RESUMEN
A mutation (C890Y) introduced into the M70 primase gene of murine cytomegalovirus (MCMV) resulted in reduced viral replication in murine embryo fibroblasts at 40°C and the mutant was severely attenuated in vivo. The attenuated replication of the M70 mutant was also observed in Raw 264.7 macrophages at 37°C, demonstrating that the mutation produced a defective rather than an unstable protein possibly reducing the amount of functional protein under different environmental conditions. Many synonymous mutations were introduced into this ORF by changing codon preferences that should reduce the efficiency of gene translation, but not change protein sequence or structure. Two Bacterial Artificial Chromosome (BAC) constructs were produced with 155 codons (at the distal third of the M70 gene) changed to MCMV less preferred codons and with either cysteine (BAC70(155Cys) ) or tyrosine (BAC70(155Tyr) ) at residue 890. Upon transfection of these BACs into NIH 3T3 cells, only BAC70(155Cys) produced virus and this mutant Mt70(155Cys) replicated similarly to its revertant and the wt MCMV K181 (Perth) variant. A metagenomic analysis of the protein structure of the primase using PredictProtein showed that the change from cysteine (M70Cys) to tyrosine (M70Tyr) has a marked effect on protein structure. However, when the cysteine residue was replaced by serine (M70Ser) or methionine (M70Met), which produced mutant viruses with a wild-type phenotype, the predicted structure was similar to the wild-type structure. J. Med. Virol. 88:1613-1621, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Cisteína/genética , ADN Primasa/genética , Muromegalovirus/genética , Mutación , Sistemas de Lectura Abierta/genética , Tirosina/genética , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Primasa/química , Replicación del ADN , ADN Viral , Fibroblastos/virología , Macrófagos/virología , Metagenómica , Ratones , Fenotipo , Temperatura , Replicación Viral/genéticaRESUMEN
Cytomegalovirus (CMV) infection leads to the development of adaptive and humoral immune responses that are among the largest for any pathogen, and intriguingly, the magnitude of the immune response increases with age, a phenomenon termed "memory inflation." Elevated CMV-specific immunity has been correlated with an increased mortality rate in elderly individuals and with impaired vaccination responses. The latent phase of CMV infection is characterized by intermittent episodes of subclinical viral reactivation and the production of immunogenic transcripts that may maintain memory inflation of virus-specific cytotoxic lymphocytes. However, the relative importance of CMV reactivation in the development of memory inflation is uncertain, as is the potential for antiviral treatment to reverse this effect. Here, we administered valaciclovir for up to 12 months in mice with established murine CMV (MCMV) infection. Treatment reduced the magnitude of the MCMV-specific CD8(+) T-lymphocyte response by 80%, and the residual MCMV tetramer-specific lymphocytes exhibited a less differentiated phenotype. In addition, latent MCMV infection suppressed the proportion of naïve CD8(+) T cells by 60% compared to antiviral-treated mice or MCMV-negative animals. Furthermore, treatment led to a reduction in influenza A viral loads following a challenge in elderly MCMV-infected animals and also reduced the differentiation of influenza virus-specific cytotoxic lymphocytes. These observations demonstrate that MCMV-specific memory inflation is maintained by viral replication and that therapeutic intervention could lead to improved immune function.
