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1.
Nat Immunol ; 16(10): 1025-33, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26343537

RESUMEN

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.


Asunto(s)
ADN Complementario/química , ADN Viral/química , ADN Viral/inmunología , VIH-1/genética , VIH-1/inmunología , Interferón-alfa/inmunología , Nucleotidiltransferasas/genética , Animales , Línea Celular , Células Cultivadas , ADN Complementario/genética , ADN Complementario/inmunología , ADN Viral/genética , Células HEK293 , Humanos , Inmunización , Ratones
2.
EMBO J ; 41(17): e111608, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35833542

RESUMEN

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Antivirales/farmacología , Inflamación/tratamiento farmacológico , Metiltransferasas/metabolismo , Ratones , Caperuzas de ARN/metabolismo , ARN Viral/genética , Ribosa , Proteínas no Estructurales Virales/genética
3.
J Am Soc Nephrol ; 2024 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-39441656

RESUMEN

BACKGROUND: The renal epithelial sodium channel (ENaC) is essential for sodium balance and blood pressure control. ENaC undergoes complex proteolytic activation by not yet clearly identified tubular proteases. Here, we examined a potential role of transmembrane serine protease 2 (TMPRSS2). METHODS: Murine ENaC and TMPRSS2 were (co-)expressed in Xenopus laevis oocytes. ENaC cleavage and function were studied in TMPRSS2-deficient murine cortical collecting duct (mCCDcl1) cells and TMPRSS2-knockout (Tmprss2-/-) mice. Short-circuit currents (ISC) were measured to assess ENaC-mediated transepithelial sodium transport of mCCDcl1 cells. The mCCDcl1 cell transcriptome was studied using RNA sequencing. The effect of low-sodium diet with or without high potassium were compared in Tmprss2-/- and wildtype mice using metabolic cages. ENaC-mediated whole-cell currents were recorded from microdissected tubules of Tmprss2-/- and wildtype mice. RESULTS: In oocytes, co-expression of murine TMPRSS2 and ENaC resulted in fully cleaved γ-ENaC and ∼2-fold stimulation of ENaC currents. High baseline expression of TMPRSS2 was detected in mCCDcl1 cells without a stimulatory effect of aldosterone on its function or transcription. TMPRSS2 knockout in mCCDcl1 cells compromised γ-ENaC cleavage and reduced baseline and aldosterone-stimulated ISC which could be rescued by chymotrypsin. A compensatory transcriptional upregulation of other proteases was not observed. Tmprss2-/- mice kept on standard diet exhibited no apparent phenotype, but renal γ-ENaC cleavage was altered. In response to a low-salt diet, particularly with high potassium intake, Tmprss2-/- mice increased plasma aldosterone significantly more than wildtype mice to achieve a similar reduction of renal sodium excretion. Importantly, the stimulatory effect of trypsin on renal tubular ENaC currents was much more pronounced in Tmprss2-/- mice than that in wildtype mice. This indicated the presence of incompletely cleaved and less active channels at the cell surface of TMPRSS2-deficient tubular epithelial cells. CONCLUSIONS: TMPRSS2 contributes to proteolytic ENaC activation in mouse kidney in vivo.

4.
EMBO Rep ; 23(12): e55648, 2022 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-36285486

RESUMEN

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , ARN , Metiltransferasas/genética , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras Transductoras de Señales/genética
5.
J Biol Chem ; 298(6): 102004, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35504352

RESUMEN

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, ß-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αßγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C-Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.


Asunto(s)
Canales Epiteliales de Sodio , Oocitos , Serina Endopeptidasas , Animales , Canales Epiteliales de Sodio/metabolismo , Humanos , Transporte Iónico/fisiología , Oocitos/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Xenopus laevis/genética
6.
Proc Natl Acad Sci U S A ; 117(30): 17965-17976, 2020 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-32651277

