RESUMEN
A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.
Asunto(s)
Macrófagos/efectos de los fármacos , Macrófagos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Vidarabina/análogos & derivados , Adenina/administración & dosificación , Adenina/análogos & derivados , Animales , Terapia Antirretroviral Altamente Activa , Antivirales/administración & dosificación , Cercocebus atys , Portadores de Fármacos , Eritrocitos/metabolismo , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Agonistas Mieloablativos/administración & dosificación , Organofosfonatos/administración & dosificación , Factor de Transcripción STAT1/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Subgrupos de Linfocitos T/efectos de los fármacos , Tenofovir , Vidarabina/administración & dosificación , Viremia/tratamiento farmacológico , Viremia/prevención & control , Viremia/virología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: The ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. Certain simian immunodeficiency viruses (SIVcpz, SIVsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the HIV-1 and HIV-2 epidemics. Although SIV diversification has been studied on a population level, the essential substrates for cross-species transmission, namely SIV sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. To characterize this intra-host SIV diversity, we performed sequence analyses of clonal viral envelope (env) V1V2 and gag p27 variants present in individual SIVsm-infected sooty mangabeys over time. RESULTS: SIVsm demonstrated extensive intra-animal V1V2 length variation and amino acid diversity (le38%), and continual variation in V1V2 N-linked glycosylation consensus sequence frequency and location. Positive selection was the predominant evolutionary force. Temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. In contrast, gag p27 was predominantly under purifying selection. SIVsm V1V2 sequence diversification is at least as great as that in HIV-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to AIDS. CONCLUSION: Positive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable SIV/HIV envelope protein. These studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission.
Asunto(s)
Evolución Molecular , Virus de la Inmunodeficiencia de los Simios/genética , Proteínas del Envoltorio Viral/genética , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Viral/genética , Variación Genética , VIH-1/genética , Humanos , Filogenia , Primates/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Virus de la Inmunodeficiencia de los Simios/clasificación , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , ZoonosisRESUMEN
In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.
Asunto(s)
Ciclo Celular/fisiología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Subgrupos de Linfocitos T/fisiología , Animales , Apoptosis , Biomarcadores , Proteína Quinasa CDC2/metabolismo , Cercocebus atys , Ciclina B/metabolismo , Leucocitos Mononucleares , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Macaca mulatta , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Especificidad de la Especie , Subgrupos de Linfocitos T/metabolismo , Carga Viral , NucleolinaRESUMEN
Although CTL escape has been well documented in pathogenic simian immunodeficiency virus (SIV) infection, there is no information on CTL escape in nonpathogenic SIV infection in nonhuman primate hosts like the sooty mangabeys. CTL responses and sequence variation in the SIV nef gene were evaluated in one sooty mangabey and one rhesus macaque inoculated together with the same stock of cloned SIVmac239. Each animal developed an immunodominant response to a distinct CTL epitope in Nef, aa 157-167 in the macaque and aa 20-28 in the mangabey. Nonsynonymous mutations in their respective epitopes were observed in both animals and resulted in loss of CTL recognition. These mutations were present in the majority of proviral DNA sequences at 16 weeks post infection in the macaque and >2 years post infection in the mangabey. These results document the occurrence of CTL escape in a host that does not develop AIDS, and adds to the growing body of evidence that CTL exert significant selective pressure in SIV infection.
Asunto(s)
Mutación , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Cercocebus atys , Epítopos de Linfocito T , Macaca mulatta , Datos de Secuencia Molecular , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Carga Viral , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/inmunologíaRESUMEN
Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the acquired immune deficiency syndrome epidemic.
Asunto(s)
Vacunas contra el SIDA/inmunología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Centro Germinal/inmunología , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , VIH-1/genética , VIH-1/inmunología , VIH-1/fisiología , Inmunidad Mucosa , Inmunización Secundaria , Memoria Inmunológica , Interferón gamma/biosíntesis , Ganglios Linfáticos/inmunología , Macaca mulatta , Vacunas contra el SIDAS/administración & dosificación , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/inmunología , Vacunas de ADN/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Carga ViralRESUMEN
African green monkeys can maintain long-term persistent infection with simian immunodeficiency viruses (SIVagm) without developing AIDS and thus provide an important model for understanding mechanisms of natural host resistance to disease. This study assessed the levels and anatomic distribution of SIVagm in healthy, naturally infected monkeys. Quantitative competitive reverse transcriptase PCR assays developed to measure SIVagm from two African green monkey subspecies demonstrated high levels of SIV RNA in plasma (>6 x 10(6) RNA copies/ml) in sabaeus and vervet monkeys. Infectious virus was readily recovered from plasma and peripheral blood mononuclear cells and shown to be highly cytopathic in human cell lines and macrophages. SIVagm DNA levels were highest in the gastrointestinal tract, suggesting that the gut is a major site for SIVagm replication in vivo. Appreciable levels of virus were also found within the brain parenchyma and the cerebrospinal fluid (CSF), with lower levels detected in peripheral blood cells and lymph nodes. Virus isolates from the CSF and brain parenchyma readily infected macrophages in culture, whereas lymph node isolates were more restricted to growth in human T-cell lines. Comparison of env V2-C4 sequences showed extensive amino acid diversity between SIVagm recovered from the central nervous system and that recovered from lymphoid tissues. Homology between brain and CSF viruses, macrophage tropism, and active replication suggest compartmentalization in the central nervous system without associated neuropathology in naturally infected monkeys. These studies provide evidence that the nonpathogenic nature of SIVagm in the natural host can be attributed neither to more effective host control over viral replication nor to differences in the tissue and cell tropism from those for human immunodeficiency virus type 1-infected humans or SIV-infected macaques.