Asunto(s)
Antivirales/administración & dosificación , Infecciones por Citomegalovirus/veterinaria , Memoria Inmunológica , Muromegalovirus/inmunología , Muromegalovirus/patogenicidad , Activación Viral , Latencia del Virus , Aciclovir/administración & dosificación , Aciclovir/análogos & derivados , Animales , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Valaciclovir , Valina/administración & dosificación , Valina/análogos & derivadosRESUMEN
Cytomegalovirus (CMV) is a widely prevalent herpesvirus that is well tolerated by an immune competent host yet establishes a state of chronic infection. The virus is thought to undergo frequent subclinical episodes of reactivation which leads to an unusually large accumulation of CMV-specific CD8(+) T lymphocytes in the peripheral blood, a phenomenon termed "memory inflation." The high magnitude of the CMV T cell response has been implicated in impaired immunity to heterologous pathogens such as EBV, influenza and West Nile virus. Here, using murine CMV (MCMV), we show that memory inflation of virus-specific CD8(+) T cells is avoided if mice are infected with a replication defective virus called temperature-sensitive mutant 5 (tsm5), which carries an attenuating mutation within the DNA primase gene. Mice infected with tsm5 do generate primary T cell responses towards viral proteins but these do not amass to skew the memory repertoire of CD8(+) T cells. Therefore, attenuation of the virus replication machinery may be valuable in future CMV vaccine designs because the virus remains immunogenic but does not contribute to CMV associated T cell immune senescence.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Memoria Inmunológica , Muromegalovirus/inmunología , Animales , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/inmunología , Modelos Animales de Enfermedad , Femenino , Infecciones por Herpesviridae/virología , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Mutación , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Virulencia , Replicación ViralRESUMEN
A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40°C, failed to replicate to detectable levels in mice. A total of 18 non-synonymous mutations have been identified in tsm5. In a previous study, a mutation (C890Y) identified in the M70 primase gene, when introduced into the wt M70 primase, resulted in a mutant with reduced viral replication at 40°C in vitro and which was severely attenuated in vivo. Five other previously identified mutations may also contribute to the tsm5 phenotype: (1) an A658S mutation in a protein expressed by the M27 ORF; (2) a V54I mutation in M36; (3) a Y565* mutation in m139; (4) a V195M mutation in m141; and (5) an M232I mutation in m143. In the present study, the above-mentioned mutations were introduced individually (M27, M36, m139, m141, m143) or together (M27/M36) into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. Growth in culture revealed that, apart from the double mutant (M27 and M36) and the m139 mutant, the introduced mutations in the above-mentioned genes did not show a temperature-sensitive phenotype in MEF or Raw 264.7 macrophage cells compared to their revertants or the wt virus. In contrast, replication of the M27/M36 double mutant was drastically reduced in MEFs at 40°C and in macrophages at 37°C. Replication of the m139 mutant was reduced in MEF cells at 40°C but not in macrophages. Thus, at least three further mutations contribute to the tsm5 phenotype.
Asunto(s)
Muromegalovirus/crecimiento & desarrollo , Muromegalovirus/genética , Mutación Missense , Sistemas de Lectura Abierta , Replicación Viral/efectos de la radiación , Animales , Células Cultivadas , ADN Viral/química , ADN Viral/genética , Fibroblastos/virología , Masculino , Ratones , Muromegalovirus/efectos de la radiación , Fenotipo , Análisis de Secuencia de ADN , TemperaturaRESUMEN
Prior to acquiring a memory phenotype, antigen-activated CD8(+) T cells need to expand and then undergo a contraction phase. Utilizing two different antigenic stimuli, we provide evidence that the tumor necrosis factor receptors OX40 and CD30 integrate synergistic signals during the expansion phase to help maintain CD8(+) effectors. Thus, double deficiency in OX40 and CD30 leads to CD8(+) cell loss during expansion after immunization either with OVA or with murine CMV. Following their contraction, OX40- and CD30-deficient CD8(+) T cells persist normally in CMV-infected mice. In contrast, persistence after OVA challenge is dependent on OX40 and CD30. Collectively, our data define the important role of both OX40 and CD30 during CD8(+) T-cell activation, and show that long-term CD8 persistence after contraction is regulated not only by stimulatory receptors but also by the nature of the antigen or how the antigen is presented.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno Ki-1/fisiología , Receptores OX40/fisiología , Transducción de Señal , Traslado Adoptivo , Animales , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Proliferación Celular , Citometría de Flujo , Inmunización Secundaria , Memoria Inmunológica/inmunología , Interferón gamma/metabolismo , Antígeno Ki-1/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores OX40/genéticaRESUMEN
The pathogenic outcomes of viral infection are often reminiscent of a dysfunctional immune system. Thus, cytomegalovirus (CMV) causes disruption of the lymphoid architecture and the functionality of lymphocytes, both of which are features of CD30 deficiency. It was therefore plausible that CD30 might interfere with CMV infection. The present study identifies CD30 as an inducible NK-cell receptor critical for innate immunity against CMV. Expression of CD30 integrates survival signals to NK cells that allow them to prevent viral spread and subsequent disintegration of secondary lymphoid tissue. Deficiency in CD30 results in exaggerated NK cell death and complete abrogation of the lymphoid architecture. Our data define the necessity of NK cells for protection of secondary lymphoid organs and describe a mechanism by which this protection is conferred.
