RESUMO
Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.
Assuntos
Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/virologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Infecções por Herpesviridae/metabolismo , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Muromegalovirus/metabolismo , Mutação , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas G/genética , Glândulas Salivares/virologia , Transdução de Sinais , Proteínas Virais/genética , Viremia/metabolismo , Viremia/virologia , Ativação Viral/genéticaRESUMO
Cytomegaloviruses (CMVs) establish systemic infections across diverse cell types. Glycoproteins that alter tropism can potentially guide their spread. Glycoprotein O (gO) is a nonessential fusion complex component of both human CMV (HCMV) and murine CMV (MCMV). We tested its contribution to MCMV spread from the respiratory tract. In vitro, MCMV lacking gO poorly infected fibroblasts and epithelial cells. Cell binding was intact, but penetration was delayed. In contrast, myeloid infection was preserved, and in the lungs, where myeloid and type 2 alveolar epithelial cells are the main viral targets, MCMV lacking gO showed a marked preference for myeloid infection. Its poor epithelial cell infection was associated with poor primary virus production and reduced virulence. Systemic spread, which proceeds via infected CD11c+ myeloid cells, was initially intact but then diminished, because less epithelial infection led ultimately to less myeloid infection. Thus, the tight linkage between peripheral and systemic MCMV infections gave gO-dependent infection a central role in host colonization.IMPORTANCE Human cytomegalovirus is a leading cause of congenital disease. This reflects its capacity for systemic spread. A vaccine is needed, but the best viral targets are unclear. Attention has focused on the virion membrane fusion complex. It has 2 forms, so we need to know what each contributes to host colonization. One includes the virion glycoprotein O. We used murine cytomegalovirus, which has equivalent fusion complexes, to determine the importance of glycoprotein O after mucosal infection. We show that it drives local virus replication in epithelial cells. It was not required to infect myeloid cells, which establish systemic infection, but poor local replication reduced systemic spread as a secondary effect. Therefore, targeting glycoprotein O of human cytomegalovirus has the potential to reduce both local and systemic infections.
Assuntos
Células Epiteliais/virologia , Fibroblastos/virologia , Infecções por Herpesviridae/virologia , Pulmão/virologia , Glicoproteínas de Membrana/metabolismo , Muromegalovirus/patogenicidade , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Infecções por Herpesviridae/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Internalização do VírusRESUMO
Cytomegaloviruses (CMVs) persistently and systemically infect the myeloid cells of immunocompetent hosts. Persistence implies immune evasion, and CMVs evade CD8+ T cells by inhibiting MHC class I-restricted antigen presentation. Myeloid cells can also interact with CD4+ T cells via MHC class II (MHC II). Human CMV (HCMV) attacks the MHC II presentation pathway in vitro, but what role this evasion might play in host colonization is unknown. We show that Murine CMV (MCMV) down-regulates MHC II via M78, a multi-membrane spanning viral protein that captured MHC II from the cell surface and was necessary although not sufficient for its degradation in low pH endosomes. M78-deficient MCMV down-regulated MHC I but not MHC II. After intranasal inoculation, it showed a severe defect in salivary gland colonization that was associated with increased MHC II expression on infected cells, and was significantly rescued by CD4+ T cell loss. Therefore MCMV requires CD4+ T cell evasion by M78 to colonize the salivary glands, its main site of long-term shedding.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Evasão da Resposta Imune , Muromegalovirus/fisiologia , Proteólise , Glândulas Salivares/imunologia , Glândulas Salivares/virologia , Animais , Células 3T3 BALB , Células Cultivadas , Cricetinae , Embrião de Mamíferos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/imunologia , Células NIH 3T3 , Glândulas Salivares/metabolismo , Glândulas Salivares/patologiaRESUMO
Human cytomegalovirus (HCMV) colonizes blood-borne dendritic cells (DCs). They express US28, a viral G protein-coupled receptor (GPCR). In vitro functions have been described for US28, but how it contributes to host colonization has been unclear. The murine CMV (MCMV) M33 GPCR promotes DC recirculation. We show that US28 shares this function. Thus, DC recirculation is also available to HCMV via US28, and inhibiting US28 G protein-dependent signalling has the potential to reduce systemic infection. We show that M33 also promotes systemic infection through infected DC extravasation.
