RESUMEN
Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Núcleo Celular/metabolismo , Infecciones por VIH/prevención & control , VIH-1/inmunología , Macrófagos/inmunología , Proteínas Nucleares/metabolismo , Factor de Transcripción Sp1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Núcleo Celular/genética , ADN Viral/genética , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Células Hep G2 , Humanos , Inmunidad Innata/inmunología , Inflamasomas/genética , Inflamasomas/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/genética , Factor de Transcripción Sp1/genética , Replicación ViralRESUMEN
Human cytomegalovirus (HCMV) persistently infects 40% to 100% of the human population worldwide. Experimental and clinical evidence indicates that humoral immunity to HCMV plays an important role in restricting virus dissemination and protecting the infected host from disease. Specific immunoglobulin preparations from pooled plasma of adults selected for high titers of HCMV antibodies have been used for the prevention of CMV disease in transplant recipients and pregnant women. Even though incubation of HCMV particles with these preparations leads to the neutralization of viral infectivity, it is still unclear whether the antibody-treated HCMV particles (referred to here as HCMV-Ab) enter the cells and modulate antiviral immune responses. Here we demonstrate that HCMV-Ab did enter macrophages. HCMV-Ab did not initiate the expression of immediate early antigens (IEAs) in macrophages, but they induced an antiviral state and rendered the cells less susceptible to HCMV infection upon challenge. Resistance to HCMV infection seemed to be due to the activation of intrinsic restriction factors and was independent of interferons. In contrast to actively infected cells, autologous NK cells did not degranulate against HCMV-Ab-treated macrophages, suggesting that these cells may not be eliminated by innate effector cells. Interestingly, HCMV-Ab-treated macrophages stimulated the proliferation of autologous adaptive CD4+ and CD8+ T cells. Our findings not only expand the current knowledge on virus-antibody immunity but may also be relevant for future vaccination strategies.IMPORTANCE Human cytomegalovirus (HCMV), a common herpesvirus, establishes benign but persistent infections in immunocompetent hosts. However, in subjects with an immature or dysfunctional immune system, HCMV is a major cause of morbidity and mortality. Passive immunization has been used in different clinical settings with variable clinical results. Intravenous hyperimmune globulin preparations (IVIg) are obtained from pooled adult human plasma selected for high anti-CMV antibody titers. While HCMV neutralization can be shown in vitro using different systems, data are lacking regarding the cross-influence of IVIg administration on the cellular immune responses. The aim of this study was to evaluate the effects of IVIg on distinct components of the immune response against HCMV, including antigen presentation by macrophages, degranulation of innate natural killer cells, and proliferation of adaptive CD4+ and CD8+ T cells.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Inmunidad Celular , Macrófagos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Infecciones por Citomegalovirus/patología , Femenino , Humanos , Inmunidad Innata , Macrófagos/patología , Macrófagos/virología , MasculinoRESUMEN
Infectious diseases and cancers are leading causes of death and pose major challenges to public health. The human peptidome encompasses millions of compounds that display an enormous structural and functional diversity and represents an excellent source for the discovery of endogenous agents with antimicrobial and/or anticancer activity. Here, we discuss how to exploit the human peptidome for novel antimicrobial and anticancer agents through the generation of peptide libraries from human body fluids and tissues and stepwise purification of bioactive compounds.
Asunto(s)
Antiinfecciosos/farmacología , Antineoplásicos/farmacología , Descubrimiento de Drogas/métodos , Biblioteca de Péptidos , Péptidos/farmacología , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Enfermedades Transmisibles/tratamiento farmacológico , Humanos , Neoplasias/tratamiento farmacológico , Péptidos/química , Péptidos/aislamiento & purificación , Proteómica/métodosRESUMEN
PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.
