RESUMO
Worldwide, the incidence of both preterm births and chronic lung disease of infancy, or bronchopulmonary dysplasia, remains high. Infants with bronchopulmonary dysplasia have larger and fewer alveoli, a lung pathology that can persist into adulthood. Although recent data point to a role for hypoxia-inducible factor-1α (HIF-1α) in mediating pulmonary angiogenesis and alveolarization, the cell-specific role of HIF-1α remains incompletely understood. Thus, we hypothesized that HIF-1α, in a distinct subset of mesenchymal cells, mediates postnatal alveolarization. To test the hypothesis, we generated mice with a cell-specific deletion of HIF-1α by crossing SM22α promoter-driven Cre mice with HIF-1αflox/flox mice (SM22α-HIF-1α-/-), determined SM-22α-expressing cell identity using single-cell RNA sequencing, and interrogated samples from preterm infants. Deletion of HIF-1α in SM22α-expressing cells had no effect on lung structure at day 3 of life. However, at 8 days, there were fewer and larger alveoli, a difference that persisted into adulthood. Microvascular density, elastin organization, and peripheral branching of the lung vasculature were decreased in SM22α-HIF-1α-/- mice, compared with control mice. Single-cell RNA sequencing demonstrated that three mesenchymal cell subtypes express SM22α: myofibroblasts, airway smooth muscle cells, and vascular smooth muscle cells. Pulmonary vascular smooth muscle cells from SM22α-HIF-1α-/- mice had decreased angiopoietin-2 expression and, in coculture experiments, a diminished capacity to promote angiogenesis that was rescued by angiopoietin-2. Angiopoietin-2 expression in tracheal aspirates of preterm infants was inversely correlated with overall mechanical ventilation time, a marker of disease severity. We conclude that SM22α-specific HIF-1α expression drives peripheral angiogenesis and alveolarization in the lung, perhaps by promoting angiopoietin-2 expression.
Assuntos
Angiopoietina-2 , Displasia Broncopulmonar , Subunidade alfa do Fator 1 Induzível por Hipóxia , Animais , Humanos , Recém-Nascido , Camundongos , Angiopoietina-2/metabolismo , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Recém-Nascido Prematuro , Pulmão/patologiaRESUMO
Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2-/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2+/+ than in SM22α-PHD1/2-/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2+/+ compared with PHD1/2-/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2-/- compared with SM22α-PHD1/2+/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH.NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , Hipertensão Pulmonar Primária Familiar/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Prolil Hidroxilases/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Remodelação VascularRESUMO
Though survival rates for preterm infants are improving, the incidence of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Histologically, BPD is characterized by larger and fewer alveoli. Hypoxia-inducible factors (HIFs) may be protective in the context of hyperoxia-induced lung injury, but the cell-specific effects of HIF expression in neonatal lung injury remain unknown. Thus, we sought to determine whether HIF stabilization in SM22α-expressing cells can limit hyperoxia-induced neonatal lung injury. We generated SM22α-specific HIF-1α-stabilized mice (SM22α-PHD1/2-/- mice) by cross-breeding SM22α-promotor-driven Cre recombinase mice with prolyl hydroxylase PHD1flox/flox and PHD2flox/flox mice. Neonatal mice were randomized to 21% O2 (normoxia) or 80% O2 (hyperoxia) exposure for 14 days. For the hyperoxia recovery studies, neonatal mice were recovered from normoxia for an additional 10 wk. SM22α-specific HIF-1α stabilization mitigated hyperoxia-induced lung injury and preserved microvessel density compared with control mice for both neonates and adults. In SM22α-PHD1/2-/- mice, pulmonary artery endothelial cells (PAECs) were more proliferative and pulmonary arteries expressed more collagen IV compared with control mice, even under hyperoxic conditions. Angiopoietin-2 (Ang2) mRNA expression in pulmonary artery smooth muscle cells (PASMC) was greater in SM22α-PHD1/2-/- compared with control mice in both normoxia and hyperoxia. Pulmonary endothelial cells (PECs) cocultured with PASMC isolated from SM22α-PHD1/2-/- mice formed more tubes and branches with greater tube length compared with PEC cocultured with PASMC isolated from SM22α-PHD1/2+/+ mice. Addition of Ang2 recombinant protein further augmented tube formation for both PHD1/2+/+ and PHD1/2-/- PASMC. Cell-specific deletion of PHD1 and 2 selectively increases HIF-1α expression in SM22α-expressing cells and protects neonatal lung development despite prolonged hyperoxia exposure. HIF stabilization in SM22α-expressing cells preserved endothelial cell proliferation, microvascular density, increased angiopoietin-2 expression, and lung structure, suggesting a role for cell-specific HIF-1α stabilization to prevent neonatal lung injury.
