RESUMO
Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.
Assuntos
Dependovirus , Terapia Genética , Humanos , Dependovirus/genética , Distribuição Tecidual , Desenvolvimento de Medicamentos , Reação em Cadeia da PolimeraseRESUMO
The clinical use of many adenovirus vaccine vectors (AdVs) is limited by the presence of pre-existing antibodies in human populations, which prevent common AdVs from transducing cells and expressing immunogenic gene products. Rare serotype AdVs, such as HAdV-28D can bypass pre-existing immunity. However, rare AdVs stimulate high-levels of type I interferon (IFN), which suppresses antigenic gene expression and therefore limits immunogenicity. Recent studies identified Gas6 as a factor that connects enveloped viruses to host-cell receptor tyrosine kinases, in turn generating signaling cascades that antagonize type I IFN responses. We discovered that Gas6 bound to the fiber proteins of common AdV serotypes, such as HAdV-5C, with a higher affinity than rare HAd-28D fibers. AdV-associated Gas6 suppressed IFN production by common AdVs and enhanced long-term expression of AdV encoded genes. We hypothesize that rare AdV serotypes might be engineered to include Gas6 binding motifs, thereby generating novel vectors that are more effective.
Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon beta/metabolismo , Adenoviridae/classificação , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferon beta/genética , Ligação Proteica , SorogrupoRESUMO
BACKGROUND: An effective malaria transmission-blocking vaccine (TBV) would be a major advance in the current efforts to eliminate and, ultimately, eradicate malaria. Antibodies against Plasmodium falciparum surface protein, Pfs25, are known to block parasite development in the mosquito vector. However, in initial clinical trials the limited immunogenicity of recombinant Pfs25 protein-in-adjuvant vaccines has been a challenge. METHODS: Novel human adenovirus type 5 (Ad5) vectors were used in heterologous prime boost vaccination strategies to augment the immune response against Pfs25. Specifically, an Ad5 vector that directs expression of full-length, membrane-bound Pfs25 was used as a priming immunization followed by a boost with Ad5 viral particles displaying only the Pfs25 epitope targeted by transmission-blocking antibodies 4B7 and 1D2 (Pfs25 aa 122-134) in hypervariable region 5 of the hexon capsid protein. RESULTS: This heterologous prime-boost vaccine strategy induced antibodies that significantly inhibit P. falciparum transmission to mosquitoes in a standard membrane-feeding assay. Further, immunized mice generated a robust anti-Pfs25 antibody response characterized by higher titer, higher relative avidity and a broader IgG subclass profile than observed with a homologous prime-boost with recombinant Pfs25/alum. CONCLUSION: The data suggest that focusing the immune response against defined epitopes displayed on the viral capsid is an effective strategy for transmission-blocking vaccine development.
Assuntos
Vacinas contra Adenovirus/genética , Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Epitopos/química , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Malária Falciparum/transmissão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/químicaRESUMO
Autophagy is an essential metabolic program that is also used for clearing intracellular pathogens. This mechanism, also termed selective autophagy, is well characterized for invasive bacteria but remains poorly documented for viral infections. Here we highlight our recent work showing that endosomolytic adenoviruses trigger autophagy when entering cells. Our study revealed how adenoviruses exploit a capsid-associated small PPxY peptide motif to manipulate the autophagic machinery to prevent autophagic degradation and to promote endosomal escape and nuclear trafficking.
Assuntos
Adenoviridae/imunologia , Autofagia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Peptídeos/metabolismoRESUMO
Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.
Assuntos
Infecções por Adenovirus Humanos/metabolismo , Autofagia/fisiologia , Proteínas do Capsídeo/metabolismo , Galectinas/metabolismo , Internalização do Vírus , Adenoviridae , Infecções por Adenovirus Humanos/imunologia , Motivos de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal , Microscopia Eletrônica de TransmissãoRESUMO
As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (Moyer and Nemerow, 2011, Curr. Opin. Virol., 1, 44-49), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus (HAdV).
Assuntos
Adenoviridae/fisiologia , Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Proteínas Virais/metabolismo , Internalização do Vírus , Animais , HumanosRESUMO
Imaging host-pathogen interactions in real time can provide significant insight into dynamic processes and provide information about time and space of their occurences. Here, we present detailed experimental instructions on how to image the membrane penetration process of the non-enveloped adenovirus in real time. The system is based on a cell line stably expressing the lectin galectin-3 fused to a fluorophore. Membrane-lytic events during adenovirus cell entry can be monitored by the recruitment of galectin-3 to galactose-containing membrane glycoproteins on the exo-surface of ruptured membranes. The simultaneous use of fluorescently labeled adenoviral capsids allows to image the events in unmatched temporal resolution.
