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HIV-1 hijacks host proteins involved in membrane trafficking, endocytosis, and autophagy that are critical for virus replication. Molecular details are lacking but are essential to inform on the development of alternative antiviral strategies. Despite their potential as clinical targets, only a few membrane trafficking proteins have been functionally characterized in HIV-1 replication. To further elucidate roles in HIV-1 replication, we performed a CRISPR-Cas9 screen on 140 membrane trafficking proteins. We identified phosphatidylinositol-binding clathrin assembly protein (PICALM) that influences not only infection dynamics but also CD4+ SupT1 biology. The knockout (KO) of PICALM inhibited viral entry. In CD4+ SupT1 T cells, KO cells exhibited defects in intracellular trafficking and increased abundance of intracellular Gag and significant alterations in autophagy, immune checkpoint PD-1 levels, and differentiation markers. Thus, PICALM modulates a variety of pathways that ultimately affect HIV-1 replication, underscoring the potential of PICALM as a future target to control HIV-1.
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This article reviews tried-and-tested methodologies that have been employed in the first studies on phase separating properties of structural, RNA-binding and catalytic proteins of HIV-1. These are described here to stimulate interest for any who may want to initiate similar studies on virus-mediated liquid-liquid phase separation. Such studies serve to better understand the life cycle and pathogenesis of viruses and open the door to new therapeutics.
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HIV-1 , Replicação Viral , Humanos , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Separação de Fases , RNA Viral/genéticaRESUMO
Worldwide, almost 40 million people are currently living with HIV-1. The implementation of cART inhibits HIV-1 replication and reduces viremia but fails to eliminate HIV-1 from latently infected cells. These cells are considered viral reservoirs from which HIV-1 rebounds if cART is interrupted. Several efforts have been made to identify these cells and their niches. There has been little success in diminishing the pool of latently infected cells, underscoring the urgency to continue efforts to fully understand how HIV-1 establishes and maintains a latent state. Reactivating HIV-1 expression in these cells using latency-reversing agents (LRAs) has been successful, but only in vitro. This review aims to provide a broad view of HIV-1 latency, highlighting Canadian contributions toward these aims. We will summarize the research efforts conducted in Canadian labs to understand the establishment of latently infected cells and how this informs curative strategies, by reviewing how HIV latency is established, which cells are latently infected, what methodologies have been developed to characterize them, how new compounds are discovered and evaluated as potential LRAs, and what clinical trials aim to reverse latency in people living with HIV (PLWH).
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Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Latência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Canadá , Ativação ViralRESUMO
Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.
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Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Retroviridae , Humanos , Regiões 5' não Traduzidas , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Sítios Internos de Entrada Ribossomal/genética , Fosforilação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Retroviridae/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.
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Condensados Biomoleculares , HIV-1 , Interações Hospedeiro-Patógeno , RNA Viral , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/genética , HIV-1/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Genoma Viral , HumanosRESUMO
A rapidly evolving understanding of phase separation in the biological and physical sciences has led to the redefining of virus-engineered replication compartments in many viruses with RNA genomes. Condensation of viral, host and genomic and subgenomic RNAs can take place to evade the innate immunity response and to help viral replication. Divergent viruses prompt liquid-liquid phase separation (LLPS) to invade the host cell. During HIV replication there are several steps involving LLPS. In this review, we characterize the ability of individual viral and host partners that assemble into biomolecular condensates (BMCs). Of note, bioinformatic analyses predict models of phase separation in line with several published observations. Importantly, viral BMCs contribute to function in key steps retroviral replication. For example, reverse transcription takes place within nuclear BMCs, called HIV-MLOs while during late replication steps, retroviral nucleocapsid acts as a driver or scaffold to recruit client viral components to aid the assembly of progeny virions. Overall, LLPS during viral infections represents a newly described biological event now appreciated in the virology field, that can also be considered as an alternative pharmacological target to current drug therapies especially when viruses become resistant to antiviral treatment.
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HIV-1 , Replicação Viral , Humanos , Condensados Biomoleculares , Núcleo Celular/metabolismo , RNA Subgenômico/genética , HIV-1/genética , HIV-1/fisiologiaRESUMO
Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 + T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.
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The leap of retroviruses and coronaviruses from animal hosts to humans has led to two ongoing pandemics and tens of millions of deaths worldwide. Retrovirus and coronavirus nucleocapsid proteins have been studied extensively as potential drug targets due to their central roles in virus replication, among which is their capacity to bind their respective genomic RNAs for packaging into nascent virions. This review focuses on fundamental studies of these nucleocapsid proteins and how their intrinsic abilities to condense through liquid-liquid phase separation (LLPS) contribute to viral replication. Therapeutic targeting of these condensates and methodological advances are also described to address future questions on how phase separation contributes to viral replication.