Assuntos
Hiperóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Lesão Pulmonar , Angiopoietina-2/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Células Endoteliais/metabolismo , Humanos , Hiperóxia/metabolismo , Hiperóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Recém-Nascido , Recém-Nascido Prematuro , Pulmão/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/prevenção & controle , CamundongosRESUMO
Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.
Assuntos
Herpesvirus Humano 3/fisiologia , Proteínas Inibidoras de Apoptose/genética , Fator de Transcrição STAT3/genética , Ativação Transcricional/fisiologia , Replicação Viral/fisiologia , Ácidos Aminossalicílicos/farmacologia , Animais , Benzenossulfonatos/farmacologia , Citometria de Fluxo , Humanos , Medições Luminescentes , Camundongos , Camundongos SCID , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Pele/metabolismo , Pele/virologia , Survivina , Ativação Transcricional/genética , Replicação Viral/genéticaRESUMO
The tropism of herpes simplex virus (HSV-1) for human sensory neurons infected in vivo was examined using dorsal root ganglion (DRG) xenografts maintained in mice with severe combined immunodeficiency (SCID). In contrast to the HSV-1 lytic infectious cycle in vitro, replication of the HSV-1 F strain was restricted in human DRG neurons despite the absence of adaptive immune responses in SCID mice, allowing the establishment of neuronal latency. At 12 days after DRG inoculation, 26.2% of human neurons expressed HSV-1 protein and 13.1% expressed latency-associated transcripts (LAT). Some infected neurons showed cytopathic changes, but HSV-1, unlike varicella-zoster virus (VZV), only rarely infected satellite cells and did not induce fusion of neuronal and satellite cell plasma membranes. Cell-free enveloped HSV-1 virions were observed, indicating productive infection. A recombinant HSV-1-expressing luciferase exhibited less virulence than HSV-1 F in the SCID mouse host, enabling analysis of infection in human DRG xenografts for a 61-day interval. At 12 days after inoculation, 4.2% of neurons expressed HSV-1 proteins; frequencies increased to 32.1% at 33 days but declined to 20.8% by 61 days. Frequencies of LAT-positive neurons were 1.2% at 12 days and increased to 40.2% at 33 days. LAT expression remained at 37% at 61 days, in contrast to the decline in neurons expressing viral proteins. These observations show that the progression of HSV-1 infection is highly restricted in human DRG, and HSV-1 genome silencing occurs in human neurons infected in vivo as a consequence of virus-host cell interactions and does not require adaptive immune control.
Assuntos
Gânglios Espinais/virologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Imunodeficiência Combinada Severa/virologia , Tropismo Viral , Aciclovir/administração & dosagem , Aciclovir/análogos & derivados , Aciclovir/farmacologia , Animais , Gânglios Espinais/patologia , Expressão Gênica , Herpes Simples/tratamento farmacológico , Herpes Simples/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 3 , Humanos , Luciferases/biossíntese , Camundongos , Camundongos SCID , Células Satélites Perineuronais/virologia , Transplante Heterólogo , Valaciclovir , Valina/administração & dosagem , Valina/análogos & derivados , Valina/farmacologia , Proteínas Virais/metabolismo , Latência Viral , Replicação ViralRESUMO
The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.