Asunto(s)
Infecciones por Herpesviridae/inmunología , Inmunidad Innata/fisiología , Antígeno Ki-1/fisiología , Células Asesinas Naturales/inmunología , Tejido Linfoide/inmunología , Muromegalovirus/inmunología , Traslado Adoptivo , Animales , Apoptosis/genética , Apoptosis/inmunología , Antígeno CD11c/metabolismo , Ligando CD30/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Proteínas de Homeodominio/genética , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/trasplante , Antígenos Comunes de Leucocito/metabolismo , Tejido Linfoide/patología , Tejido Linfoide/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Bazo/virología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Studies with a murine cytomegalovirus mutant tsm5 suggested two possible approaches to producing a live attenuated human cytomegalovirus vaccine. One approach would be to use a combination of five to six mutants where an attenuating mutation in the gene of one mutant is compensated by the wild-type version in a second mutant, which in turn has a mutation in a different gene compensated by the wild-type version in a third mutant, etc. Important genes in this approach could include those involved in DNA replication. The importance of the carboxy terminase of the primase gene (M70/UL70) for its function suggested a second approach where some of the natural codons in this region could be substituted with synonymous non-preferred (minor) codons that would reduce the replication fitness of the mutant.
RESUMEN
Identification of mutations in mutants derived chemically is a difficult and relatively random process. NimbleGen Comparative Genome Sequencing (CGS) was assessed as an inexpensive, rapid method of identifying mutations in the temperature-sensitive mutant tsm5 of the K181 (Birmingham) variant of murine cytomegalovirus (MCMV). This genome resequencing approach requires an established genome sequence as a reference. Comparison of tsm5 and the K181 (Birmingham) variant with the published K181 (Perth) MCMV genomic sequence revealed a total of 10 synonymous and 15 non-synonymous SNPs in tsm5 and 14 of the latter were confirmed by sequencing. Thus, while CGS cannot be relied upon to identify correctly all mutations it was helpful for identifying a large number of mutations for further investigation that could contribute to the ts phenotype of tsm5.
Asunto(s)
Genes Esenciales , Genes Virales , Genoma Viral , Muromegalovirus/genética , Mutación Missense , ADN Viral/química , ADN Viral/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Influenza virus is a major human pathogen that causes epidemics and pandemics with increased morbidity and, especially in the elderly and those with pre-existing medical conditions, increased mortality. Influenza is characterised by respiratory symptoms and constitutional symptoms. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced considerably of late we still know relatively little about other aspects of influenza virus virulence. In this review, we will explore what is known about the role of apoptosis in respiratory epithelial cell damage and the role of cytokines in inflammation and constitutional symptoms with particular emphasis on the link between apoptosis, inflammation, fever and cytokine production.
Asunto(s)
Apoptosis/fisiología , Citocinas/fisiología , Regulación de la Expresión Génica , Orthomyxoviridae/fisiología , Citocinas/genética , Citocinas/inmunología , Humanos , Gripe Humana/inmunología , Morbilidad , Orthomyxoviridae/genética , Orthomyxoviridae/inmunologíaRESUMEN
Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-alpha2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-alpha alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-alphas currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-alpha2b.
Asunto(s)
Antivirales/química , Hepatitis C Crónica/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Interferón-alfa/genética , Secuencia de Aminoácidos , Antivirales/uso terapéutico , Línea Celular , Epítopos de Linfocito T/análisis , Hepacivirus/efectos de los fármacos , Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Interferón alfa-2 , Interferón-alfa/química , Interferón-alfa/uso terapéutico , Datos de Secuencia Molecular , Péptidos/genética , Mutación Puntual , Proteínas Recombinantes de Fusión/inmunología , Proteínas RecombinantesRESUMEN
Twenty-six non-synonymous and synonymous mutations have been identified in the temperature-sensitive (ts) mutant (tsm5) of the K181 (Birmingham) variant of murine cytomegalovirus that is deficient in DNA synthesis, processing and packaging at the non-permissive temperature and produces undetectable levels of infectious virus in mice. Non-synonymous mutations identified in the M70 (primase), M56 (terminase) and M98 (nuclease) ORFs were introduced individually and in combination into the K181 (Perth) variant using BAC technology to examine their role in the ts phenotype. The M56 (G439R) and M98 (P324S) mutations had no evident role in the ts phenotype. However, the C890Y M70 mutation alone and in combination with the M56 and/or M98 mutations rendered the virus ts, unable to replicate in mice and highly defective in DNA synthesis. Reversion of the tyrosine mutation to cysteine or introduction of C890M (experimentally) or C890S (naturally) restored the wt phenotype.