Asunto(s)
VIH-1 , Virus de la Leucemia Murina , Fosfoproteínas , Animales , Ratones , Humanos , VIH-1/inmunología , VIH-1/genética , Virus de la Leucemia Murina/genética , Virus de la Leucemia Murina/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/inmunología , Replicación Viral , Línea Celular , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/virologíaRESUMEN
Repeated emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased fitness underscores the value of rapid detection and characterization of new lineages. We have developed PyR0, a hierarchical Bayesian multinomial logistic regression model that infers relative prevalence of all viral lineages across geographic regions, detects lineages increasing in prevalence, and identifies mutations relevant to fitness. Applying PyR0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase fitness, including previously identified spike mutations and many nonspike mutations within the nucleocapsid and nonstructural proteins. PyR0 forecasts growth of new lineages from their mutational profile, ranks the fitness of lineages as new sequences become available, and prioritizes mutations of biological and public health concern for functional characterization.
Asunto(s)
COVID-19 , Aptitud Genética , SARS-CoV-2 , Teorema de Bayes , COVID-19/virología , Genoma Viral , Humanos , Mutación , Análisis de Regresión , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Repeated emergence of SARS-CoV-2 variants with increased fitness necessitates rapid detection and characterization of new lineages. To address this need, we developed PyR 0 , a hierarchical Bayesian multinomial logistic regression model that infers relative prevalence of all viral lineages across geographic regions, detects lineages increasing in prevalence, and identifies mutations relevant to fitness. Applying PyR 0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase fitness, including previously identified spike mutations and many non-spike mutations within the nucleocapsid and nonstructural proteins. PyR 0 forecasts growth of new lineages from their mutational profile, identifies viral lineages of concern as they emerge, and prioritizes mutations of biological and public health concern for functional characterization. ONE SENTENCE SUMMARY: A Bayesian hierarchical model of all SARS-CoV-2 viral genomes predicts lineage fitness and identifies associated mutations.
RESUMEN
IFI16 is an important player of the host intrinsic immune response. Among others, it has been reported to sense intermediate products of HIV-1 reverse transcription in the cytosol and to sequester the transcription factor Sp1 in the nucleus to attenuate viral gene expression. Here, we present three different methods to reduce IFI16 protein expression levels in HIV-1 primary target cells. These techniques can be adapted for the investigation of other cellular factors in primary macrophages and CD4+ T lymphocytes. For complete details on the use and execution of this protocol, please refer to Hotter et al. (2019) and Bosso et al. (2020).
Asunto(s)
Linfocitos T CD4-Positivos , Técnicas de Silenciamiento del Gen , Infecciones por VIH , VIH-1/metabolismo , Macrófagos , Proteínas Nucleares , Fosfoproteínas , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMEN
Polyoxometalates (POMs), molecular metal oxide anions, are inorganic clusters with promising antiviral activity. Herein we report increased anti-HIV-1 activity of a POM when electrostatically combined with organic counter-cations. To this end, Keggin-type cerium tungstate POMs have been combined with organic methyl-caffeinium (Caf) cations, and their cytotoxicity, antiviral activity and mode of action have been studied. The novel compound, Caf4 K[ß2 -CeSiW11 O39 ]×H2 O, exhibits sub-nanomolar antiviral activity and inhibits HIV-1 infectivity by acting on an early step of the viral infection cycle. This work demonstrates that combination of POM anions and organic bioactive cations can be a powerful new strategy to increase antiviral activity of these inorganic compounds.
Asunto(s)
Aniones/farmacología , Fármacos Anti-VIH/farmacología , Cafeína/farmacología , VIH/efectos de los fármacos , Polielectrolitos/farmacología , Aniones/síntesis química , Aniones/química , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Cafeína/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Polielectrolitos/síntesis química , Polielectrolitos/químicaRESUMEN
Subtype C is the most prevalent clade of human immunodeficiency virus type 1 (HIV-1) worldwide. The reasons for this are poorly understood. Here, we demonstrate that a characteristic additional third nuclear factor κB (NF-κB) binding site in the long terminal repeat (LTR) promoter allows subtype C HIV-1 strains to evade restriction by nuclear PYHIN proteins, which sequester the transcription factor Sp1. Further, other LTR alterations are responsible for rare PYHIN resistance of subtype B viruses. Resistance-conferring mutations generally reduce the dependency of HIV-1 on Sp1 for virus production and render LTR transcription highly responsive to stimulation by NF-κB/p65. A third NF-κB binding site increases infectious virus yield in primary CD4+ T cells in an γ-interferon-inducible protein 16 (IFI16)-dependent manner. Comprehensive sequence analyses suggest that the frequency of circulating PYHIN-resistant HIV-1 strains is increasing. Our finding that an additional NF-κB binding site in the LTR confers resistance to nuclear PYHIN proteins helps to explain the dominance of clade C HIV-1 strains.
Asunto(s)
VIH-1/genética , FN-kappa B/química , Proteínas Nucleares/metabolismo , Sitios de Unión , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Susceptibilidad a Enfermedades , Genotipo , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Humanos , FN-kappa B/metabolismo , Fosfoproteínas/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Secuencias Repetidas Terminales/genética , Replicación ViralRESUMEN
Innate immune sensors and restriction factors are cellular proteins that synergize to build an effective first line of defense against viral infections. Innate sensors are usually constitutively expressed and capable of detecting pathogen-associated molecular patterns (PAMPs) via specific pattern recognition receptors (PRRs) to stimulate the immune response. Restriction factors are frequently upregulated by interferons (IFNs) and may inhibit viral pathogens at essentially any stage of their replication cycle. Members of the Pyrin and hematopoietic interferon-inducible nuclear (HIN) domain (PYHIN) family have initially been recognized as important sensors of foreign nucleic acids and activators of the inflammasome and the IFN response. Accumulating evidence shows, however, that at least three of the four members of the human PYHIN family restrict viral pathogens independently of viral sensing and innate immune activation. In this review, we provide an overview on the role of human PYHIN proteins in the innate antiviral immune defense and on viral countermeasures.
Asunto(s)
Resistencia a la Enfermedad , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Virosis/metabolismo , Animales , Resistencia a la Enfermedad/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Familia de Multigenes , Proteínas Nucleares/genética , Virosis/inmunología , Virosis/virologíaRESUMEN
Restriction factors are structurally and functionally diverse cellular proteins that constitute a first line of defense against viral pathogens. Exceptions exist, but typically these proteins are upregulated by interferons (IFNs), target viral components, and are rapidly evolving due to the continuous virus-host arms race. Restriction factors may target HIV replication at essentially each step of the retroviral replication cycle, and the suppression of viral transcription and the degradation of viral RNA transcripts are emerging as major innate immune defense mechanisms. Recent data show that some antiviral factors, such as the tripartite motif-containing protein 22 (TRIM22) and the g-IFN-inducible protein 16 (IFI16), do not target HIV-1 itself but limit the availability of the cellular transcription factor specificity protein 1 (Sp1), which is critical for effective viral gene expression. In addition, several RNA-interacting cellular factors including RNAse L, the NEDD4-binding protein 1 (N4BP1), and the zinc finger antiviral protein (ZAP) have been identified as important immune effectors against HIV-1 that may be involved in the maintenance of the latent viral reservoirs, representing the major obstacle against viral elimination and cure. Here, we review recent findings on specific cellular antiviral factors targeting HIV-1 transcription or viral RNA transcripts and discuss their potential role in viral latency.
Asunto(s)
Infecciones por VIH/metabolismo , VIH-1/genética , ARN Viral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Replicación ViralRESUMEN
The interferon γ-inducible protein 16 (IFI16) is known as immune sensor of retroviral DNA intermediates. We show that IFI16 restricts HIV-1 independently of immune sensing by binding and inhibiting the host transcription factor Sp1 that drives viral gene expression. This antiretroviral activity and ability to bind Sp1 require the N-terminal pyrin domain and nuclear localization of IFI16, but not the HIN domains involved in DNA binding. Highly prevalent clade C HIV-1 strains are more resistant to IFI16 and less dependent on Sp1 than other HIV-1 subtypes. Furthermore, inhibition of Sp1 by IFI16 or pharmacologically by Mithramycin A suppresses reactivation of latent HIV-1 in CD4+ T cells. Finally, IFI16 also inhibits retrotransposition of LINE-1, known to engage Sp1, and murine IFI16 homologs restrict Friend retrovirus replication in mice. Thus, IFI16 restricts retroviruses and retrotransposons by interfering with Sp1-dependent gene expression, and evasion from this restriction may facilitate spread of HIV-1 subtype C.