RESUMEN
The susceptibility of genetically divergent HIV-1 strains (HIV-1 non-M) from groups O, N, and P to the CCR5 co-receptor antagonist, maraviroc (MVC), was investigated among a large panel of 45 clinical strains, representative of the viral genetic diversity. The results were compared to the reference strains of HIV-1 group M (HIV-1/M) with known tropism. Among the non-M strains, a wide range of phenotypic susceptibilities to MVC were observed. The large majority of HIV-1/O strains (40/42) displayed a high susceptibility to MVC, with median and mean IC50 values of 1.23 and 1.33 nM, respectively, similar to the HIV-1/M R5 strain (1.89 nM). However, the two remaining HIV-1/O strains exhibited a lower susceptibility (IC50 at 482 and 496 nM), in accordance with their dual/mixed (DM) tropism. Interestingly, the two HIV-1/N strains demonstrated varying susceptibility patterns, despite always having relatively low IC50 values (2.87 and 47.5 nM). This emphasized the complexity of determining susceptibility solely based on IC50 values. Our study examined the susceptibility of all HIV-1 non-M groups to MVC and correlated these findings with virus tropism (X4, R5, or DM). The results confirm the critical significance of tropism determination before initiating MVC treatment in patients infected with HIV-1 non-M. Furthermore, we advocate for the consideration of additional parameters, such as the slope of inhibition curves, to provide a more thorough characterization of phenotypic susceptibility profiles. IMPORTANCE: Unlike HIV-1 group M, the scarcity of studies on HIV-1 non-M groups (O, N, and P) presents challenges in understanding their susceptibility to antiretroviral treatments, particularly due to their natural resistance to non-nucleoside reverse transcriptase inhibitors. The TROPI-CO study logically complements our prior investigations into integrase inhibitors and anti-gp120 efficacy. The largest panel of 45 non-M strains existing so far yielded valuable results on maraviroc (MVC) susceptibility. The significant variations in MVC IC50 reveal a spectrum of susceptibilities, with most strains displaying R5 tropism. Notably, the absence of MVC-resistant strains suggests a potential therapeutic avenue. The study also employs a robust novel cell-based phenotropism assay and identifies distinct groups of susceptibilities based on inhibition curve slopes. Our findings emphasize the importance of determining tropism before initiating MVC and provide crucial insights for selecting effective therapeutic strategies in the delicate context of HIV-1 non-M infections.
Asunto(s)
Antagonistas de los Receptores CCR5 , Infecciones por VIH , VIH-1 , Maraviroc , Tropismo Viral , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-1/fisiología , Maraviroc/farmacología , Humanos , Antagonistas de los Receptores CCR5/farmacología , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , Concentración 50 Inhibidora , Triazoles/farmacología , Fenotipo , Pruebas de Sensibilidad Microbiana , Receptores CCR5/metabolismo , Receptores CCR5/genética , Fármacos Anti-VIH/farmacología , Ciclohexanos/farmacología , Farmacorresistencia Viral/genética , Inhibidores de Fusión de VIH/farmacologíaRESUMEN
The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , TYK2 Quinasa , Garrapatas , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo , Interferones/metabolismo , Garrapatas/metabolismo , TYK2 Quinasa/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , HumanosRESUMEN
Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.
Asunto(s)
ARN Helicasas DEAD-box , VIH-1 , Virus ARN Monocatenarios Positivos , Replicación Viral , Humanos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , VIH-1/fisiología , Virus ARN Monocatenarios Positivos/fisiología , SARS-CoV-2/fisiologíaRESUMEN
OBJECTIVES: HIV-1 group O (HIV-1/O) is one of the four HIV-1 groups and is endemic in Cameroon, representing 1% of HIV-1 infections in the population. Around 50% of the strains of this group naturally show a mutation (Y181C) providing them with resistance to NNRTIs and making therapeutic management more difficult. Today, the WHO recommends the use of integrase strand transfer inhibitors (INSTIs) as first-line treatment. Bictegravir and cabotegravir are the two most recent INSTIs. Because of the genetic polymorphism of HIV-1/O, studies are required to evaluate their phenotypic susceptibility to these two drugs. PATIENTS AND METHODS: We performed a phenotypic study on a large panel including 41 HIV-1/O clinical isolates and other rare non-group M HIV-1 (2 HIV-1/N and 1 HIV-1/P) to evaluate in vitro susceptibility to bictegravir and cabotegravir. RESULTS: The results showed an overall susceptibility of non-group M strains to the two drugs compared with HIV-1 group M. There was no difference between the mean (min-max) IC50 of HIV-1/M [1.86 (0.93-4.12) and 5.24 (1.76-12.41) nM for bictegravir and cabotegravir, respectively] and HIV-1/non-M [2.17 (0.03-9.47) and 4.88 (0.02-15.64) nM for bictegravir and cabotegravir, respectively]. However, we found a significant difference between IC50 values for bictegravir and cabotegravir in the whole panel (P value <â0.001). CONCLUSIONS: This study has shown encouraging results regarding the clinical use of these drugs in HIV-1/non-M-infected patients, which will need to be confirmed with clinical data.
