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1.
Viruses ; 16(3)2024 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-38543698

RESUMO

The human genome is estimated to encode more than 500 proteases performing a wide range of important physiological functions. They digest proteins in our food, determine the activity of hormones, induce cell death and regulate blood clotting, for example. During viral infection, however, some proteases can switch sides and activate viral glycoproteins, allowing the entry of virions into new target cells and the spread of infection. To reduce unwanted effects, multiple protease inhibitors regulate the proteolytic processing of self and non-self proteins. This review summarizes our current knowledge of endogenous protease inhibitors, which are known to limit viral replication by interfering with the proteolytic activation of viral glycoproteins. We describe the underlying molecular mechanisms and highlight the diverse strategies by which protease inhibitors reduce virion infectivity. We also provide examples of how viruses evade the restriction imposed by protease inhibitors. Finally, we briefly outline how cellular protease inhibitors can be modified and exploited for therapeutic purposes. In summary, this review aims to summarize our current understanding of cellular protease inhibitors as components of our immune response to a variety of viral pathogens.


Assuntos
Inibidores de Proteases , Vírus , Humanos , Inibidores de Proteases/farmacologia , Glicoproteínas/metabolismo , Proteólise , Vírus/metabolismo , Peptídeo Hidrolases/metabolismo
2.
Front Immunol ; 15: 1303089, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38348040

RESUMO

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family by conducting evolutionary analyses and performing a molecular and functional characterization of rabbit GBPs. We observed that lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or to the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, from an evolutionary perspective, lagomorph GBPs experienced multiple gain and loss events, and the molecular and functional characteristics of ocGBP suggest a role in innate immunity.


Assuntos
Lagomorpha , Animais , Coelhos , Humanos , Camundongos , Lagomorpha/metabolismo , Proteínas de Transporte , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Imunidade Inata/genética , Interferons/metabolismo
3.
Viruses ; 15(12)2023 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-38140605

RESUMO

Coronavirus infection induces interferon-stimulated genes, one of which encodes Tetherin, a transmembrane protein inhibiting the release of various enveloped viruses from infected cells. Previous studies revealed that SARS-CoV encodes two Tetherin antagonists: the Spike protein (S), inducing lysosomal degradation of Tetherin, and ORF7a, altering its glycosylation. Similarly, SARS-CoV-2 has also been shown to use ORF7a and Spike to enhance virion release in the presence of Tetherin. Here, we directly compare the abilities and mechanisms of these two viral proteins to counteract Tetherin. Therefore, cell surface and total Tetherin levels upon ORF7a or S expression were investigated using flow cytometry and Western blot analysis. SARS-CoV and SARS-CoV-2 S only marginally reduced Tetherin cell surface levels in a cell type-dependent manner. In HEK293T cells, under conditions of high exogenous Tetherin expression, SARS-CoV-2 S and ORF7a reduced total cellular Tetherin levels much more efficiently than the respective counterparts derived from SARS-CoV. Nevertheless, ORF7a from both species was able to alter Tetherin glycosylation. The ability to decrease total protein levels of Tetherin was conserved among S proteins from different SARS-CoV-2 variants (α, γ, δ, ο). While SARS-CoV-2 S and ORF7a both colocalized with Tetherin, only ORF7a directly interacted with the restriction factor in a two-hybrid assay. Despite the presence of multiple Tetherin antagonists, SARS-CoV-2 replication in Caco-2 cells was further enhanced upon Tetherin knockout. Altogether, our data show that endogenous Tetherin restricts SARS-CoV-2 replication and that the antiviral activity of Tetherin is only partially counteracted by viral antagonists with differential and complementary modes of action.


Assuntos
Antígeno 2 do Estroma da Médula Óssea , COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Células CACO-2 , COVID-19/metabolismo , COVID-19/virologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo
4.
J Virol ; 97(10): e0080323, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37712707

RESUMO

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.


Assuntos
Proteínas Ligadas por GPI , Galliformes , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Evolução Biológica , Antígeno 2 do Estroma da Médula Óssea/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Galliformes/genética , Evolução Molecular , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo
5.
J Virol ; 97(6): e0029423, 2023 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-37272794

RESUMO

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.


Assuntos
Furina , HIV-1 , Serpinas , Humanos , Linhagem Celular , Produtos do Gene env do Vírus da Imunodeficiência Humana , Furina/metabolismo , HIV-1/fisiologia , Serpinas/química , Serpinas/metabolismo , Serpinas/farmacologia , Replicação Viral
6.
Viruses ; 15(5)2023 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-37243275

RESUMO

HIV-1 envelope glycoproteins (Envs) mediate viral entry and represent a target of choice for small molecule inhibitors. One of them, temsavir (BMS-626529) prevents the interaction of the host cell receptor CD4 with Env by binding the pocket under the ß20-ß21 loop of the Env subunit gp120. Along with its capacity to prevent viral entry, temsavir stabilizes Env in its "closed" conformation. We recently reported that temsavir affects glycosylation, proteolytic processing, and overall conformation of Env. Here, we extend these results to a panel of primary Envs and infectious molecular clones (IMCs), where we observe a heterogeneous impact on Env cleavage and conformation. Our results suggest that the effect of temsavir on Env conformation is associated with its capacity to decrease Env processing. Indeed, we found that the effect of temsavir on Env processing affects the recognition of HIV-1-infected cells by broadly neutralizing antibodies and correlates with their capacity to mediate antibody-dependent cellular cytotoxicity (ADCC).