Assuntos
Movimento Celular , Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Células Dendríticas/virologia , Interações Hospedeiro-Patógeno , Linfonodos/virologia , Receptores de Quimiocinas/metabolismo , Proteínas Virais/metabolismo , Estruturas Animais/virologia , Animais , Células Cultivadas , Citomegalovirus/crescimento & desenvolvimento , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/veterinária , Células Dendríticas/imunologia , Humanos , Linfonodos/imunologia , Camundongos Endogâmicos BALB C , Muromegalovirus/crescimento & desenvolvimentoRESUMO
Cytomegaloviruses (CMVs) establish chronic, systemic infections. Peripheral infection spreads via lymph nodes, which are also a focus of host defence. Thus, this is a point at which systemic infection spread might be restricted. Subcapsular sinus macrophages (SSM) captured murine CMV (MCMV) from the afferent lymph and poorly supported its replication. Blocking the type I interferon (IFN-I) receptor (IFNAR) increased MCMV infection of SSM and of the fibroblastic reticular cells (FRC) lining the subcapsular sinus, and accelerated viral spread to the spleen. Little splenic virus derived from SSM, arguing that they mainly induce an anti-viral state in the otherwise susceptible FRC. NK cells also limited infection, killing infected FRC and causing tissue damage. They acted independently of IFN-I, as IFNAR blockade increased NK cell recruitment, and NK cell depletion increased infection in IFNAR-blocked mice. Thus SSM restricted MCMV infection primarily though IFN-I, with NK cells providing a second line of defence. The capacity of innate immunity to restrict MCMV escape from the subcapsular sinus suggested that enhancing its recruitment might improve infection control.
Assuntos
Infecções por Herpesviridae/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Animais , Linfonodos/virologia , Macrófagos/virologia , Camundongos , Muromegalovirus/imunologiaRESUMO
Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.
Assuntos
Tronco Encefálico/fisiologia , Rede Nervosa/fisiologia , Técnicas de Rastreamento Neuroanatômico/métodos , Prosencéfalo/fisiologia , Células Receptoras Sensoriais/fisiologia , Traqueia/fisiologia , Animais , Tronco Encefálico/química , Herpesvirus Humano 1 , Masculino , Rede Nervosa/química , Prosencéfalo/química , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/química , Sinapses/química , Sinapses/fisiologia , Traqueia/química , Traqueia/inervaçãoRESUMO
Cytomegaloviruses (CMVs) establish persistent, systemic infections and cause disease by maternal-foetal transfer, suggesting that their dissemination is a key target for antiviral intervention. Late clinical presentation has meant that human CMV (HCMV) dissemination is not well understood. Murine CMV (MCMV) provides a tractable model. Whole mouse imaging of virus-expressed luciferase has proved a useful way to track systemic infections. MCMV, in which the abundant lytic gene M78 was luciferase-tagged via a self-cleaving peptide (M78-LUC), allowed serial, unbiased imaging of systemic and peripheral infection without significant virus attenuation. Ex vivo luciferase imaging showed greater sensitivity than plaque assay, and revealed both well-known infection sites (the lungs, lymph nodes, salivary glands, liver, spleen and pancreas) and less explored sites (the bone marrow and upper respiratory tract). We applied luciferase imaging to tracking MCMV lacking M33, a chemokine receptor conserved in HCMV and a proposed anti-viral drug target. M33-deficient M78-LUC colonized normally in peripheral sites and local draining lymph nodes but spread poorly to the salivary gland, suggesting a defect in vascular transport consistent with properties of a chemokine receptor.
Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Luciferases/genética , Tropismo Viral , Animais , Citomegalovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Feminino , Genes Reporter , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Imagem Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency, phenotypes partially rescued by the human cytomegalovirus CKR US28. Herein, we demonstrate that complementation of salivary gland tropism is mediated predominantly by G protein-dependent signaling conserved with that of M33; in contrast, both G protein-dependent and -independent pathways contribute to the latency phenotypes. A novel M33-dependent replication phenotype in cultured bone marrow macrophages is also described.
Assuntos
Citomegalovirus/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Glândulas Salivares/virologia , Transdução de Sinais/fisiologia , Tropismo Viral/fisiologia , Ativação Viral/fisiologia , Análise de Variância , Animais , Células COS , Chlorocebus aethiops , Citomegalovirus/genética , Citomegalovirus/metabolismo , Células HEK293 , Humanos , Luciferases , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Octoxinol , Fenótipo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Latência Viral/fisiologiaRESUMO
Acyclovir (ACV) as a model antiviral microbicide, was incorporated in controlled-release polycaprolactone (PCL) matrices designed for application as intra-vaginal ring inserts (IVRs). Microporous materials incorporating acyclovir up to a level of ~10 % w/w were produced by rapidly cooling suspensions of drug powder in PCL solution followed by solvent extraction from the hardened matrices. Around 21, 50 and 78 % of the drug content was gradually released from matrices over 30 days in simulated vaginal fluid at 37 °C, corresponding to drug loadings of 5.9, 7.0 and 9.6 % w/w. The release behaviour of matrices having the lowest drug loading followed a zero order model, whereas, the release kinetics of 7.0 and 9.6 % ACV-loaded PCL matrices could be described effectively by the Higuchi model, suggesting that Fickian diffusion is controlling drug release. Corresponding values of the diffusion co-efficient for ACV in the PCL matrices of 3.16 × 10(-9) and 1.07 × 10(-8) cm(2)/s were calculated. Plaque reduction assays provided an IC50 value of 1.09 µg/mL for acyclovir against HSV-2 and confirmed the antiviral activity of released acyclovir against HSV-2 replication in primate kidney cells (Vero) at levels ~70 % that of non-formulated acyclovir at day 30. Estimated minimum in vivo acyclovir concentrations produced by a PCL IVR (19 µg/mL) exceeded by a factor of 20 the IC50 value against HSV-2 and the reported ACV vaginal concentrations in women (0.5-1.0 µg/mL) following oral administration. These findings recommend further investigations of PCL matrices for vaginal delivery of antiviral agents in the treatment and prevention of sexually transmitted infections such as AIDS.
Assuntos
Aciclovir/administração & dosagem , Antivirais/administração & dosagem , Sistemas de Liberação de Medicamentos , Poliésteres/química , Vagina/efeitos dos fármacos , Aciclovir/farmacocinética , Administração Intravaginal , Antivirais/farmacocinética , Preparações de Ação Retardada , Feminino , Dureza , Herpesvirus Humano 2 , Humanos , Concentração Inibidora 50 , Teste de Materiais , Solventes/química , Vagina/virologia , Viroses/prevenção & controleRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world population and causes lifelong latent infection. HCMV has been shown to exacerbate cardiovascular diseases, including myocarditis, vascular sclerosis, and transplant vasculopathy. Recently, we have shown that murine CMV (MCMV) recapitulates the cardiovascular dysfunction observed in patients with HCMV-induced myocarditis. To understand the viral mechanisms involved in CMV-induced heart dysfunction, we further characterized cardiac function in response to MCMV and examined virally encoded G-protein-coupled receptor homologs (vGPCRs) US28 and M33 as potential factors that promote infection in the heart. We hypothesized that the CMV-encoded vGPCRs could exacerbate cardiovascular damage and dysfunction. Three viruses were used to evaluate the role of vGPCRs in cardiac dysfunction: wild-type MCMV, a M33-deficient virus (∆M33), and a virus with the M33 open reading frame (ORF) replaced with US28, an HCMV vGPCR (i.e., US28+). Our in vivo studies revealed that M33 plays a role in promoting cardiac dysfunction by increasing viral load and heart rate during acute infection. During latency, ΔM33-infected mice demonstrated reduced calcification, altered cellular gene expression, and less cardiac hypertrophy compared with wild-type MCMV-infected mice. Ex vivo viral reactivation from hearts was less efficient in ΔM33-infected animals. HCMV protein US28 expression restored the ability of the M33-deficient virus to reactivate from the heart. US28+ MCMV infection caused damage to the heart comparable with wild-type MCMV infection, suggesting that the US28 protein is sufficient to complement the function of M33 in the heart. Altogether, these data suggest a role for vGPCRs in viral pathogenesis in the heart and thus suggest that vGPCRs promote long-term cardiac damage and dysfunction.