Asunto(s)
Infecciones por VIH , Inhibidores de Integrasa VIH , Integrasa de VIH , VIH-1 , Amidas , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/uso terapéutico , VIH-1/genética , Compuestos Heterocíclicos con 3 Anillos , Humanos , Piperazinas , Piridonas/farmacologíaRESUMEN
BACKGROUND: Reverse transcription-quantitative PCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method but exhibits imperfect sensitivity. METHODS: We developed a multiplex reverse transcription-digital droplet PCR (RT-ddPCR) assay, targeting 6 SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate SARS-CoV2 infection. RESULTS: For the nasopharyngeal swab samples, the results obtained using the 6-plex RT-ddPCR and RT-qPCR assays were all concordant. The 6-plex RT-ddPCR assay was more sensitive than RT-qPCR (85% versus 62%) on saliva samples from patients with positive nasopharyngeal swabs. CONCLUSION: Multiplex RT-ddPCR represents an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results. It can also be applied to saliva for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Nasofaringe/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Saliva/virología , COVID-19/sangre , Humanos , Límite de Detección , ARN Viral/sangre , Reproducibilidad de los Resultados , SARS-CoV-2/química , Manejo de Especímenes/instrumentaciónRESUMEN
Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response.IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.
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Proteína 58 DEAD Box/metabolismo , Virus de la Fiebre Amarilla/metabolismo , eIF-2 Quinasa/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinoma Hepatocelular , Línea Celular , Línea Celular Tumoral , Citocinas/metabolismo , Proteína 58 DEAD Box/deficiencia , Proteína 58 DEAD Box/genética , ADN Helicasas/genética , Técnicas de Silenciamiento del Gen , Haplorrinos , Hepatocitos/virología , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Helicasas/genética , Proteínas con Motivos de Reconocimiento de ARN/genética , ARN Interferente Pequeño , ARN Viral/genética , Proteínas de Unión al ARN/genética , Receptores Inmunológicos , Antígeno Intracelular 1 de las Células T/genética , Transcriptoma , eIF-2 Quinasa/genéticaRESUMEN
BACKGROUND: To be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus, which includes human viruses transmitted by Aedes mosquitoes, such as dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. CONCLUSIONS/SIGNIFICANCE: The two barriers associated with Ae. aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector-pathogen interactions and may also aid in the development of non-transmissible live virus vaccines.
Asunto(s)
Aedes/virología , Tracto Gastrointestinal/virología , Replicación Viral/efectos de los fármacos , Vacuna contra la Fiebre Amarilla/farmacología , Virus de la Fiebre Amarilla/efectos de los fármacos , Virus de la Fiebre Amarilla/crecimiento & desarrollo , Animales , Línea Celular , Tracto Gastrointestinal/fisiología , Interacciones Huésped-Patógeno/fisiología , Mosquitos Vectores , Glándulas Salivales , Vacunas Atenuadas , Carga Viral , Virus de la Fiebre Amarilla/genéticaRESUMEN
Flaviviruses, such as dengue (DENV), West Nile (WNV), yellow fever (YFV) and Zika (ZIKV) viruses, are mosquito-borne pathogens that present a major risk to global public health. To identify host factors that promote flavivirus replication, we performed a genome-wide gain-of-function cDNA screen for human genes that enhance the replication of flavivirus reporter particles in human cells. The screen recovered seventeen potential host proteins that promote viral replication, including the previously known dolichyl-diphosphooligosaccharide--protein glycosyltransferase non-catalytic subunit (DDOST). Using silencing approaches, we validated the role of four candidates in YFV and WNV replication: ribosomal protein L19 (RPL19), ribosomal protein S3 (RPS3), DDOST and importin 9 (IPO9). Applying a panel of virological, biochemical and microscopic methods, we validated further the role of RPL19 and DDOST as host factors required for optimal replication of YFV, WNV and ZIKV. The genome-wide gain-of-function screen is thus a valid approach to advance our understanding of flavivirus replication.
Asunto(s)
Flavivirus/genética , Interacciones Huésped-Patógeno/genética , Replicación Viral , Animales , Línea Celular , Culicidae/virología , Virus del Dengue/genética , Flavivirus/fisiología , Biblioteca de Genes , Hexosiltransferasas/genética , Humanos , Carioferinas/genética , Proteínas de la Membrana/genética , Proteínas Ribosómicas/genética , Virus del Nilo Occidental/genética , Virus Zika/genéticaRESUMEN
The flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. In vitro production of fragments of the DENV genome confirmed the NGS data and revealed that the 5' end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Virus del Dengue/genética , Genoma Viral , Virus Zika/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Dengue/virología , Células HEK293 , Humanos , Receptores Inmunológicos , Replicación Viral/fisiologíaRESUMEN
Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact- dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.