RESUMEN

Mobile genetic elements have significantly shaped our genomic landscape. LINE-1 retroelements are the only autonomously active elements left in the human genome. Since new insertions can have detrimental consequences, cells need to efficiently control LINE-1 retrotransposition. Here, we demonstrate that the intrinsic immune factor TRIM5α senses and restricts LINE-1 retroelements. Previously, rhesus TRIM5α has been shown to efficiently block HIV-1 replication, while human TRIM5α was found to be less active. Surprisingly, we found that both human and rhesus TRIM5α efficiently repress human LINE-1 retrotransposition. TRIM5α interacts with LINE-1 ribonucleoprotein complexes in the cytoplasm, which is essential for restriction. In line with its postulated role as pattern recognition receptor, we show that TRIM5α also induces innate immune signaling upon interaction with LINE-1 ribonucleoprotein complexes. The signaling events activate the transcription factors AP-1 and NF-κB, leading to the down-regulation of LINE-1 promoter activity. Together, our findings identify LINE-1 as important target of human TRIM5α, which restricts and senses LINE-1 via two distinct mechanisms. Our results corroborate TRIM5α as pattern recognition receptor and shed light on its previously undescribed activity against mobile genetic elements, such as LINE-1, to protect the integrity of our genome.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Factores de Restricción Antivirales , Expresión Génica , Genes Reporteros , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Macaca mulatta , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Transducción de Señal , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética
7.
PLoS Pathog ; 16(8): e1008752, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32760121

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 Viral
8.
Retrovirology ; 12: 103, 2015 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-26667483

RESUMEN

BACKGROUND: Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies. RESULTS: We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA. CONCLUSION: Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.


Asunto(s)
VIH-1/fisiología , Virus de la Leucemia Murina/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Infecciones por Retroviridae/virología , Transcripción Reversa , Animales , Línea Celular , Células Cultivadas , Humanos , Macrófagos/virología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Fosforilación , ARN Viral/genética , ARN Viral/metabolismo , Proteína 1 que Contiene Dominios SAM y HD , Treonina/fisiología , Replicación Viral
9.
Retrovirology ; 10: 26, 2013 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-23497255

RESUMEN

BACKGROUND: SAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined. RESULTS: We show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction. CONCLUSION: The results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.


Asunto(s)
Macrófagos/virología , Proteínas de Unión al GTP Monoméricas/farmacología , Células Mieloides/virología , Retroviridae/efectos de los fármacos , Retroviridae/patogenicidad , Replicación Viral/efectos de los fármacos , Línea Celular , Células Dendríticas/metabolismo , Células Dendríticas/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Células Jurkat , Macrófagos/inmunología , Proteínas de Unión al GTP Monoméricas/metabolismo , Células Mieloides/metabolismo , Retroviridae/clasificación , Retroviridae/fisiología , Proteína 1 que Contiene Dominios SAM y HD
10.
Front Immunol ; 14: 1168589, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37180175

RESUMEN

The intracellular restriction factor TRIM5α inhibits endogenous LINE-1 retroelements. It induces innate immune signaling cascades upon sensing of cytoplasmic LINE-1 complexes, thereby underlining its importance for protecting the human genome from harmful retrotransposition events. Here, we show that a frequent SNP within the RING domain of TRIM5α, resulting in the variant H43Y, blocks LINE-1 retrotransposition with higher efficiency compared to TRIM5α WT. Upon sensing of LINE-1 complexes in the cytoplasm, TRIM5α H43Y activates both NF-κB and AP-1 signaling pathways more potently than TRIM5α WT, triggering a strong block of the LINE-1 promoter. Interestingly, the H43Y allele lost its antiviral function suggesting that its enhanced activity against endogenous LINE-1 elements is the driving force behind its maintenance within the population. Thus, our study suggests that the H43Y variant of the restriction factor and sensor TRIM5α persists within the human population since it preserves our genome from uncontrolled LINE-1 retrotransposition with higher efficiency.


Asunto(s)
Elementos de Nucleótido Esparcido Largo , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Factores de Restricción Antivirales , Inmunidad Innata/genética
11.
J Med Virol ; 83(11): 1938-50, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21915869

RESUMEN

Herpesvirus saimiri (HVS) causes acute lymphoma and leukemia upon experimental infection of various monkey species. HVS strain C488 is also capable of transforming human T-lymphocytes to stable growth in culture. The most susceptible species for oncogenesis are New World primates, in particular the cottontop tamarin (Saguinus oedipus). However, Old World monkeys such as macaques are the most used animal model for the close-to-human situation. The limited data on HVS infection in Old World monkeys prompted us to investigate susceptibility to infection and disease induction by HVS in macaques. After having established that rhesus macaques can be infected productively, and that rhesus T-cells can be transformed in vivo by HVS, we observed induction of lymphoma in all inoculated animals. Pre-existing humoral immunity in part of the rhesus colony capable of blocking HVS infection could be overcome by preselecting rhesus macaques for lack of this immunity of unknown origin. HVS infection of rhesus macaques as compared to that of New World monkeys has the advantages that disease progression is more prolonged, and larger blood volumes can be collected, which allows more extended analyses. Also, rhesus monkeys are the best immunologically and immunogenetically characterized primate species next to humans. This model could be useful for the evaluation of candidate tumor vaccines and to test novel approaches for cancer immunotherapy. In addition, HVS infection of macaques could eventually be useful as a surrogate model to address certain questions in rhadinovirus-induced human cancer such as effusion lymphoma or Kaposi's sarcoma.