Assuntos
Infecções por Citomegalovirus , Cardiopatias , Muromegalovirus , Miocardite , Humanos , Animais , Camundongos , Muromegalovirus/fisiologia , Receptores de Quimiocinas/genética , Proteínas Virais/metabolismo , Citomegalovirus/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The human cytomegalovirus (CMV) proteins US28 and UL33 are homologous to chemokine receptors (CKRs). Knockout of the mouse CMV M33 protein (UL33 homologue) results in substantial attenuation of salivary gland infection/replication and reduced efficiency of reactivation from tissue explants. M33-mediated G protein-coupled signaling is critical for the salivary gland phenotype. In this report, we demonstrate that US28 and (to a lesser degree) UL33 restore reactivation from tissue explants and partially restore replication in salivary glands (compared to a signaling-deficient M33 mutant). These studies provide a novel small animal model for evaluation of therapies targeting the human CMV CKRs.
Assuntos
Citomegalovirus/fisiologia , Modelos Animais de Doenças , Muromegalovirus/fisiologia , Receptores de Quimiocinas/metabolismo , Proteínas Virais/metabolismo , Animais , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Feminino , Infecções por Herpesviridae/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Muromegalovirus/metabolismo , Especificidade de Órgãos , Receptores de Quimiocinas/genética , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia , Proteínas Virais/genética , Ativação Viral , Latência Viral , Replicação ViralRESUMO
Human cytomegalovirus (HCMV) encodes four homologs of G protein coupled receptors (vGPCRs), of which two, designated UL33 and US28, signal constitutively. UL33 and US28 are also conserved with chemokine receptors: US28 binds numerous chemokine classes, including the membrane bound chemokine, fractalkine; whereas UL33 remains an orphan receptor. There is emerging data that UL33 and US28 each contribute to HCMV associated disease, although no studies to date have reported their potential contribution to aberrant placental physiology that has been detected with HCMV congenital infection. We investigated the signaling repertoire of UL33 and US28 and their potential to enable trophoblast mobilization in vitro. Results demonstrate the constitutive activation of CREB by each vGPCR in ACIM-88 and HTR-8SVneo trophoblasts; constitutive NF-kB activation was detected for US28 only. Constitutive signaling by each vGPCR enabled trophoblast migration. For US28, fractalkine exhibited inverse agonist activity and dampened trophoblast migration. UL33 stimulated expression of both p38 mitogen activated (MAP) and Jun N-terminal (JNK) kinases; while p38 MAP kinase stimulated CREB, JNK was inhibitory, suggesting that UL33 dependent CREB activation was regulated by p38/JNK crosstalk. Given that chemokines and their receptors are important for placental development, these data point to the potential of HCMV UL33 and US28 to interfere with trophoblast responses which are important for normal placental development.
Assuntos
Citomegalovirus/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Quimiocina CX3CL1/metabolismo , Citomegalovirus/fisiologia , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Introduction: Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. Methods: Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. Results: We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. Discussion: Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.