Assuntos
Infecções por HIV , HIV-1 , Humanos , Antígenos CD4/metabolismo , Conformação Proteica , Proteólise , Peptídeo Hidrolases/metabolismo , Anticorpos Anti-HIV , Anticorpos Neutralizantes , Proteína gp120 do Envelope de HIV/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana
7.
J Med Virol ; 95(1): e28124, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36056630

RESUMO

Host cell proteases such as TMPRSS2 are critical determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and pathogenesis. Here, we show that antithrombin (AT), an endogenous serine protease inhibitor regulating coagulation, is a broad-spectrum inhibitor of coronavirus infection. Molecular docking and enzyme activity assays demonstrate that AT binds and inhibits TMPRSS2, a serine protease that primes the Spike proteins of coronaviruses for subsequent fusion. Consequently, AT blocks entry driven by the Spikes of SARS-CoV, MERS-CoV, hCoV-229E, SARS-CoV-2 and its variants of concern including Omicron, and suppresses lung cell infection with genuine SARS-CoV-2. Thus, AT is an endogenous inhibitor of SARS-CoV-2 that may be involved in COVID-19 pathogenesis. We further demonstrate that activation of AT by anticoagulants, such as heparin or fondaparinux, increases the anti-TMPRSS2 and anti-SARS-CoV-2 activity of AT, suggesting that repurposing of native and activated AT for COVID-19 treatment should be explored.


Assuntos
COVID-19 , Humanos , Antitrombinas/farmacologia , Linhagem Celular , Tratamento Farmacológico da COVID-19 , Simulação de Acoplamento Molecular , SARS-CoV-2/metabolismo , Internalização do Vírus , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Glicoproteína da Espícula de Coronavírus/metabolismo , Serina Endopeptidases/genética
8.
PLoS Pathog ; 17(11): e1009728, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34780577

RESUMO

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.


Assuntos
Evolução Molecular , Infecções por HIV/metabolismo , Lentivirus/genética , Proteínas Proto-Oncogênicas c-hck/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-hck/genética , Homologia de Sequência de Aminoácidos , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
9.
Front Microbiol ; 11: 591778, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193257

RESUMO

The rhadinoviruses Kaposi's Sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus (MHV-68) persist in infected hosts in a latent state that is characterized by the absence of virus production and by restricted viral gene expression. Their major latency protein, the latency-associated nuclear antigen (kLANA for KSHV and mLANA for MHV-68), is essential for viral genome maintenance and replication and involved in transcriptional regulation. Both kLANA and mLANA interact with cellular chromatin-associated proteins, among them the Bromodomain and Extra Terminal domain (Brd/BET) proteins, which recruit cellular and viral proteins to acetylated histones through their bromodomains and modulate cellular gene expression. Brd/BET proteins also play a role in the tethering, replication, segregation or integration of a diverse group of viral DNA genomes. In this study we explored if Brd/BET proteins influence the localization of the LANAs to preferential regions in the host chromatin and thereby contribute to kLANA- or mLANA-mediated transcriptional regulation. Using ChIP-Seq, we revealed a genome-wide co-enrichment of kLANA with Brd2/4 near cellular and viral transcriptional start sites (TSS). Treatment with I-BET151, an inhibitor of Brd/BET, displaced kLANA and Brd2/4 from TSS in the viral and host chromatin, but did not affect the direct binding of kLANA to kLANA-binding sites (LBS) in the KSHV latent origin of replication. Similarly, mLANA, but not a mLANA mutant deficient for binding to Brd2/4, also associated with cellular TSS. We compared the transcriptome of KSHV-infected with uninfected and kLANA-expressing human B cell lines, as well as a murine B cell line expressing mLANA or a Brd2/4-binding deficient mLANA mutant. We found that only a minority of cellular genes, whose TSS are occupied by kLANA or mLANA, is transcriptionally regulated by these latency proteins. Our findings extend previous reports on a preferential deposition of kLANA on cellular TSS and show that this characteristic chromatin association pattern is at least partially determined by the interaction of these viral latency proteins with members of the Brd/BET family of chromatin modulators.

10.
Exp Eye Res ; 201: 108258, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32980316

RESUMO

The transforming growth factor-beta (TGFB) plays an essential role in the pathogenesis of some ophthalmologic diseases, including neovascular age-related macular degeneration (nAMD) and proliferative vitreoretinopathy (PVR). TGFB activates the transcription factors SMAD2 and SMAD3 via the TGFB receptor, which together activate several genes, including VEGFA. TGFB treated ARPE-19 cells show an increased proliferation rate and undergo epithelial to mesenchymal transition (EMT). Since microRNAs (miRNAs) are capable of inhibiting the translation of multiple genes, we screened for miRNAs that regulate the TGFB signalling pathways at multiple levels. In this study, we focused on two miRNAs, miR-302d and miR-93, which inhibit TGFB signalling pathway and therefore TGFB-induced EMT transition as well as VEGFA secretion from ARPE-19 cells. Furthermore, we could show that both miRNAs can retransform TGFB-stimulated mesenchymal ARPE-19 cells towards the morphological epithelial-like state. Taken together, transient overexpression of these miRNAs in RPE cells might be a promising approach for further translational strategies.


Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica , MicroRNAs/genética , Epitélio Pigmentado da Retina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Degeneração Macular Exsudativa/genética , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , MicroRNAs/biossíntese , RNA/genética , Epitélio Pigmentado da Retina/patologia , Fator de Crescimento Transformador beta/biossíntese , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologia
11.
J Virol ; 90(16): 7046-7065, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27194770

RESUMO

UNLABELLED: Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency. IMPORTANCE: Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined.


Assuntos
Regulação Viral da Expressão Gênica , Repetição Terminal Longa de HIV/genética , HIV-1/fisiologia , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Fator de Transcrição Sp1/metabolismo , Infecções por HIV/virologia , Humanos , Células Jurkat , NF-kappa B/genética , Ligação Proteica , Fator de Transcrição Sp1/genética , Transcrição Gênica , Replicação Viral
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