Assuntos
Adenoviridae/fisiologia , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Imagem Molecular , Endossomos/virologia , Citometria de Fluxo , Humanos , Microscopia Confocal , Imagem Molecular/métodosRESUMO
α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.
Assuntos
Catepsinas/metabolismo , Endocitose/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , alfa-Sinucleína/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Inflamassomos/metabolismo , Mutação , Multimerização Proteica , Transporte Proteico , Ratos , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1(+)) early endosomes or LAMP-1(+) late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3(+) endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3(+) endosomes, pVI appears to remain associated with the gal3(+) ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events.
Assuntos
Adenoviridae/metabolismo , Capsídeo/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Galectina 3/biossíntese , Células HEK293 , Células HeLa , Humanos , Cinética , Lisossomos/metabolismo , Microscopia de Fluorescência/métodos , Fatores de Tempo , Proteínas de Transporte Vesicular/biossínteseRESUMO
Adenovirus relies on numerous interactions between viral and host cell proteins to efficiently enter cells. Undoubtedly, post-translational modifications of host and cellular proteins can impact the efficiency of this cell entry process. Ubiquitylation, once simply thought of as a modification targeting proteins for proteasomal degradation, is now known to regulate protein trafficking within cells, protein-protein interactions and cell signalling pathways. Accumulating evidence suggests that protein ubiquitylation can influence all stages of the life cycle of other viruses such as cell entry, replication and egress. Until recently, the influence of ubiquitylation has only been documented during adenovirus replication. This review highlights the most recent evidence demonstrating direct engagement of host ubiquitylation and SUMOylation machinery by adenovirus during cell entry. Additionally, potential roles for host protein ubiquitylation and the potential for adenovirus regulation of host ubiquitylation machinery during cell entry are discussed.
Assuntos
Adenoviridae/fisiologia , Proteínas do Capsídeo/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Internalização do Vírus , Adenoviridae/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Receptores Virais/metabolismo , Sumoilação , UbiquitinaçãoRESUMO
In response to viral infection, reactive oxygen species (ROS) mediate innate immune signaling or generate danger signals to activate immune cells. The mechanisms of virally induced ROS are poorly defined, however. We demonstrate that ROS are produced within minutes of adenovirus type 5 (Ad5) infection of macrophages and that oxidative stress supports Ad5-induced cytokine secretion. We show that short hairpin RNA (shRNA) knockdown of TLR9 has no effect on ROS production despite observed decreases in Ad-induced cytokine secretion. A major source of ROS in macrophages is NADPH oxidase. However, shRNA knockdown of the NADPH oxidase subunit NOX2 does not attenuate Ad-induced ROS. Induction of ROS is not observed in cells infected with a temperature-sensitive mutant of Ad2, ts1, which is defective in endosomal membrane penetration during cell entry. Further, Ad5, but not ts1, induces the release of lysosomal cathepsin B into the cytoplasm of infected cells. In agreement with this finding, we observe a loss of mitochondrial membrane potential upon Ad infection which requires Ad endosomal membrane penetration and cathepsin B activity. Overexpression of Bcl-2 attenuates Ad5-induced ROS, further supporting the role for mitochondrial membrane destabilization as the source of ROS in response to Ad5 infection. Together, these data suggest that ROS produced in response to Ad5 infection depends on the virally induced endosomal membrane rupture to release lysosomal cathepsins. Furthermore, the release of cathepsins leads to mitochondrial membrane disruption and thus the release of ROS from the mitochondria.
Assuntos
Adenoviridae/imunologia , Catepsina B/metabolismo , Lisossomos/enzimologia , Lisossomos/virologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Potencial da Membrana Mitocondrial , Membranas Mitocondriais/fisiologiaRESUMO
The identification of the adenovirus (AdV) protein that mediates endosome penetration during infection has remained elusive. Several lines of evidence from previous studies suggest that the membrane lytic factor of AdV is the internal capsid protein VI. While these earlier results imply a role for protein VI in endosome disruption, direct evidence during cell entry has not been demonstrated. To acquire more definitive proof, we engineered random mutations in a critical N-terminal amphipathic α-helix of VI in an attempt to generate AdV mutants that lack efficient membrane penetration and infection. Random mutagenesis within the context of the AdV genome was achieved via the development of a novel technique that incorporates both error-prone PCR and recombineering. Using this system, we identified a single mutation, L40Q, that significantly reduced infectivity and selectively impaired endosome penetration. Furthermore, we obtained biophysical data showing that the lack of efficient endosomalysis is associated with reduced insertion of the L40Q mutation in protein VI (VI-L40Q) into membranes. Our studies indicate that protein VI is the critical membrane lytic factor of AdV during cellular entry and reveal the biochemical basis for its membrane interactions.