Assuntos
Varicela/virologia , Herpesvirus Humano 3/metabolismo , Fases de Leitura Aberta , Proteínas de Ligação a RNA/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Linhagem Celular , Herpesvirus Humano 3/genética , Humanos , Camundongos , Camundongos SCID , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas Estruturais Virais/genéticaRESUMO
Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Células-Tronco Mesenquimais , Recém-Nascido , Lactente , Humanos , Células Endoteliais , PulmãoRESUMO
Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-DeltaP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-DeltaY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.
Assuntos
Varicela/fisiopatologia , Herpesvirus Humano 3/patogenicidade , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular Tumoral , Varicela/metabolismo , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiologia , Humanos , Camundongos , Camundongos SCID , Mutagênese , Estrutura Terciária de Proteína , Pele/citologia , Pele/patologia , Pele/virologia , Dermatopatias/patologia , Dermatopatias/virologia , Transplante de Pele , Linfócitos T/imunologia , Linfócitos T/virologia , Transplante Heterólogo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Replicação Viral/genéticaRESUMO
At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.
RESUMO
MxA is an antiviral protein induced by interferon (IFN)-α/ß that is known to inhibit the replication of many RNA viruses. In these experiments, the 76-kDa MxA protein expressed in IFN-α-treated cells was shown to have antiviral activity against herpes simplex virus-1 (HSV-1), a human DNA virus. However, MxA was expressed as a 56-kDa protein in HSV-1-infected cells in the absence of IFN-α. This previously unrecognized MxA isoform was produced from an alternatively spliced MxA transcript that had a deletion of Exons 14-16 and a frame shift altering the C-terminus. The variant MxA (varMxA) isoform was associated with HSV-1 regulatory proteins and virions in nuclear replication compartments. varMxA expression enhanced HSV-1 infection as shown by a reduction in infectious virus titers from cells in which MxA had been inhibited by RNA interference and by an increase in HSV-1 titers when the 56-kDa varMxA was expressed constitutively. Thus, the human MxA gene encodes two MxA isoforms, which are expressed differentially depending on whether the stimulus is IFN-α or HSV-1. These findings show that alternative splicing of cellular mRNA can result in expression of a novel isoform of a host defense gene that supports instead of restricting viral infection.
Assuntos
Proteínas de Ligação ao GTP/genética , Herpesvirus Humano 1/fisiologia , Replicação Viral/fisiologia , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/ultraestrutura , Humanos , Interferon-alfa/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/virologia , Dados de Sequência Molecular , Proteínas de Resistência a Myxovirus , Biossíntese de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-beta production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-beta secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.
Assuntos
Herpesvirus Humano 3/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas Imediatamente Precoces/fisiologia , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/antagonistas & inibidores , Fator Regulador 3 de Interferon/imunologia , Transativadores/fisiologia , Proteínas do Envelope Viral/fisiologia , Fatores de Virulência/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Células Cultivadas , Regulação para Baixo , Fibroblastos/virologia , Perfilação da Expressão Gênica , Genes Reporter , Herpesvirus Humano 3/imunologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/antagonistas & inibidores , Luciferases/genética , Luciferases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA , Serina/metabolismo , Fatores de Transcrição/biossínteseRESUMO
Varicella-zoster virus (VZV) is a medically important human alphaherpesvirus that causes varicella and zoster. VZV initiates primary infection by inoculation of the respiratory mucosa. In the course of primary infection, VZV establishes a life-long persistence in sensory ganglia; VZV reactivation from latency may result in zoster in healthy and immunocompromised patients. The VZV genome has at least 70 known or predicted open reading frames (ORFs), but understanding how these gene products function in virulence is difficult because VZV is a highly human-specific pathogen. We have addressed this obstacle by investigating VZV infection of human tissue xenografts in the severe combined immunodeficiency mouse model. In studies relevant to the pathogenesis of primary VZV infection, we have examined VZV infection of human T cell (thymus/liver) and skin xenografts. This work supports a new paradigm for VZV pathogenesis in which VZV T cell tropism provides a mechanism for delivering the virus to skin. We have also shown that VZV-infected T cells transfer VZV to neurons in sensory ganglia. The construction of infectious VZV recombinants that have deletions or targeted mutations of viral genes or their promoters and the evaluation of VZV mutants in T cell and skin xenografts has revealed determinants of VZV virulence that are important for T cell and skin tropism in vivo.