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HIV-1 , Proteínas do Nucleocapsídeo , SARS-CoV-2 , Replicação Viral , Humanos , Proteínas do Nucleocapsídeo de Coronavírus , COVID-19 , SARS-CoV-2/fisiologia , HIV-1/fisiologiaRESUMO
Membraneless biomolecular condensates (BMCs) contribute to the replication of a growing number of viruses but remain to be functionally characterized. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) proteins phase separated into condensates regulating virus assembly. Here we discover that intrinsically disordered human immunodeficiency virus-type 1 (HIV-1) core proteins condense with the viral genomic RNA (vRNA) to assemble as BMCs attaining a geometry characteristic of viral reverse transcription complexes. We explore the predisposition, mechanisms, and pharmacologic sensitivity of HIV-1 core BMCs in living cells. HIV-1 vRNA-interacting NC condensates were found to be scaffolds onto which client capsid, reverse transcriptase, and integrase condensates assemble. HIV-1 core BMCs exhibit fundamental characteristics of BMCs and are drug-sensitive. Lastly, protease-mediated maturation of Gag and Gag-Pol precursor proteins yield abundant and visible BMCs in cells. This study redefines HIV-1 core components as fluid BMCs and advances our understanding of the nature of viral cores during ingress.
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HIV-1 , Condensados Biomoleculares , HIV-1/genética , Humanos , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologiaRESUMO
Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5'untranslated region (5'UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.
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Proteínas do Citoesqueleto/metabolismo , HIV-1/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células HCT116 , Células HEK293 , HumanosRESUMO
Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.
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Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Infecção por Zika virus/patologia , Zika virus/patogenicidade , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo , Embrião de Mamíferos/virologia , Perfilação da Expressão Gênica , Camundongos , MicroRNAs/genética , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.
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Cobre/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Nucleocapsídeo/metabolismo , Príons/metabolismo , Zinco/metabolismo , Biologia Computacional , Metanálise como Assunto , Simulação de Dinâmica Molecular , Doenças Neurodegenerativas/virologia , Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/genética , Príons/genética , Domínios Proteicos , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The full-length mRNAs of the human immunodeficiency virus type-1 (HIV-1), the human T-cell lymphotropic virus type-1 (HTLV-1), and the mouse mammary tumor virus (MMTV) harbor IRESs. The activity of the retroviral-IRESs requires IRES-transacting factors (ITAFs), being hnRNP A1, a known ITAF for the HIV-1 IRES. In this study, we show that hnRNP A1 is also an ITAF for the HTLV-1 and MMTV IRESs. The MMTV IRES proved to be more responsive to hnRNP A1 than either the HTLV-1 or the HIV-1 IRESs. The impact of post-translational modifications of hnRNP A1 on HIV-1, HTLV-1 and MMTV IRES activity was also assessed. Results show that the HIV-1 and HTLV-1 IRESs were equally responsive to hnRNP A1 and its phosphorylation mutants S4A/S6A, S4D/S6D and S199A/D. However, the S4D/S6D mutant stimulated the activity from the MMTV-IRES to levels significantly higher than the wild type hnRNP A1. PRMT5-induced symmetrical di-methylation of arginine residues of hnRNP A1 enabled the ITAF to stimulate the HIV-1 and HTLV-1 IRESs while reducing the stimulatory ability of the ITAF over the MMTV IRES. We conclude that retroviral IRES activity is not only dependent on the recruited ITAFs but also relies on how these proteins are modified at the post-translational level.
