RESUMO
The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the C377,409S double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding.IMPORTANCE FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Produtos do Gene gag/metabolismo , HIV-1/fisiologia , Vírus da Imunodeficiência Felina/fisiologia , Precursores de Proteínas/metabolismo , Liberação de Vírus , Animais , Proteínas de Ligação ao Cálcio/genética , Gatos , Proteínas de Ciclo Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Produtos do Gene gag/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Domínios Proteicos , Precursores de Proteínas/genética , Dedos de ZincoRESUMO
Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Herpesvirus Humano 1/patogenicidade , Interações Hospedeiro-Patógeno , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Humanos , Proteases Específicas de UbiquitinaRESUMO
Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2(504) and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2(504) is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2(504) failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2(504) was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2(504) also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction.
Assuntos
Gatos/metabolismo , Síndrome de Imunodeficiência Adquirida Felina/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Gatos/genética , Linhagem Celular , Síndrome de Imunodeficiência Adquirida Felina/genética , Síndrome de Imunodeficiência Adquirida Felina/virologia , Proteínas Ligadas por GPI/genética , Humanos , Vírus da Imunodeficiência Felina/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Liberação de VírusRESUMO
INTRODUCTION: We analyzed a cohort of patients with cancer and Sars-Cov-2 infection from the Veneto Oncology Network registry across two pandemic time periods. MATERIALS AND METHODS: 761 patients with cancer and SARS-CoV-2 infection were included. RESULTS: 198 patients were diagnosed during the first pandemic time period (TP1; February 2020 September 2020), 494 during TP2 before the vaccination campaign (TP2/pre-vaccination; September 2020-21 February 2021) and 69 in TP2/post-vaccination (22 February 2021-15 May 2021). TP2 vs TP1 patients were younger (p = 0.004), showed more frequently a good performance status (p < 0.001) and <2 comorbidities (p = 0.002), were more likely to be on active anticancer therapy (p = 0.006). Significantly fewer patients in TP2 (3-4%) vs TP1 (22%) had an in-hospital potential source of infection (p < 0.001). TP2 patients were more frequently asymptomatic (p = 0.003). Significantly fewer patients from TP2 were hospitalized (p < 0.001) or admitted to intensive care unit (p = 0.006). All-cause mortality decreased from 30.3% in TP1, to 8.9% and 8.7% in the two TP2 periods (p < 0.001), reflected by a significant reduction in Sars-Cov-2-related mortality (15.2%, 7.5% and 5.8% in the three consecutive time periods, p = 0.004). CONCLUSIONS: Differences in clinical characteristics and features of Sars-Cov-2 infection between TP1 and TP2 reflect the effects of protective measures and increased testing capacity. The lower mortality in TP2 is in line with a less frail population. However, the vast majority of death events in TP2 were related to COVID-19, reinforcing the priority to protect cancer patients.
Assuntos
COVID-19 , Neoplasias , COVID-19/epidemiologia , Hospitalização , Humanos , Neoplasias/epidemiologia , Pandemias , SARS-CoV-2RESUMO
Monosodium Glutamate (MSG) is used as flavour enhancer, with potential beneficial effects due to its nutritional value. Given the decline in kidney functions during aging, we investigated the impact of MSG voluntary intake on the kidney of male mice, aged 6 or 18 months. For 2 months, they freely consumed water (control group), sodium chloride (0.3% NaCl) or MSG (1% MSG) in addition to standard diet. Young animals consuming sodium chloride presented signs of proteinuria, hyperfiltration, enhanced expression and excretion of Aquaporin 2 and initial degenerative reactions suggestive of fibrosis, while MSG-consuming mice were similar to controls. In old mice, aging-related effects including proteinuria and increased renal corpuscle volume were observed in all groups. At an advanced age, MSG caused no adverse effects on the kidney compared to controls, despite the presence of a sodium moiety, similar to sodium chloride. These data show that prolonged MSG intake in mice has less impact on kidney compared to sodium chloride, that already in young animals induced some effects on kidney, possibly related to hypertension.