Assuntos
Varicela/imunologia , Varicela/virologia , Herpes Zoster/virologia , Herpesvirus Humano 3/imunologia , Tecido Linfoide/imunologia , Dermatopatias Infecciosas/virologia , Linfócitos T/imunologia , Animais , Herpes Zoster/imunologia , Humanos , Tecido Linfoide/virologia , Camundongos , Camundongos SCID , Pele/imunologia , Pele/virologia , Dermatopatias Infecciosas/imunologia , Linfócitos T/virologiaRESUMO
At birth, the lungs rapidly transition from a pathogen-free, hypoxic environment to a pathogen-rich, rhythmically distended air-liquid interface. Although many studies have focused on the adult lung, the perinatal lung remains unexplored. Here, we present an atlas of the murine lung immune compartment during early postnatal development. We show that the late embryonic lung is dominated by specialized proliferative macrophages with a surprising physical interaction with the developing vasculature. These macrophages disappear after birth and are replaced by a dynamic mixture of macrophage subtypes, dendritic cells, granulocytes, and lymphocytes. Detailed characterization of macrophage diversity revealed an orchestration of distinct subpopulations across postnatal development to fill context-specific functions in tissue remodeling, angiogenesis, and immunity. These data both broaden the putative roles for immune cells in the developing lung and provide a framework for understanding how external insults alter immune cell phenotype during a period of rapid lung growth and heightened vulnerability.
Assuntos
Pulmão/crescimento & desenvolvimento , Pulmão/imunologia , Animais , Células Dendríticas/imunologia , Granulócitos/imunologia , Homeostase , Imunomodulação , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , FenótipoRESUMO
The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.
Assuntos
Herpesvirus Humano 3/patogenicidade , Família Multigênica , Fases de Leitura Aberta , Dermatopatias Infecciosas/etiologia , Replicação Viral , Animais , Linhagem Celular Tumoral , Células Cultivadas , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma/ultraestrutura , Melanoma/virologia , Camundongos , Camundongos SCID , Transplante de Pele , Transplante HeterólogoRESUMO
VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.
Assuntos
Herpesvirus Humano 3/genética , Proteínas Imediatamente Precoces/genética , Sinais de Localização Nuclear/genética , Vírus Reordenados/genética , Transativadores/genética , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Citoplasma/metabolismo , Citoplasma/virologia , Expressão Gênica , Genes Reporter , Herpesvirus Humano 3/metabolismo , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Luciferases/genética , Luciferases/metabolismo , Mutação , Sinais de Localização Nuclear/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Vírus Reordenados/metabolismo , Transativadores/química , Transativadores/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.
