catalytic triad and the key residues involved in drug resistance. Each of the G118R, Y143R, Q148R, R263K, and G140S/Q148R mutations, when introduced into SIV, impaired infectiousness and replication fitness compared to wild-type virus. Using TZM-bl cells, we demonstrated that the Q148R and N155H mutational pathways conferred resistance to EVG (36- and 62-fold, respectively), whereas R263K also displayed moderate resistance to EVG (12-fold). In contrast, Y143R, Q148R, and N155H all yielded low levels of resistance to RAL. The combination of G140S/Q148R conferred high-level resistance to both RAL and EVG (>300- and 286-fold, respectively). DTG remained fully effective against all site-directed mutants except G118R and R263K. Thus, HIV INSTI mutations, when inserted into SIV, resulted in a similar phenotype. These findings suggest that SIV and HIV may share similar resistance pathways profiles and that SIVmac239 could be a useful nonhuman primate model for studies of HIV resistance to INSTIs. IMPORTANCE: The goal of our project was to establish whether drug resistance against integrase inhibitors in SIV are likely to be the same as those responsible for drug resistance in HIV. Our data answer this question in the affirmative and show that SIV can probably serve as a good animal model for studies of INSTIs and as an early indicator for possible emergent mutations that may cause treatment failure. An SIV-primate model remains an invaluable tool for investigating questions related to the potential role of INSTIs in HIV therapy, transmission, and pathogenesis, and the present study will facilitate each of the above.
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Antivirais/farmacologia , Farmacorresistência Viral , Integrase de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/enzimologia , Substituição de Aminoácidos , Animais , Células Cultivadas , Integrase de HIV/metabolismo , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Macaca mulatta , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxazinas , Piperazinas , Piridonas , Pirrolidinonas/farmacologia , Quinolonas/farmacologia , Raltegravir Potássico , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Replicação ViralRESUMO
HIV resistance to current anti-HIV drugs and drug toxicity have created a need for new anti-HIV agents. We have examined and characterized a synthetic resveratrol analog, termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (M8), for potential anti-HIV activity. Here, we demonstrate that M8 possesses potent anti-HIV activity against several HIV variants with EC50 values in the low µM range. M8 was shown to act at a very early step of HIV entry prior to fusion to host cells. These results demonstrate that this novel resveratrol derivative possesses potent anti-HIV-1 activity and may have a mechanism of action that is different from current anti-HIV-1 drugs including entry inhibitors. Further structure-guided design might lead to the development of newer improved resveratrol derivatives that could have value either in therapy or as microbicides to prevent the sexual transmission of HIV-1.
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Antivirais/farmacologia , HIV-1/efeitos dos fármacos , Pirogalol/análogos & derivados , Estilbenos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Pirogalol/farmacologiaRESUMO
BACKGROUND: First-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL. RESULTS: Using biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation. CONCLUSIONS: Since the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.
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Farmacorresistência Viral , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Mutação de Sentido Incorreto , Replicação Viral , Substituição de Aminoácidos , Fármacos Anti-HIV/metabolismo , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Oxazinas , Piperazinas , PiridonasRESUMO
HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.
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Inibidores de Integrase de HIV/química , Integrase de HIV/química , HIV-1/química , Pirrolidinonas/química , Região 3'-Flanqueadora , Sítios de Ligação , Ligação Competitiva , Clonagem Molecular , Farmacorresistência Viral , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Integrase de HIV/classificação , Integrase de HIV/genética , HIV-1/enzimologia , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Raltegravir Potássico , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. METHODS: We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. RESULTS: We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. CONCLUSIONS: The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic.
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Farmacorresistência Viral/genética , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mutação , Replicação Viral/efeitos dos fármacos , Substituição de Aminoácidos , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Integrase de HIV/química , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/química , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Oxazinas , Piperazinas , Ligação Proteica , Piridonas , Integração Viral/genéticaRESUMO
The immunological signatures driving the severity of coronavirus disease 19 (COVID-19) in Ghanaians remain poorly understood. We performed bulk transcriptome sequencing of nasopharyngeal samples from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected Ghanaians with mild and severe COVID-19, as well as healthy controls to characterize immune signatures at the primary SARS-CoV-2 infection site and identify drivers of disease severity. Generally, a heightened antiviral response was observed in SARS-CoV-2-infected Ghanaians compared with uninfected controls. COVID-19 severity was associated with immune suppression, overexpression of proinflammatory cytokines, including CRNN, IL1A, S100A7, and IL23A, and activation of pathways involved in keratinocyte proliferation. SAMD9L was among the differentially regulated interferon-stimulated genes in our mild and severe disease cohorts, suggesting that it may play a critical role in SARS-CoV-2 pathogenesis. By comparing our data with a publicly available dataset from a non-African (Indians) (GSE166530), an elevated expression of antiviral response-related genes was noted in COVID-19-infected Ghanaians. Overall, the study describes immune signatures driving COVID-19 severity in Ghanaians and identifies immune drivers that could serve as potential prognostic markers for future outbreaks or pandemics. It further provides important preliminary evidence suggesting differences in antiviral response at the upper respiratory interface in sub-Saharan Africans (Ghanaians) and non-Africans, which could be contributing to the differences in disease outcomes. Further studies using larger datasets from different populations will expand on these findings.
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COVID-19 , Humanos , COVID-19/genética , Gana , SARS-CoV-2 , Perfilação da Expressão Gênica , Epitélio , Antivirais , TranscriptomaRESUMO
Background: The highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. Methods: To generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. Results: We successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. Significance: This assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.
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Ghana and other parts of West Africa have experienced lower COVID-19 mortality rates than other regions. This phenomenon has been hypothesized to be associated with previous exposure to infections such as malaria. This study investigated the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the influence of previous malaria exposure. Blood samples were collected from individuals with asymptomatic or symptomatic COVID-19 (n = 217). A variety of assays were used to characterize the SARS-CoV-2-specific immune response, and malaria exposure was quantified using Plasmodium falciparum ELISA. The study found evidence of attenuated immune responses to COVID-19 among asymptomatic individuals, with elevated proportions of non-classical monocytes and greater memory B cell activation. Symptomatic patients displayed higher P. falciparum-specific T cell recall immune responses, whereas asymptomatic individuals demonstrated elevated P. falciparum antibody levels. Summarily, this study suggests that P. falciparum exposure-associated immune modulation may contribute to reduced severity of SARS-CoV-2 infection among people living in malaria-endemic regions.
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BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown. RESULTS: Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays. CONCLUSION: These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.
Assuntos
HIV-1/imunologia , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/imunologia , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Linhagem Celular , Cromatografia Líquida , Estabilidade Enzimática , Humanos , Espectrometria de Massas , Metilação , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genéticaRESUMO
BACKGROUND: Clinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naïve patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naïve patients to date. RESULTS: We now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication. CONCLUSIONS: Since H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Replicação Viral , Integrase de HIV/genética , HIV-1/enzimologia , Humanos , Mutação de Sentido Incorreto , Oxazinas , Piperazinas , Mutação Puntual , Piridonas , Seleção GenéticaRESUMO
Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3' processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility.
Assuntos
Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Farmacorresistência Viral/genética , Ativação Enzimática/efeitos dos fármacos , Integrase de HIV/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Oxazinas , Piperazinas , PiridonasRESUMO
PURPOSE OF REVIEW: Integrase strand transfer inhibitors (INSTIs) have become a key component of antiretroviral therapy since the approval of twice-daily raltegravir in 2007 and the more recent approval of elvitegravir in 2012. At the same time, a third compound, dolutegravir, is in late-phase clinical trials, being tested as part of a multidrug once-daily formulation comprising this INSTI and two other antiretroviral (ARV) drugs. This review focuses on the factors leading to the development of drug resistance mutations (DRMs) against INSTIs, evidence of cross-resistance among them, and the results of regimen simplification in regard to this topic. RECENT FINDINGS: Sequencing data show that DRMs are highly dynamic in patients failing INSTI therapy. Considerations of viral fitness and drug resistance can together determine the evolution of drug resistance mutations, and in this regard the Y143 and Q148 pathways are superior to the N155 pathway in the promotion of resistance. Preventing the emergence of DRMs requires that effective reverse transcriptase or other inhibitors be used together with INSTIs and that high-level adherence to treatment be maintained. SUMMARY: Because of the susceptibility to drug resistance, INSTIs should always be used together with other effective ARV drugs.
Assuntos
Farmacorresistência Viral Múltipla/genética , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , HIV-1/genética , Mutação , Análise Mutacional de DNA , Combinação de Medicamentos , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HumanosRESUMO
OBJECTIVES: HIV-1 protease inhibitors (PIs) are key components of HIV therapy. PL-100 is a novel lysine sulphonamide that demonstrates potent antiviral activity against multiresistant HIV-1 strains as well as a higher genetic barrier for development of resistance mutations compared with first-generation PIs. In the present study, we compared the antiviral activity of PL-100 against HIV-1 subtype B with that of darunavir. METHODS: We used tissue culture experiments to evaluate the in vitro development of resistance to PL-100 and tested the antiviral activity of several clinically approved PIs against PL-100-selected resistant variants. Structural modelling was also used to compare the binding of PL-100 and darunavir to the HIV-1 protease (PR) enzyme. RESULTS: PL-100-resistant variants that emerged within 8-48 weeks showed low- to high-level resistance (3.5- to 21.6-fold) to PL-100, but commonly retained susceptibility to darunavir, which, in contrast, did not select for resistance mutations over a period of 40 weeks. Structural modelling demonstrated that binding of PL-100 was predominantly based on polar interactions and delocalized hydrophobic interactions through its diphenyl groups, while darunavir has numerous interactions with PR that include hydrogen bonding to PR backbone oxygens at amino acid positions A28, D29 and D30 via di-tetrahydrofuran (di-THF) groups. CONCLUSIONS: Hydrogen-bonding contacts and the di-THF group in darunavir, as well as the hydrophobic nature of PL-100, contribute to PI binding and a high genetic barrier for resistance. Redesigning the structure of PL-100 to include a di-THF group might improve it.