RESUMEN
Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.
Asunto(s)
Glicoproteínas/metabolismo , Virus Lassa/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Señales de Clasificación de Proteína , Proteínas del Envoltorio Viral/metabolismo , Animales , Chlorocebus aethiops , Glicoproteínas/química , Concentración de Iones de Hidrógeno , Virus Lassa/química , Virus Lassa/ultraestructura , Proteínas de Membrana de los Lisosomas/química , Modelos Moleculares , Conformación Molecular , Complejos Multiproteicos , Unión Proteica , Estructura Terciaria de Proteína , Células Vero , Proteínas del Envoltorio Viral/química , Virión , Internalización del VirusRESUMEN
The activation and expansion of effector CD8(+) T cells are essential for controlling viral infections and tumor surveillance. During an immune response, T cells encounter extrinsic and intrinsic factors, including oxidative stress, nutrient availability, and inflammation, that can modulate their capacity to activate, proliferate, and survive. The dependency of T cells on autophagy for in vitro and in vivo activation, expansion, and memory remains unclear. Moreover, the specific signals and mechanisms that activate autophagy in T effector cells and their survival are not known. In this study, we generated a novel inducible autophagy knockout mouse to study T cell effector responses during the course of a virus infection. In response to influenza infection, Atg5(-/-) CD8(+) T cells had a decreased capacity to reach the peak effector response and were unable to maintain cell viability during the effector phase. As a consequence of Atg5 deletion and the impairment in effector-to-memory cell survival, mice fail to mount a memory response following a secondary challenge. We found that Atg5(-/-) effector CD8(+) T cells upregulated p53, a transcriptional state that was concomitant with widespread hypoxia in lymphoid tissues of infected mice. The onset of p53 activation was concurrent with higher levels of reactive oxygen species (ROS) that resulted in ROS-dependent apoptotic cell death, a fate that could be rescued by treating with the ROS scavenger N-acetylcysteine. Collectively, these results demonstrate that effector CD8(+) T cells require autophagy to suppress cell death and maintain survival in response to a viral infection.
Asunto(s)
Autofagia/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Animales , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Femenino , Expresión Génica , Hipoxia/metabolismo , Memoria Inmunológica , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Especies Reactivas de Oxígeno/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Tumors and the immune system are intertwined in a competition where tilting the fine balance between tumor-specific immunity and tolerance can ultimately decide the fate of the host. Defensive and suppressive immunological responses to cancer are exquisitely sensitive to metabolic features of rapidly growing tumors, such as hypoxia, low nutrient availability, and aberrant growth factor signaling. As a result, clinical therapies impacting these properties change the in situ antitumor immune response by virtue of disrupting the tumor environment. To compensate for disruptions in cellular metabolism, cells activate autophagy to promote survival. On the basis of this notion, strategies designed to block autophagy in tumor cells are currently being tested in several human clinical trials. However, therapies that impair tumor metabolism must also take into account their effect on lymphocytes activated in the immune response to cancer. Given that a strong antitumor immune response is a positive prognostic factor in overall patient survival, identifying ways to block essential processes in tumor cells and suppressive immune cells while promoting those that are important for a robust immune response are of critical importance. Herein, we review the effects of anti-cancer agents that impact metabolism administered concurrently with autophagy inhibitors on immune cells and consider the implications for patient response to therapy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Cloroquina/farmacología , Hidroxicloroquina/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Inhibidores de la Angiogénesis/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Metabolismo Energético/efectos de los fármacos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Neoplasias/metabolismo , Inhibidores de Proteasoma/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The epithelium plays a key role in the spread of Lassa virus. Transmission from rodents to humans occurs mainly via inhalation or ingestion of droplets, dust, or food contaminated with rodent urine. Here, we investigated Lassa virus infection in cultured epithelial cells and subsequent release of progeny viruses. We show that Lassa virus enters polarized Madin-Darby canine kidney (MDCK) cells mainly via the basolateral route, consistent with the basolateral localization of the cellular Lassa virus receptor alpha-dystroglycan. In contrast, progeny virus was efficiently released from the apical cell surface. Further, we determined the roles of the glycoprotein, matrix protein, and nucleoprotein in directed release of nascent virus. To do this, a virus-like-particle assay was developed in polarized MDCK cells based on the finding that, when expressed individually, both the glycoprotein GP and matrix protein Z form virus-like particles. We show that GP determines the apical release of Lassa virus from epithelial cells, presumably by recruiting the matrix protein Z to the site of virus assembly, which is in turn essential for nucleocapsid incorporation into virions.
Asunto(s)
Células Epiteliales/virología , Virus Lassa/fisiología , Proteínas Virales/fisiología , Internalización del Virus , Animales , Células CHO , Polaridad Celular , Células Cultivadas , Cricetinae , Cricetulus , Glicoproteínas/análisis , Glicoproteínas/fisiología , Humanos , Nucleoproteínas/análisis , Nucleoproteínas/fisiología , Proteínas de la Matriz Viral/análisis , Proteínas de la Matriz Viral/fisiología , Virión/fisiologíaRESUMEN
Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.
Asunto(s)
Colesterol/farmacología , Glicoproteínas , Virus Lassa/metabolismo , Virus Lassa/fisiología , Proteínas del Envoltorio Viral , Replicación Viral , Animales , Línea Celular , Centrifugación por Gradiente de Densidad , Chlorocebus aethiops , Colesterol/metabolismo , Cricetinae , Reactivos de Enlaces Cruzados , Dimerización , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Virus Lassa/efectos de los fármacos , Virus Lassa/patogenicidad , Conformación Proteica , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Hypoxia is a signature feature of growing tumors. This cellular state creates an inhospitable condition that impedes the growth and function of all cells within the immediate and surrounding tumor microenvironment. To adapt to hypoxia, cells activate autophagy and undergo a metabolic shift increasing the cellular dependency on anaerobic metabolism. Autophagy upregulation in cancer cells liberates nutrients, decreases the buildup of reactive oxygen species, and aids in the clearance of misfolded proteins. Together, these features impart a survival advantage for cancer cells in the tumor microenvironment. This observation has led to intense research efforts focused on developing autophagy-modulating drugs for cancer patient treatment. However, other cells that infiltrate the tumor environment such as immune cells also encounter hypoxia likely resulting in hypoxia-induced autophagy. In light of the fact that autophagy is crucial for immune cell proliferation as well as their effector functions such as antigen presentation and T cell-mediated killing of tumor cells, anticancer treatment strategies based on autophagy modulation will need to consider the impact of autophagy on the immune system.
RESUMEN
The Lassa virus glycoprotein consists of an ectodomain, a transmembrane anchor, and a cytoplasmic domain. It is synthesized as an inactive precursor and cleaved within the ectodomain to yield the mature form. Here, we show that this maturation cleavage can be abolished by mutation of single conserved amino acids within the cytoplasmic domain at the carboxy-terminus of the glycoprotein. Moreover, substitutions and deletions of multiple amino acids result in destabilization of the glycoprotein oligomers. These results indicate that conformation changes in the cytoplasmic domain travel across the membrane and subsequently abolish the maturation cleavage. Therefore, we postulate that the cytoplasmic domain is an important maturation factor stabilizing the overall conformation of the glycoprotein.