Asunto(s)
ADN Primasa/genética , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Muromegalovirus/enzimología , Muromegalovirus/genética , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , ADN Viral/biosíntesis , ADN Viral/genética , Genes Virales , Variación Genética , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/fisiología , Mutación , Sistemas de Lectura Abierta , Fenotipo , Temperatura , Técnicas de Cultivo de Tejidos , Replicación ViralRESUMEN
A viable virus could not be recovered from a mutant murine cytomegalovirus (MCMV) BAC in which the M34 ORF had been deleted (BACDeltaM34). In contrast, an M34 mutant virus (RcM34), in which the M34 ORF was interrupted by transposon insertion at nt 44,827 of the Smith MCMV BAC, was attenuated in replication both in tissue culture and in SCID mice. Similarly, mutant virus Rc3'DeltaM34, in which the 3'-end was deleted from nt 44,724 to nt 45,647, produced similar replication kinetics in tissue culture to RcM34 while BAC5'DeltaM34, in which the 5'-end from nt 43,083 up to nt 44,896 was deleted, was non-viable like BACDeltaM34. A transcript analysis of wt and RcM34 virus-infected cells showed that a truncated transcript encoding a putative protein of 624 amino acids was produced by RcM34, of which the amino terminal 582 amino acids would be identical to the predicted wt 854 amino acids product. Recent, re-annotations of the MCMV genome have identified three putative M34 overlapping ORFs (m33.1, m34.1, and m34.2) that may be interrupted in the above mutants. All three were transcribed in RcM34 virus-infected cells confirming that the RcM34 virus phenotype was probably due to interruption of the M34 ORF. These results suggest that M34, like human CMV UL34, is an essential gene.
Asunto(s)
Muromegalovirus/genética , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Eliminación de Secuencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Femenino , Fibroblastos/virología , Infecciones por Herpesviridae/virología , Ratones , Ratones SCID , Datos de Secuencia Molecular , Muromegalovirus/fisiología , Fenotipo , Transcripción Genética , Replicación ViralRESUMEN
In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients.
Asunto(s)
Antígenos Ly/metabolismo , Quimiotaxis de Leucocito/inmunología , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C/metabolismo , Bazo/virología , Animales , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/metabolismo , Citometría de Flujo , Inmunohistoquímica , Rayos Láser , Ratones , Microdisección , Microscopía Confocal , Muromegalovirus/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK , Receptores CXCR3/metabolismo , Receptores Similares a Lectina de Células NK , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Bazo/patología , Linfocitos T/inmunologíaRESUMEN
Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2-3 log(10) p.f.u. ml(-1)) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced ( approximately 1 log(10) p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.
Asunto(s)
Antígenos Virales/fisiología , Proteínas Inmediatas-Precoces/fisiología , Muromegalovirus/fisiología , Replicación Viral , Secuencia de Aminoácidos , Estructuras Animales/virología , Animales , Antígenos Virales/química , Antígenos Virales/genética , Secuencia de Bases , Codón sin Sentido , Infecciones por Herpesviridae/virología , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Datos de Secuencia Molecular , Muromegalovirus/genética , Mutagénesis , Células 3T3 NIH , Sistemas de Lectura Abierta , Mutación Puntual , Análisis de Supervivencia , Factores de Tiempo , Ensayo de Placa ViralRESUMEN
A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3' or 5' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.