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Ribonucleoproteína Nuclear Heterogênea A1/genética , Sítios Internos de Entrada Ribossomal/genética , Iniciação Traducional da Cadeia Peptídica , Processamento de Proteína Pós-Traducional/genética , Animais , Regulação Viral da Expressão Gênica/genética , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Fosforilação/genética , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genéticaRESUMO
Nucleoside analogs have proven effective for the inhibition of viral polymerases and are the foundation of many antiviral therapies. In this work, the antiretroviral potential of 6-azauracil analogs was assessed using activity-based protein profiling techniques and functional assays. Probes based on the 6-azauracil scaffold were examined and found to bind to HCV polymerase and HIV-1 reverse transcriptase through covalent modification of residues near the active site. The modified sites on the HIV-1 RT were examined using a mass spectrometry approach, and it was discovered that the azauracil moieties modified the enzyme in proximity to its active site. However, these scaffolds gave little or no inhibition of enzyme activity. Instead, a bifunctional inhibitor was prepared using click chemistry to link the 6-azauracil moiety to azidothymidine (AzT) and the corresponding triphosphate (AzTTP). These bifunctional inhibitors were found to have potent inhibitory function through a mode of action that includes both alkylation and chain termination. An in vitro assay demonstrated that the bifunctional inhibitor was 23-fold more effective in inhibiting HIV-1 RT activity than the parent AzTTP. The bifunctional inhibitor was also tested in HIV-1 permissive T cells where it decreased Gag expression similarly to the front-line drug Efavirenz with no evidence of cytotoxicity. This new bifunctional scaffold represents an interesting tool for inhibiting HIV-1 by covalently anchoring a chain-terminating nucleoside analog in the active site of the reverse transcriptase, preventing its removal and abolishing enzymatic activity, and represents a novel mode of action for inhibiting polymerases including reverse transcriptases.
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Transcriptase Reversa do HIV/antagonistas & inibidores , Nucleosídeos/química , Nucleosídeos/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Domínio Catalítico , Química Click , Desenho de Fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Modelos MolecularesRESUMO
Amyotrophic lateral sclerosis (ALS) arises from an interplay of genetic mutations and environmental factors. ssRNA viruses are possible ALS risk factors, but testing their interaction with mutations such as in FUS, which encodes an RNA-binding protein, has been difficult due to the lack of a human disease model. Here, we use isogenic induced pluripotent stem cell (iPSC)-derived spinal neurons (SNs) to investigate the interaction between ssRNA viruses and mutant FUS. We find that rabies virus (RABV) spreads ALS phenotypes, including the formation of stress granules (SGs) with aberrant composition due to increased levels of FUS protein, as well as neurodegeneration and reduced restriction activity by FUS mutations. Consistent with this, iPSC-derived SNs harboring mutant FUS are more sensitive to human immunodeficiency virus (HIV-1) and Zika viruses (ZIKV). We demonstrate that RABV and HIV-1 exacerbate cytoplasmic mislocalization of FUS. Our results demonstrate that viral infections worsen ALS pathology in SNs with genetic risk factors, suggesting a novel role for viruses in modulating patient phenotypes.
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The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1-/- gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1-/- cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.
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Grânulos Citoplasmáticos/virologia , Proteínas do Citoesqueleto/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Vírion/genética , Transporte Biológico , Grânulos Citoplasmáticos/metabolismo , Proteínas do Citoesqueleto/deficiência , Regulação da Expressão Gênica , Células HCT116 , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Viral/metabolismo , Transdução de Sinais , Transfecção , Vírion/metabolismo , Montagem de Vírus/genética , Replicação Viral/genéticaRESUMO
BACKGROUND: Mammalian cells harbour RNA quality control and degradative machineries such as nonsense-mediated mRNA decay that target cellular mRNAs for clearance from the cell to avoid aberrant gene expression. The role of the host mRNA decay pathways in macrophages in the context of human immunodeficiency virus type 1 (HIV-1) infection is yet to be elucidated. Macrophages are directly infected by HIV-1, mediate the dissemination of the virus and contribute to the chronic activation of the inflammatory response observed in infected individuals. Therefore, we characterized the effects of four host mRNA decay proteins, i.e., UPF1, UPF2, SMG6 and Staufen1, on viral replication in HIV-1-infected primary monocyte-derived macrophages (MDMs). RESULTS: Steady-state expression levels of these host mRNA decay proteins were significantly downregulated in HIV-1-infected MDMs. Moreover, UPF2 and SMG6 inhibited HIV-1 gene expression in macrophages to a similar level achieved by SAMHD1, by directly influencing viral genomic RNA levels. Staufen1, a host protein also involved in UPF1-dependent mRNA decay and that acts at several HIV-1 replication steps, enhanced HIV-1 gene expression in MDMs. CONCLUSIONS: These results provide new evidence for roles of host mRNA decay proteins in regulating HIV-1 replication in infected macrophages and can serve as potential targets for broad-spectrum antiviral therapeutics.
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HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Macrófagos/virologia , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Replicação Viral , Células Cultivadas , Regulação da Expressão Gênica , HumanosRESUMO
Continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. Close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. There is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory T cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. Here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. To observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. We focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory T cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates.