RESUMEN
This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K-->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-->K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K-->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F-->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.
Asunto(s)
Glicoproteínas/genética , Coriomeningitis Linfocítica/microbiología , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/patogenicidad , Macrófagos/microbiología , ADN Polimerasa Dirigida por ARN/genética , Proteínas Virales/genética , Animales , Variación Genética , Macrófagos del Hígado/microbiología , Virus de la Coriomeningitis Linfocítica/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mutación , Análisis de Secuencia , Bazo/microbiología , Virulencia , Replicación ViralRESUMEN
This study demonstrates cell-specific selection of viral variants during persistent lymphocytic choriomeningitis virus infection in its natural host. We have analyzed viral isolates obtained from CD4+ T cells and macrophages of congenitally infected carrier mice and found that three types of variants are present in individual carrier mice: (i) macrophage-tropic, (ii) lymphotropic, and (iii) amphotropic. The majority of the isolates were amphotropic and exhibited enhanced growth in both lymphocytes and macrophages. However, some of the lymphocyte-derived isolates grew well in lymphocytes but poorly in macrophages, and a macrophage-derived isolate replicated well in macrophages but not in lymphocytes. In striking contrast, the original wild-type (wt) Armstrong strain of lymphocytic choriomeningitis virus that was used to initiate the chronic infection and from which the variants are derived grew poorly in both lymphocytes and macrophages. These three types of variants also differed from the parental virus in their ability to establish a chronic infection in immunocompetent hosts. Adult mice infected with the wt Armstrong strain cleared the infection within 2 weeks, whereas adult mice infected with the variants harbored virus for several months. These results suggest that the ability of the variants to persist in adult mice is due to enhanced replication in macrophages and/or lymphocytes. This conclusion is further strengthened by the finding that the variants and the parental wt virus grew equally well in mouse fibroblasts and that the observed growth differences were specific for cells of the immune system.
Asunto(s)
Linfocitos T CD4-Positivos/microbiología , Coriomeningitis Linfocítica/microbiología , Virus de la Coriomeningitis Linfocítica/crecimiento & desarrollo , Macrófagos/microbiología , Animales , Antígenos de Diferenciación de Linfocitos T/análisis , Antígenos CD8 , Fibroblastos/microbiología , Técnica del Anticuerpo Fluorescente , Virus de la Coriomeningitis Linfocítica/clasificación , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos BALB C , Subgrupos de Linfocitos T/microbiología , Replicación ViralRESUMEN
This study has identified a single amino acid change in the viral glycoprotein that profoundly affects the ability of lymphocytic choriomeningitis virus (LCMV) to persist in its natural host. Adult immunocompetent mice infected with a variant of the Armstrong strain, spleen isolate clone 13 (svA/svA), harbor virus for several months and exhibit suppressed T cell responses. In contrast, adult mice infected with a reassortant virus (svA/wtA) that contains the L segment of the spleen variant and the S segment of the parental wt Armstrong, make potent LCMV-specific CTL responses and clear the infection within 2-4 wk. These two viruses, spleen variant clone 13 and the reassortant svA/wtA, are identical in their noncoding regions and show no amino acid changes in any of their viral genes except for one substitution in the glycoprotein. The reassortant virus svA/wtA has a phenylalanine at amino acid residue 260 of the glycoprotein, whereas the spleen variant clone 13 has a leucine at this position. This study constitutes one of the first reports defining the genetic basis of viral persistence at the whole animal level, and identifying a single mutation that markedly increases the ability of a virus to persist in its natural host.
Asunto(s)
Glicoproteínas/química , Virus de la Coriomeningitis Linfocítica/genética , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Viral variants with different biological properties are present in the central nervous systems (CNS) and lymphoid tissues of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Viral isolates from the CNS are similar to the original Armstrong LCMV strain and induce potent virus-specific T-cell responses in adult mice, and the infection is rapidly cleared. In contrast, LCMV isolates derived from spleens of carrier mice cause persistent infections in adult mice. This chronic infection is associated with low levels of antiviral T-cell responses. In this study, we genetically characterized two independently derived spleen variants by making recombinants (reassortants) between the spleen isolates and wild-type (wt) LCMV and showed that the ability to persist in adult mice and the associated suppression of T-cell responses segregates with the large (L) RNA segment. In addition, we analyzed a revertant (isolated from the CNS) derived from one of the spleen variants. By comparing the biological properties of three reassortants that contained the same S segment but had the L segment of either the original wt Armstrong LCMV, the spleen variant derived from it, or the CNS revertant derived from the spleen variant, we were able to show unequivocally that biologically relevant mutations occurred in the L segment not only during generation of the spleen variant from wt LCMV but also in reversion of the spleen variant to the wt phenotype. Thus, our results showed that (i) genetic alterations in the L genomic segment were involved in organ-specific selection of viral variants, and (ii) these mutations profoundly affected the ability of LCMV to cause chronic infections in adult mice.
Asunto(s)
Virus de la Coriomeningitis Linfocítica/genética , ARN Viral/genética , Bazo/microbiología , Virosis/microbiología , Animales , Encéfalo/microbiología , Línea Celular , Enfermedad Crónica , Citotoxicidad Inmunológica , Genes Virales , Variación Genética , Genotipo , Hipersensibilidad Tardía , Tejido Linfoide/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Hibridación de Ácido Nucleico , Especificidad de Órganos , Linfocitos T Citotóxicos/inmunología , Células VeroRESUMEN
Amyloglucosidase (glucoamylase; EC 3.2.1.3) has been purified from the culture filtrates of Aspergillus candidus Link var. aureus using hydrophobic interaction chromatography and DEAE-cellulose treatment. The enzyme thus obtained has a specific activity of 329 units/mg protein with the overall recoveries between 70 and 75%. The process appears to be of industrial promise.