Asunto(s)
Empaquetamiento del ADN/genética , Replicación del ADN/genética , Viabilidad Microbiana/genética , Muromegalovirus/genética , Muromegalovirus/fisiología , Mutación , Sustitución de Aminoácidos , Animales , Cápside/química , Cápside/ultraestructura , Línea Celular , Análisis Mutacional de ADN , ADN Viral/química , ADN Viral/genética , Infecciones por Herpesviridae/virología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación Missense , Mutación Puntual , Recombinación Genética , Análisis de Secuencia de ADN , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
In this report, we identify an important function for CD30 signals in the effective segregation of B and T lymphocytes in the murine spleen, additional to the recognized requirement for lymphotoxin signals. We show that CD30 signals are not required for transcription or protein expression of homeostatic chemokines, but CD30-deficient mice display impaired B/T segregation. This defect correlates with defective expression as detected by Abs of the transmembrane mucin-type protein podoplanin on T zone stroma, although expression at other sites is normal. Defective segregation is not intrinsic to CD30-deficient lymphocytes which segregate normally following transfer into RAG-deficient mice and significantly up-regulate the expression of both CCL21 and podoplanin on T zone stroma of RAG-deficient mice. During development, induction of expression of the CD30 ligand by lymphoid tissue inducer cells and podoplanin by T zone stroma are temporally linked, and the spatial association of these cells suggests that lymphoid tissue inducer cells are capable of providing the CD30 signals. Finally, we show that the appearance of podoplanin on T zone stroma in development is associated with B/T segregation of splenic white pulp areas. Our studies indicate that homeostatic chemokine expression by itself is not sufficient for B/T segregation and our data point to a significant role for podoplanin or molecules associated with podoplanin expressing stroma in the effective segregation of lymphocytes.
Asunto(s)
Linfocitos B/inmunología , Antígeno Ki-1/inmunología , Bazo/inmunología , Linfocitos T/inmunología , Animales , Ligando CD30/genética , Ligando CD30/inmunología , Quimiocinas/genética , Quimiocinas/inmunología , Citometría de Flujo , Perfilación de la Expresión Génica , Antígeno Ki-1/deficiencia , Antígeno Ki-1/genética , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Modelos Inmunológicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Three influenza viruses, A/Puerto Rico/8/34-A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to the herpes simplex virus tegument protein, VP22. However, this did not reflect viral protein-protein-RNA interactions known to occur within infected cells. To explore the role of viral proteins in apoptosis under infection conditions, recombinant viruses with single or triple gene exchanges were generated using A/Victoria or clone 7a as the background virus. Inserting the A/Fiji NS or PB2 gene into A/Victoria or clone 7a significantly reduced the level of apoptosis compared to the parent virus while clone 7a PA or NP genes increased apoptosis. Inserting A/Fiji NA or HA or clone 7a NS, M, NA, or HA genes individually into A/Victoria had no significant effect on apoptosis. Surprisingly, inserting the M, NA, and HA genes of A/Fiji together into clone 7a reduced apoptosis, whereas inserting clone 7a M, NA, and HA together into A/Fiji increased apoptosis. These results suggest that no single virus protein induces apoptosis and that the combination of genes required may be strain specific, highlighting the difficulty of predicting the virulence of new strains that arise in nature. No support for the view that apoptosis is essential for high virus yields was obtained as high virus yields were obtained with viruses that induced both high and low levels of apoptosis.
Asunto(s)
Apoptosis , Ingeniería Genética , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Recombinante/genética , Perros , Genes Virales/genética , Humanos , Datos de Secuencia Molecular , Proteínas Virales/química , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Previously, we showed that two temperature-sensitive mutants of murine cytomegalovirus (tsm5 and tsm30) expressed immediate-early (IE-1), early (E-1), and late (gB) phase genes in the tissues of immunocompetent Balb/c mice, yet failed to produce infectious progeny virus in any tissue at any time at 1-21 days post-infection. Mice inoculated intraperitoneally with tsm5 became latently infected, but this latent virus could not be reactivated as an infectious virus after immunosuppression, although all three transcripts were produced. Immunocompetent mice infected with tsm30 did not become latently infected. In the present study, immunodeficient SCID mice supported productive infection of both mutants, suggesting that low-level viral replication does occur in immunocompetent mice, but that it is limited by the host immune response. This is supported by the observation that immunocompetent mice were protected against virulent K181 challenge even after immunisation with as few as 40 pfu of mutant virus, whereas UV-inactivated mutant or K181 virus was not immunoprotective at doses of 40,000 pfu. Immunity induced by subcutaneous inoculation was also protective, whereas that induced by intragastric immunisation was not. Protection was lifelong (18 months). Although tsm5 induced high antibody titres, there was little evidence of an antibody response to tsm30. In contrast, a significant CD8(+) CTL response to the Balb/c immunodominant IE-1 nonapeptide (YPHFMPTNL) was elicited by both mutants, as determined by an interferon-gamma ELISPOT assay, although this response was lower than that induced by K181 infection. In addition, CTLs specific for m04 (YGPSLYRRF) and M84 (AYAGLFTPL) peptides could be detected at low frequency after K181, tsm5, and tsm30 immunisation. Such protective immunity did not prevent the challenge K181 virus from entering the latent state, but it appeared to reduce the frequency of reactivation.
Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , Infecciones por Herpesviridae/inmunología , Muromegalovirus/inmunología , Mutación , Vacunas Virales/inmunología , Animales , Femenino , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/virología , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Vacunas Virales/administración & dosificación , Virulencia , Activación Viral , Latencia del VirusRESUMEN
Infection of Balb/c mice with murine cytomegalovirus (MCMV) has been used extensively as a model system with which to study host mechanisms of immunity to cytomegaloviruses. In this model, the cytotoxic T-lymphocyte (CTL) response crucial for clearing infected cells is dominated by CTLs specific for the MCMV nonapeptide 168YPHFMPTNL176 encoded by the immediate-early 1 (IE-1) gene. The intradermal injection of plasmid pcDNA89 encoding IE-1 has been shown to offer some protection from viral challenge. In the present studies, the protective efficacy of immunisation with pcDNA89 given by intradermal injection was compared with particle-mediated DNA delivery (PMDD) and contrasted with that induced by injection with the K181 MCMV strain and with temperature-sensitive mutants (tsm) derived from the K181 strain. Modest protection was afforded by pcDNA89 immunisation given by PMDD, but none was observed after intradermal injection. PMDD immunisation induced a frequency of 168YPHFMPTNL176-specific interferon-gamma (IFN-gamma)-secreting splenocytes, which was equivalent to that after K181 infection and significantly higher than tsm immunisation. Whereas tsm-immunised mice were completely protected from MCMV challenge, PMDD-immunised mice were only weakly protected. Tsm immunisation protected mice completely against challenge with natural isolates having sequence variation in the IE-1 nonapeptide, while PMDD-immunised mice were weakly protected from isolates encoding 168YPHFMPTNL176 and were not protected against isolates encoding 168YPHFMPPSL176 or 168YLDFMPPNL176. Thus, while IE-1-specific IFN-gamma-secreting splenocytes do contribute to immunity from MCMV challenge, their presence in isolation is insufficient to provide complete protection and they may not be involved in the protection observed against MCMV isolates having IE-1 sequence variation.
Asunto(s)
Infecciones por Herpesviridae/prevención & control , Proteínas Inmediatas-Precoces/inmunología , Interferón gamma/metabolismo , Muromegalovirus/inmunología , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Transactivadores/inmunología , Células 3T3 , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales , Vacunas contra Citomegalovirus/administración & dosificación , Vacunas contra Citomegalovirus/genética , Vacunas contra Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/genética , Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Péptidos/química , Bazo/citología , Bazo/inmunología , Transactivadores/química , Transactivadores/genética , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunologíaRESUMEN
Infection of cells with influenza A virus results in cell death with apoptotic characteristics. Apoptosis is regarded as a non-inflammatory process. However, during influenza an inflammatory response occurs in the airway epithelium. An examination of this apparent paradox was made using influenza A virus infection of human nasal and bronchiolar epithelial cells. Some cytokine genes (IL-18, CCL2 and CCL5) were expressed constitutively in nasal cells but no cytokine was released. In bronchiolar cells, IL-1 beta, IL-6 and CXCL8 expression was constitutive, whilst CCL2 and CCL5 expression was upregulated following influenza virus infection. IL-6, CXCL8 and CCL5 were released but IL-1 beta and CCL2 were not. In bronchiolar cells, cell death was inhibited by the caspase-8 (Z-IETD-fmk) and pan-caspase (Z-VAD-fmk) inhibitors and these inhibitors enhanced expression of CCL5 and increased the levels of the three secreted cytokines significantly. Thus, the amount of each cytokine released from bronchiolar cells is reduced during cell death, implying that the observed inflammatory response in influenza would be greater if cell death did not occur. Reduced cytokine release is also associated with fragmentation of the Golgi body, as the caspase inhibitors also rescued influenza A virus-induced fragmentation of the Golgi ribbon.