Assuntos
Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidade , Fases de Leitura Aberta , Proteínas de Ligação a RNA/fisiologia , Proteínas Estruturais Virais/fisiologia , Fatores de Virulência/fisiologia , Núcleo Celular/química , Citoplasma/química , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Virais , Humanos , Proteínas Imediatamente Precoces/biossíntese , Peso Molecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Pele/patologia , Pele/virologia , Transplante Heterólogo , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
Varicella-zoster virus (VZV) open reading frame 10 (ORF10) is a determinant of virulence in SCIDhu skin xenografts but not in human T cells in vivo. In this analysis of the regulation of ORF10 transcription, we have identified four ORF10-related transcripts, including a major 1.3-kb RNA spanning ORF10 only and three other read-through transcripts. Rapid-amplification-of-cDNA-ends experiments indicated that the 1.3-kb transcript of ORF10 has single initiation and termination sites. In transient expression assays, the ORF10 promoter was strongly stimulated by the major VZV transactivator, IE62. Deletion analyses revealed approximate boundaries for the full ORF10 promoter activity between -75 and -45 and between +5 and -8, relative to the ORF10 transcription start site. The recombinant virus POKA10-Deltapro, with the ORF10 promoter deletion, blocked transcription of ORF10 and also of ORF9A and ORF9 mRNAs, whereas expression of read-through ORF9A/9/10 and ORF9/10 transcripts was increased, compensating for the loss of the monocistronic mRNAs. The cellular factor USF bound specifically to its consensus site within the ORF10 promoter and was required for IE62 transactivation, whereas disrupting the predicted TATA boxes or Oct-1 binding elements had no effect. The USF binding site was disrupted in the recombinant virus, POKA10-proDeltaUSF, and no ORF10 protein was produced. Both ORF10 promoter mutants reduced VZV replication in SCIDhu skin xenografts. These observations provided further evidence of the contribution of the ORF10 protein to VZV pathogenesis in skin and demonstrated that VZV depends upon the cellular transcriptional factor USF to support its virulence in human skin in vivo.
Assuntos
Herpesvirus Humano 3/metabolismo , Herpesvirus Humano 3/patogenicidade , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Transplante de Pele/patologia , Transplante Heterólogo/patologia , Fatores Estimuladores Upstream/metabolismo , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Genes Reporter/genética , Herpesvirus Humano 3/genética , Humanos , Camundongos , Camundongos SCID , Mutação/genética , RNA/genética , Elementos de Resposta , Transativadores/genética , Transcrição Gênica/genética , Fatores Estimuladores Upstream/genética , Virulência , Replicação ViralRESUMO
The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKA delta10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKA delta10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKA delta10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.
Assuntos
Herpesvirus Humano 3/genética , Herpesvirus Humano 3/patogenicidade , Fases de Leitura Aberta , Pele/virologia , Linfócitos T/virologia , Animais , Cosmídeos , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma/ultraestrutura , Melanoma/virologia , Camundongos , Camundongos SCID , Mutação Puntual , Recombinação Genética , Pele/patologia , Pele/ultraestrutura , Fatores de Tempo , Transplante Heterólogo , Vacinas Atenuadas/imunologia , Vírion/ultraestrutura , Virulência , Replicação ViralRESUMO
Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.
Assuntos
Herpes Zoster/virologia , Herpesvirus Humano 3/fisiologia , Proteínas Quinases/fisiologia , Linfócitos T/virologia , Animais , Linhagem Celular , Herpesvirus Humano 3/patogenicidade , Humanos , Camundongos , Camundongos SCID , Fases de Leitura Aberta/fisiologia , Virulência , Replicação ViralRESUMO
Citrus tristeza virus (CTV)-infected plants contain one or more populations of defective RNAs (dRNAs), mostly with a size range of ca. 2.0 to 5.0 kb. Several CTV dRNAs have been characterized and found to consist mainly of the two termini of the genomic RNA, with extensive internal deletions. The present paper describes a new class of large ( approximately 12.0 kb) dRNAs from three different CTV isolates with two unusual features. First is their composition with intact replicase genes. These dRNAs contained a large 5' portion of the genomic RNA terminus, which apparently corresponded to the recently described 5' large single-stranded subgenomic RNA (sgRNA) of ORF1a+1b (Che et al., 2001). The 3' portion of the large dRNAs varied among the 10 different cDNA clones examined in this work. In 2 dRNAs this portion consisted of truncated ORF10 (p20), and in 5 dRNAs it contained truncated ORF11 (p23). Two dRNA molecules were found with a 3' portion that started in the exact 5' position of the intergenic region between the p20 and p23 ORFs. In one dRNA, this portion coincided with the full-length sgRNA corresponding to ORF10. The second unusual feature was their ability to be readily transmitted mechanically to citrus plants by stem slashing and also to Nicotiana benthamiana protoplasts. The possibility that these dRNAs may be encapsidated and be capable of self-replication is discussed.