Assuntos
Infecções por Citomegalovirus , Evasão da Resposta Imune , Humanos , Animais , Camundongos , Latência Viral/fisiologia , Citomegalovirus/fisiologia , Receptores Acoplados a Proteínas G , Linfócitos T CD8-Positivos , Infecções por Citomegalovirus/genéticaRESUMO
M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, DeltaM33B(T2), was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, DeltaM33B(T2) exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced DeltaM33B(T2) viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent DeltaM33B(T2) viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of DeltaM33B(T2) virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.
Assuntos
Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Receptores de Quimiocinas/metabolismo , Proteínas Virais/metabolismo , Latência Viral , Replicação Viral , Animais , Feminino , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Células NIH 3T3 , Especificidade de Órgãos , Pâncreas/virologia , Receptores de Quimiocinas/genética , Glândulas Salivares/virologia , Baço/virologia , Proteínas Virais/genéticaRESUMO
In this study, we determined the function of a novel non-ribosomal peptide synthetase (NRPS) system carried by a streptococcal integrative conjugative element (ICE), ICESe2. The NRPS shares similarity with the yersiniabactin system found in the high-pathogenicity island of Yersinia sp. and is the first of its kind to be identified in streptococci. We named the NRPS product 'equibactin' and genes of this locus eqbA-N. ICESe2, although absolutely conserved in Streptococcus equi, the causative agent of equine strangles, was absent from all strains of the closely related opportunistic pathogen Streptococcus zooepidemicus. Binding of EqbA, a DtxR-like regulator, to the eqbB promoter was increased in the presence of cations. Deletion of eqbA resulted in a small-colony phenotype. Further deletion of the irp2 homologue eqbE, or the genes eqbH, eqbI and eqbJ encoding a putative ABC transporter, or addition of the iron chelator nitrilotriacetate, reversed this phenotype, implicating iron toxicity. Quantification of (55)Fe accumulation and sensitivity to streptonigrin suggested that equibactin is secreted by S. equi and that the eqbH, eqbI and eqbJ genes are required for its associated iron import. In agreement with a structure-based model of equibactin synthesis, supplementation of chemically defined media with salicylate was required for equibactin production.
Assuntos
Proteínas de Bactérias/metabolismo , Compostos Férricos/metabolismo , Peptídeo Sintases/biossíntese , Streptococcus equi/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cloretos , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Família Multigênica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Streptococcus equi/efeitos dos fármacos , Streptococcus equi/metabolismo , Estreptonigrina/farmacologia , Especificidade por SubstratoRESUMO
The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N- and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R(340) and A(353) of the C terminus was found to be important for CREB- and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.
Assuntos
Muromegalovirus/fisiologia , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Sequência Conservada , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos , Muromegalovirus/genética , Fatores de Transcrição NFATC/metabolismo , Fosfatidilinositóis/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Receptores de Quimiocinas/genética , Glândulas Salivares/virologia , Transdução de Sinais , Proteínas Virais/genética , Replicação ViralRESUMO
Infection with equid herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurologic disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism resulting in an amino acid variation of the EHV-1 DNA polymerase (N752/D752) is significantly associated with the neuropathogenic potential of naturally occurring strains. To test the hypothesis that this single amino acid exchange by itself influences neuropathogenicity, we generated recombinant viruses with differing polymerase sequences. Here we show that the N752 mutant virus caused no neurologic signs in the natural host, while the D752 virus was able to cause inflammation of the central nervous system and ataxia. Neurologic disease induced by the D752 virus was concomitant with significantly increased levels of viremia (p = 0.01), but the magnitude of virus shedding from the nasal mucosa was similar between the N752 and D752 viruses. Both viruses replicated with similar kinetics in fibroblasts and epithelial cells, but exhibited differences in leukocyte tropism. Last, we observed a significant increase (p < 0.001) in sensitivity of the N752 mutant to aphidicolin, a drug targeting the viral polymerase. Our results demonstrate that a single amino acid variation in a herpesvirus enzyme can influence neuropathogenic potential without having a major effect on virus shedding from infected animals, which is important for horizontal spread in a population. This observation is very interesting from an evolutionary standpoint and is consistent with data indicating that the N752 DNA pol genotype is predominant in the EHV-1 population, suggesting that decreased viral pathogenicity in the natural host might not be at the expense of less efficient inter-individual transmission.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/enzimologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/genética , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Afidicolina/farmacologia , Western Blotting , Linfócitos T CD4-Positivos/virologia , Cromossomos Artificiais Bacterianos , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/efeitos dos fármacos , Feminino , Genótipo , Infecções por Herpesviridae/patologia , Doenças dos Cavalos/enzimologia , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-AtividadeRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0009681.].