Asunto(s)
Transformación Celular Viral , Modelos Animales de Enfermedad , Infecciones por Herpesviridae/patología , Herpesvirus Saimiriino 2/patogenicidad , Linfoma/patología , Linfocitos T/virología , Infecciones Tumorales por Virus/patología , Animales , Femenino , Linfoma/virología , Macaca mulatta , Masculino , Rhadinovirus/patogenicidad
12.
Nat Microbiol ; 4(12): 2273-2284, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31548683

RESUMEN

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.


Asunto(s)
Muromegalovirus/efectos de los fármacos , Fosfotransferasas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/antagonistas & inhibidores , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Animales , Antivirales/farmacología , Factores Estimulantes de Colonias/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/virología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/enzimología , Muromegalovirus/crecimiento & desarrollo , Células 3T3 NIH , Fosforilación , Proteínas Recombinantes , Proteína 1 que Contiene Dominios SAM y HD/genética , Transcriptoma , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacos
13.
Cell Host Microbe ; 25(6): 858-872.e13, 2019 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-31175045

RESUMEN

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.


Asunto(s)
VIH-1/inmunología , Factores Inmunológicos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factor de Transcripción Sp1/antagonistas & inhibidores , Transcripción Genética , Activación Viral , Latencia del Virus , Animales , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Genotipo , VIH-1/clasificación , VIH-1/genética , VIH-1/crecimiento & desarrollo , Ratones
14.
Mob DNA ; 9: 11, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29610582

RESUMEN

BACKGROUND: The restriction factor SAMHD1 regulates intracellular nucleotide level by degrading dNTPs and blocks the replication of retroviruses and DNA viruses in non-cycling cells, like macrophages or dendritic cells. In patients, inactivating mutations in samhd1 are associated with the autoimmune disease Aicardi-Goutières Syndrome (AGS). The accumulation of intracellular nucleic acids derived from endogenous retroelements thriving in the absence of SAMHD1 has been discussed as potential trigger of the autoimmune reaction. In vitro, SAMHD1 has been found to restrict endogenous retroelements, like LINE-1 elements (L1). The mechanism, however, by which SAMHD1 blocks endogenous retroelements, is still unclear. RESULTS: Here, we show that SAMHD1 inhibits the replication of L1 and other endogenous retroelements in cycling cells. By applying GFP- and neomycin-based reporter assays we found that the anti-L1 activity of SAMHD1 is regulated by phosphorylation at threonine 592 (T592). Similar to the block of HIV, the cofactor binding site and the enzymatic active HD domain of SAMHD1 proofed to be essential for restriction of L1 elements. However, phosphorylation at T592 did not correlate with the dNTP hydrolase activity of SAMHD1 in cycling 293T cells suggesting an alternative mechanism of regulation. Interestingly, we found that SAMHD1 binds to ORF2 protein of L1 and that this interaction is regulated by T592 phosphorylation. Together with the finding that the block is also active in cycling cells, our results suggest that the SAMHD1-mediated inhibition of L1 is similar but not identical to HIV restriction. CONCLUSION: Our findings show conclusively that SAMHD1 restricts the replication of endogenous retroelements in vitro. The results suggest that SAMHD1 is important for maintaining genome integrity and support the idea of an enhanced replication of endogenous retroelements in the absence of SAMHD1 in vivo, potentially triggering autoimmune diseases like AGS. Our analysis also contributes to the better understanding of the activities of SAMHD1 in antiviral defense and nucleotide metabolism. The finding that the phosphorylation of SAMHD1 at T592 regulates its activity against retroelements but not necessarily intracellular dNTP level suggests that the dNTP hydrolase activity might not be the only function of SAMHD1 important for its antiviral activity and for controlling autoimmunity.