Assuntos
Adenovírus Humanos/patogenicidade , Proteínas do Capsídeo/metabolismo , Endossomos/virologia , Membranas Intracelulares/metabolismo , Internalização do Vírus , Substituição de Aminoácidos/genética , Proteínas do Capsídeo/genética , Linhagem Celular , Análise Mutacional de DNA , Engenharia Genética/métodos , Genética Microbiana/métodos , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase/métodos , Recombinação GenéticaRESUMO
Adenovirus disrupts endosomal membranes during cell entry. The membrane lytic capsid protein VI (pVI) facilitates entry by fragmenting membranes. Although an N-terminal amphipathic α-helix (VI-Φ) possesses similar membrane affinity as pVI, truncated protein lacking VI-Φ (VIΔ54) still possesses moderate membrane affinity. We demonstrate that incorporation of nickel-NTA lipids in membranes enhances the membrane affinity and the membrane lytic activity of VIΔ54. We also demonstrate that 3 predicted pVI α-helices within residues 54-114 associate with membranes, sitting roughly parallel to the membrane surface. His-tagged VIΔ54 is capable of fragmenting membranes similar to pVI and the VI-Φ peptide. Interestingly, neither VI-Φ nor His-tagged VIΔ54 can induce tubule formation in giant lipid vesicles as observed for pVI. These data suggest cooperativity between the amphipathic α-helix and residues in VIΔ54 to induce positive membrane curvature and tubule formation. These results provide additional details regarding the mechanism of nonenveloped virus membrane penetration.
Assuntos
Adenoviridae/fisiologia , Proteínas do Capsídeo/metabolismo , Membrana Celular/metabolismo , Internalização do Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Adenovirus (Ad) membrane penetration during cell entry is poorly understood. Here we show that antibodies which neutralize the membrane lytic activity of the Ad capsid protein VI interfere with Ad endosomal membrane penetration. In vitro studies using a peptide corresponding to an N-terminal amphipathic alpha-helix of protein VI (VI-Phi), as well as other truncated forms of protein VI suggest that VI-Phi is largely responsible for protein VI binding to and lysing of membranes. Additional studies suggest that VI-Phi lies nearly parallel to the membrane surface. Protein VI fragments membranes and induces highly curved structures. Further studies suggest that protein VI induces positive membrane curvature. These data support a model in which protein VI binds membranes, inducing positive curvature strain which ultimately leads to membrane fragmentation. These results agree with previous observations of Ad membrane permeabilization during cell entry and provide an initial mechanistic description of a nonenveloped virus membrane lytic protein.
Assuntos
Adenoviridae/fisiologia , Proteínas do Capsídeo/fisiologia , Membrana Celular/metabolismo , Internalização do Vírus , Proteínas do Capsídeo/genética , Linhagem Celular , Sobrevivência Celular , Humanos , Modelos Biológicos , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Of the 53 different human adenovirus (HAdV) serotypes belonging to species A-G, a significant number are associated with acute respiratory, gastrointestinal and ocular infections. Replication-defective HAdV-5-based vectors also continue to play a significant role in gene transfer trials and in vaccine delivery efforts in the clinic. Although significant progress has been made from studies of AdV biology, we still have an incomplete understanding of AdV's structure as well as its multifactorial interactions with the host. Continuing efforts to improve knowledge in these areas, as discussed in this chapter, will be crucial for revealing the mechanisms of AdV pathogenesis and for allowing optimal use of AdV vectors for biomedical applications.
Assuntos
Adenoviridae/fisiologia , Internalização do Vírus , Adenoviridae/ultraestrutura , Animais , Capsídeo/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Sistema Imunitário/fisiologia , Receptores Virais/fisiologia , Vírion/fisiologia , Ligação ViralRESUMO
Viruses use cellular machinery to enter and infect cells. In this study we address the cell entry mechanisms of nonenveloped adenoviruses (Ads). We show that protein VI, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. We found that a PPxY motif within protein VI recruits Nedd4 E3 ubiquitin ligases to bind and ubiquitylate protein VI. We further show that this PPxY motif is involved in rapid, microtubule-dependent intracellular movement of protein VI. Ads with a mutated PPxY motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. Likewise, depletion of Nedd4 ligases attenuates nuclear accumulation of incoming Ad particles and infection. Our data provide the first evidence that virus-encoded PPxY motifs are required during virus entry, which may be of significance for several other pathogens.
Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/crescimento & desenvolvimento , Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Humanos , Pulmão/citologia , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/virologia , Microtúbulos/metabolismo , Microtúbulos/virologia , Ubiquitina-Proteína Ligases Nedd4 , Osteossarcoma , Estrutura Terciária de Proteína , Epitélio Pigmentado da Retina/citologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologiaRESUMO
The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 A by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via alphav integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/fisiologia , Adenoviridae/ultraestrutura , Mutação , Internalização do Vírus , Adenoviridae/química , Adenoviridae/genética , Capsídeo/química , Capsídeo/metabolismo , Linhagem Celular , Microscopia Crioeletrônica , Humanos , Temperatura , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/genética , Vírion/fisiologia , Vírion/ultraestrutura , Montagem de VírusRESUMO
Diverse virus families have evolved to exploit the acidification of endosomal compartments to gain entry into cells. We describe a supramolecular approach for selectively targeting and inhibiting viral infections through this central biochemical pathway. Using adenovirus as a model non-enveloped virus, we have determined that an eight-residue cyclic D,L-alpha-peptide, selected from a directed combinatorial library, can specifically prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection without an apparent adverse effect on cell viability. The likely generality of this approach against other pH-dependent viral infections is supported by the inhibition of type-A influenza virus escape from endosomes in the presence of the same peptide. Our studies suggest that self-assembling cyclic D,L-alpha-peptides hold considerable potential as a new rational supramolecular approach toward the design and discovery of broad-spectrum antiviral agents.
Assuntos
Antivirais/farmacologia , Peptídeos Cíclicos/farmacologia , Viroses/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/uso terapêutico , Linhagem Celular , Cães , Corantes Fluorescentes , Humanos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêutico , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
In contrast to enveloped viruses, the mechanisms involved in membrane penetration by nonenveloped viruses are not as well understood. In these studies, we determined the relationship between adenovirus (Ad) capsid disassembly and the development of membrane lytic activity. Exposure to low pH or heating induced conformational changes in wild-type Ad but not in temperature-sensitive Ad (ts1) particles that fail to escape the early endosome. Wild-type Ad but not ts1 particles permeabilized model membranes (liposomes) and facilitated the cytosolic delivery of a ribotoxin. Alterations in wild-type Ad capsids were associated with the exposure of a pH-independent membrane lytic factor. Unexpectedly, this factor was identified as protein VI, a 22-kDa cement protein located beneath the peripentonal hexons in the viral capsid. Recombinant protein VI and preprotein VI, but not a deletion mutant lacking an N-terminal amphipathic alpha-helix, possessed membrane lytic activity similar to partially disassembled virions. A new model of Ad entry is proposed based on our present observations of capsid disassembly and membrane penetration.
Assuntos
Adenoviridae/química , Proteínas do Capsídeo/farmacologia , Membrana Celular/efeitos dos fármacos , Vírion/fisiologia , Adenoviridae/classificação , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células Tumorais CultivadasRESUMO
Thermally induced structural changes in adenovirus type 5 (Ad) in the presence of either 2 or 10% sucrose were investigated using a variety of biophysical techniques. In solutions containing 2% sucrose, a highly cooperative transition in the structure of the virus was observed at 45 degrees C as detected by tryptophan fluorescence, derivative UV absorption spectroscopy, circular dichroism (CD), and dynamic and static light scattering. This transition resulted in (at least partial) disassembly of the virus and a concomitant increase in the accessibility of the viral DNA to the fluorescent dye, TOTO-1. Capsid disassembly was verified by transmission electron microscopy, which showed ruptured icosahedral vertices near 45 degrees C followed by complete capsid disassembly at higher temperatures. SDS-PAGE of thermally treated Ad suggests that the penton base (protein III) and protein IIIa (located in the peripentonal region) are significantly more labile than other capsid proteins and may be the initial instigators in capsid disassembly. Additional discrete structural transitions were observed in viral proteins using the aforementioned spectroscopic techniques. Thermally induced rearrangements of the condensed DNA at higher temperatures were also detected by the appearance of "psi"-like features in the CD spectra as well as a dramatic decrease in accessibility of DNA to TOTO-1. These transitions corresponded to discrete endothermic events that are also detected by differential scanning calorimetry. By increasing the concentration of sucrose to 10%, secondary and tertiary structural features of adenoviral proteins were significantly stabilized, although loss of quaternary structure at 45 degrees C was still observed.