RESUMO
Mesoporous silica nanoparticles (MSNs) are drug delivery agents that are able to incorporate drugs within their pores. Furthermore, MSNs can be functionalized by attachment of bioactive ligands on their surface to enhance their activity, and nanoparticles modified with glycosaminoglycan (GAG) mimetics inhibit the entry of herpes simplex virus (HSV) into cells. In this study, structure-activity relationships of GAGs attached to MSNs were investigated in relation to HSV-1 and HSV-2, and acyclovir was loaded into the pores of MSNs. The sulfonate group was demonstrated to be essential for antiviral activity, which was enhanced by incorporating a benzene group within the ligand. Loading acyclovir into GAG mimetic-functionalized MSNs reduced the viral infection, resulting in nanoparticles that simultaneously target two distinct viral pathways, namely, inhibition of viral entry and inhibition of DNA replication.
RESUMO
Herpesviruses have coevolved with their hosts over hundreds of millions of years and exploit fundamental features of their biology. Cytomegaloviruses (CMVs) colonize blood-borne myeloid cells, and it has been hypothesized that systemic dissemination arises from infected stem cells in bone marrow. However, poor CMV transfer by stem cell transplantation argues against this being the main reservoir. To identify alternative pathways for CMV spread, we tracked murine CMV (MCMV) colonization after mucosal entry. We show that following intranasal MCMV infection, lung CD11c+ dendritic cells (DC) migrated sequentially to lymph nodes (LN), blood, and then salivary glands. Replication-deficient virus followed the same route, and thus, DC infected peripherally traversed LN to enter the blood. Given that DC are thought to die locally following their arrival and integration into LN, recirculation into blood represents a new pathway. We examined host and viral factors that facilitated this LN traverse. We show that MCMV-infected DC exited LN by a distinct route to lymphocytes, entering high endothelial venules and bypassing the efferent lymph. LN exit required CD44 and the viral M33 chemokine receptor, without which infected DC accumulated in LN and systemic spread was greatly reduced. Taken together, our studies provide the first demonstration of virus-driven DC recirculation. As viruses follow host-defined pathways, high endothelial venules may normally allow DC to pass from LN back into blood.IMPORTANCE Human cytomegalovirus (HCMV) causes devastating disease in the unborn fetus and in the immunocompromised. There is no licensed vaccine, and preventive measures are impeded by our poor understanding of early events in host colonization. HCMV and murine CMV (MCMV) both infect blood-borne myeloid cells. HCMV-infected blood cells are thought to derive from infected bone marrow stem cells. However, infected stem cells have not been visualized in vivo nor shown to produce virus ex vivo, and hematopoietic transplants poorly transfer infection. We show that MCMV-infected dendritic cells in the lungs reach the blood via lymph nodes, surprisingly migrating into high endothelial venules. Dissemination did not require viral replication. It depended on the constitutively active viral chemokine receptor M33 and on the host hyaluronan receptor CD44. Thus, viral chemokine receptors are a possible target to limit systemic CMV infections.