Assuntos
Envelhecimento/efeitos dos fármacos , Albuminúria/patologia , Fibrose/patologia , Nefropatias/patologia , Cloreto de Sódio/toxicidade , Glutamato de Sódio/farmacologia , Albuminúria/induzido quimicamente , Animais , Ingestão de Líquidos , Fibrose/induzido quimicamente , Nefropatias/induzido quimicamente , Masculino , CamundongosRESUMO
One of the main pathological features of Parkinson's disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal-lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of "consensus" motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS's intracellular levels. Furthermore, Nac32HECT expression partially rescues aS's overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS's accumulation and aggregation in neurons.
Assuntos
Vetores Genéticos/metabolismo , Lentivirus/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Doença de Parkinson/patologia , UbiquitinaçãoRESUMO
INTRODUCTION: Coronavirus disease 2019 (COVID-19) pandemic started in Italy with clusters identified in Northern Italy. The Veneto Oncology Network (Rete Oncologica Veneta) licenced dedicated guidelines to ensure proper care minimising the risk of infection in patients with cancer. Rete Oncologica Veneta covID19 (ROVID) is a regional registry aimed at describing epidemiology and clinical course of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in patients with cancer. MATERIALS AND METHODS: Patients with cancer diagnosis and documented SARS-CoV-2 infection are eligible. Data on cancer diagnosis, comorbidities, anticancer treatments, as well as details on SARS-CoV-2 infection (hospitalisation, treatments, fate of the infection), have been recorded. Logistic regression analysis was applied to calculate the association between clinical/laboratory variables and death from any cause. RESULTS: One hundred seventy patients have been enrolled. The median age at time of the SARS-CoV infection was 70 years (25-92). The most common cancer type was breast cancer (n = 40). The majority of the patients had stage IV disease. Half of the patients had two or more comorbidities. The majority of the patients (78%) presented with COVID-19 symptoms. More than 77% of the patients were hospitalized and 6% were admitted to intensive care units. Overall, 104 patients have documented resolution of the infection. Fifty-seven patients (33%) have died. In 29 cases (17%), the cause of death was directly correlated to SARS-CoV-2 infection. Factors significantly correlated with the risk of death were the following: Eastern Cooperative Oncology Group performance status (PS), age, presence of two or more comorbidities, presence of dyspnoea, COVID-19 phenotype ≥ 3, hospitalisation, intensive care unit admission, neutrophil/lymphocyte ratio and thrombocytopenia. CONCLUSIONS: The mortality rate reported in this confirms the frailty of this population. These data reinforce the need to protect patients with cancer from SARS-CoV-2 infection.
Assuntos
COVID-19/diagnóstico , COVID-19/epidemiologia , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/complicações , COVID-19/patologia , Redes Comunitárias , Progressão da Doença , Feminino , Mortalidade Hospitalar , Hospitalização/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Neoplasias/patologia , Pandemias , Prognóstico , Sistema de Registros , SARS-CoV-2/fisiologia , Índice de Gravidade de DoençaRESUMO
RNA-enveloped viruses bud from infected cells by exploiting the multivesicular body (MVB) pathway. In this context, ubiquitination of structural viral proteins and their direct interaction with cellular factors involved in the MVB biogenesis through short proline rich regions, named late domains (L-domains), are crucial mechanisms. Here we report that, in contrast with the human immunodeficiency virus (HIV), the feline immunodeficiency virus (FIV), a non-primate lentivirus, is strictly dependent for its budding on a "PSAP"-type L-domain, mapping in the carboxy-terminal region of Gag, irrespective of a functional viral protease. Moreover, we provide evidence that FIV egress is related to Gag ubiquitination, that is, linked to the presence of an active L-domain. Finally, although FIV Gag does not contain a PPxY motif, we show that the Nedd4-2s ubiquitin ligase enhances FIV Gag ubiquitination and it is capable to rescue viral mutants lacking a functional L-domain. In conclusion, our data bring to light peculiar aspects of FIV egress, but we also demonstrate that a non-primate lentivirus shares with HIV-1 a novel mechanism of connection to the cellular budding machinery.
Assuntos
Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Gatos , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/genética , Genes gag , HIV-1/genética , HIV-1/fisiologia , Humanos , Vírus da Imunodeficiência Felina/genética , Mutagênese Sítio-Dirigida , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ubiquitinação , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013â»2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.