15.
J Virol Methods ; 141(1): 87-96, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17196267

RESUMEN

Performance of phenotypic assays and replication capacity assays require normalization of virus input. Therefore, quantitation of HIV-1 in supernatants to inoculate cell cultures is an important step. Since the gold standard for the determination of infectivity, the tissue culture infectious dose 50% (TCID50) is time-consuming, several other methods are in use. This study evaluated methods for the quantitation of drug resistant viruses in cell culture supernatants. The compared methods were based on the detection of viral structural components like genomic RNA or p24 antigen (CA-p24) (particle-based), the determination of reverse transcriptase (RT) activity, and methods based on the detection of viral infectivity like LTR-induced beta-galactosidase (beta-gal) activity and the TCID50 (infectivity-based). Significant correlations were observed between beta-gal activity and TCID50, and between CA-p24 and viral RNA. RT activity did not correlate with any other method. However, RT activity correlated significantly with infectivity when non-resistant subtype-B isolates were analyzed. In contrast to viral infectivity, CA-p24 exhibited a long half life and accumulated in cell culture, resulting in decreasing ratios of infectious virions to CA-p24 over time. As a consequence, relative replication capacities of drug resistant viruses were only determined reliably if the input virus was normalized according to infectivity. In conclusion, RT activity seems to be feasible for non-resistant subtype-B viruses but may be of limited use for non-B subtypes and for drug resistant viruses. Methods determining infectivity are most suitable for quantitation of cell culture inocula, whereas particle-based assays are more appropriate for quantitation of virus production during an experiment.


Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , Proteína p24 del Núcleo del VIH/análisis , Transcriptasa Inversa del VIH/análisis , VIH-1/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Estudios de Evaluación como Asunto , Genes Reporteros , Genoma Viral , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Luciferasas/metabolismo , Mediciones Luminiscentes , ARN Viral/análisis , beta-Galactosidasa/metabolismo
16.
Viruses ; 8(1)2015 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-26703718

RESUMEN

The promyelocytic leukemia protein (PML) is the main structural component of the nuclear matrix structures termed nuclear domain 10 (ND10) or PML nuclear bodies (PML-NBs). PML and ND10 structures have been shown to mediate an intrinsic immune response against a variety of different viruses. Their role during retroviral replication, however, is still controversially discussed. In this study, we analyzed the role of PML and the ND10 components Daxx and Sp100 during retroviral replication in different cell types. Using cell lines exhibiting a shRNA-mediated knockdown, we found that PML, but not Daxx or Sp100, inhibits HIV and other retroviruses in a cell type-dependent manner. The PML-mediated block to retroviral infection was active in primary human fibroblasts and murine embryonic fibroblasts but absent from T cells and myeloid cell lines. Quantitative PCR analysis of HIV cDNA in infected cells revealed that PML restricts infection at the level of reverse transcription. Our findings shed light on the controversial role of PML during retroviral infection and show that PML contributes to the intrinsic restriction of retroviral infections in a cell type-dependent manner.


Asunto(s)
Infecciones por VIH/inmunología , VIH-1/fisiología , Proteínas Nucleares/inmunología , Factores de Transcripción/inmunología , Proteínas Supresoras de Tumor/inmunología , Animales , Fibroblastos/inmunología , Fibroblastos/virología , Infecciones por VIH/genética , Infecciones por VIH/virología , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Especificidad de la Especie , Linfocitos T/inmunología , Linfocitos T/virología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética
17.
Exp Neurol ; 261: 685-97, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25150097

RESUMEN

Despite highly active antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) are still highly prevalent. Direct neurotoxicity of microglia activated by HIV-infected monocytes independent from viral replication may account for this observation. To investigate underlying molecular and viral determinants, human monocytoid cells (U937) transduced with HIV-particles were co-cultured with primary human microglia or astrocytes. Using genetically-engineered HIV-particles key steps of infection were examined. Levels of pro-inflammatory/neurotoxic cytokines were investigated in co-culture supernatants by flow cytometry. Neurotoxicity mediated by the supernatants was analysed using primary cortical rat neurons. To corroborate our findings, cytokine profiles in cerebrospinal fluid (CSF) of neuropsychologically asymptomatic HIV positive (HIV(+)) patients (n=45) were correlated with neurofilament H (NfH) as surrogate of neuronal/axonal degeneration. In contrast to direct exposure of HIV to microglia, only the presence of HIV-transduced monocytoid cells strongly activated human microglia as evidenced by enhanced secretion of CXCL10, CCL5, CCL2, and IL-6 (1.3-7.1-fold; p<0.01) leading to two-fold increased neurotoxicity (p<0.001). In direct comparison, astrocyte activation by HIV-transduced monocytoid cells was limited. Using different mutant HIV-particles we show that the presence of cytoplasmic HIV-RNA in monocytoid cells is the viral determinant for this unique microglial activation pattern and subsequent neuronal cell death; reverse transcription and expression of viral genes were not essential. In CSF of presymptomatic HIV(+) patients, CXCL10, CCL5 and IL-6 were correlated with NfH as surrogate marker of neurodegeneration as well as CSF-pleocytosis. In conclusion, cytosolic viral RNA in monocytes is mandatory for subsequent microglial activation and neurotoxicity; activated astrocytes may augment neuroinflammation. In addition, neuroinflammation and neurodegeneration occur even in preclinical HIV(+) patients and are associated with cytokines regulated in vitro. Our data may aid in the development of biomarkers and glia-directed therapeutic approaches of HAND.


Asunto(s)
VIH/genética , Microglía/fisiología , Monocitos/metabolismo , Monocitos/virología , ARN Viral/metabolismo , Animales , Ciclo Celular/fisiología , Muerte Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/virología , Técnicas de Cocultivo , Citocinas/metabolismo , Embrión de Mamíferos , Feto/citología , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/líquido cefalorraquídeo , Humanos , Microglía/citología , Monocitos/citología , Neuronas/fisiología , Neuronas/virología , ARN Viral/genética , Ratas
18.
Cell Rep ; 4(4): 689-96, 2013 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-23972988

RESUMEN

Aicardi-Goutières syndrome (AGS), a hereditary autoimmune disease, clinically and biochemically overlaps with systemic lupus erythematosus (SLE) and, like SLE, is characterized by spontaneous type I interferon (IFN) production. The finding that defects of intracellular nucleases cause AGS led to the concept that intracellular accumulation of nucleic acids triggers inappropriate production of type I IFN and autoimmunity. AGS can also be caused by defects of SAMHD1, a 3' exonuclease and deoxynucleotide (dNTP) triphosphohydrolase. Human SAMHD1 is an HIV-1 restriction factor that hydrolyzes dNTPs and decreases their concentration below the levels required for retroviral reverse transcription. We show in gene-targeted mice that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells. Importantly, the absence of SAMHD1 triggered IFN-ß-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types indicative of spontaneous IFN production. SAMHD1-deficient mice may be instrumental for elucidating the mechanisms that trigger pathogenic type I IFN responses in AGS and SLE.


Asunto(s)
Proteínas de Unión al GTP Monoméricas/metabolismo , Replicación Viral , Animales , Células Dendríticas/metabolismo , Células Dendríticas/virología , Desoxirribonucleótidos/metabolismo , Virus de la Leucemia Murina de Friend/metabolismo , Virus de la Leucemia Murina de Friend/fisiología , VIH-1/metabolismo , VIH-1/fisiología , Interferón beta/genética , Interferón beta/metabolismo , Linfocitos/metabolismo , Linfocitos/virología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Transcripción Reversa , Proteína 1 que Contiene Dominios SAM y HD , Transcripción Genética , Regulación hacia Arriba
19.
Virology ; 314(2): 471-87, 2003 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-14554077

RESUMEN

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.


Asunto(s)
Transformación Celular Viral , Genoma Viral , Herpesvirus Saimiriino 2/fisiología , Linfocitos T/virología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Regulación Viral de la Expresión Génica , Herpesvirus Saimiriino 2/patogenicidad , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Saimiri/virología , Análisis de Secuencia de ADN , Proteínas Virales/genética
20.
Trends Immunol ; 23(2): 89-96, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11929132

RESUMEN

A family of interleukin-10 (IL-10)-related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence paralogs, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads, through the activation of signal transducer and activator of transcription (STAT) factors, to diverse biological effects. For example, whereas IL-10 is a well-studied pleiotropic immunosuppressive and immunostimulatory cytokine, IL-22/IL-TIF mediates acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes, which has been proposed as a pathogenic mechanism of psoriasis.


Asunto(s)
Interleucina-10/inmunología , Interleucinas , Secuencia de Aminoácidos , Animales , Humanos , Interleucina-10/química , Interleucina-10/genética , Interleucina-10/uso terapéutico , Datos de Secuencia Molecular , Receptores de Interleucina/inmunología , Receptores de Interleucina-10
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