Asunto(s)
Inmunidad Innata , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Secuencia de Aminoácidos , Animales , Encéfalo/virología , Líquido Cefalorraquídeo/virología , Chlorocebus aethiops , Productos del Gen env/química , Productos del Gen env/genética , Humanos , Tejido Linfoide/virología , Macrófagos/virología , Datos de Secuencia Molecular , ARN Viral/análisis , ARN Viral/sangre , Análisis de Secuencia de ADN , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación ViralRESUMEN
To elucidate the relationship between early viral infection events and immunodeficiency virus disease progression, quantitative-competitive and branched-DNA methods of simian immunodeficiency virus (SIV) RNA quantitation were cross-validated and used to measure viremia following infection of rhesus macaques with the pathogenic SIVmac251 virus isolate. Excellent correlation between the methods suggests that both accurately approximate SIV copy number. Plasma viremia was evident 4 days postinfection, and rapid viral expansion led to peak viremia levels of 10(7) to 10(9) SIV RNA copies/ml by days 8 to 17. Limited resolution of primary viremia was accompanied by relatively short, though variable, times to the development of AIDS (81 to 630 days). The persistent high-level viremia observed following intravenous inoculation of SIVmac251 explains the aggressive disease course in this model. Survival analyses demonstrated that the disease course is established 8 to 17 days postinfection, when peak viremia is observed. The most significant predictor of disease progression was the extent of viral decline following peak viremia; larger decrements in viremia were associated with both lower steady-state viremia (P = 0.0005) and a reduced hazard of AIDS (P = 0.004). The data also unexpectedly suggested that following SIVmac251 infection, animals with the highest peak viremia were better able to control virus replication rather than more rapidly developing disease. Analysis of early viral replication dynamics should help define host responses that protect from disease progression and should provide quantitative measures to assess the extent to which protective responses may be induced by prophylactic vaccination.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Viremia/virología , Replicación Viral , Animales , Macaca mulatta , Reacción en Cadena de la Polimerasa , ARN Viral/sangreRESUMEN
Some individuals in well-defined cohorts have now been infected with HIV-1 for well over a decade and yet remain clinically asymptomatic with normal CD4 counts. To determine immunologic and virologic parameters in these individuals, we examined 10 persons from the San Francisco City Clinic with firmly documented infection of 11-15 years duration who had maintained stable CD4 counts above 500 cells/microliters. Our results indicate that long-term nonprogressors are a heterogeneous group with respect to viral load and HIV-1-specific immune responses, and that progression can occur even after 15 years of stable infection. However, in a subset of persons with the lowest viral loads and persistent nonprogressive infection, we detected strong CTL responses, whereas neutralizing antibody studies revealed weak to undetectable titers against a panel of 10 primary isolates. This study demonstrates that a vigorous in vivo activated HIV-1-specific CTL response can be part of the host immune response in stable nonprogressive HIV-1 infection, and that circulating activated CTL can be detected in the setting of an extremely low viral load. These results also indicate that long-term nonprogressing HIV-1 infection does not require the presence of broadly cross-reactive neutralizing antibodies.
Asunto(s)
Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Linfocitos T Citotóxicos/inmunología , Línea Celular Transformada , Progresión de la Enfermedad , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Humanos , Masculino , Pruebas de Neutralización , SobrevivientesRESUMEN
Little is known about the factors that govern the level of HIV-1 replication in infected individuals. Recent studies (using potent antiviral drugs) of the kinetics of HIV-1 replication in vivo have demonstrated that steady-state levels of viremia are sustained by continuous rounds of de novo infection and the associated rapid turnover of CD4+ T lymphocytes. However, no information is available concerning the biologic variables that determine the size of the pool of T cells that are susceptible to virus infection or the amount of virus produced from infected cells. Furthermore, it is not known whether all CD4+ T lymphocytes are equally susceptible to HIV-1 infection at a given time or whether the infection is focused on cells of a particular state of activation or antigenic specificity. Although HIV-1 replication in culture is known to be greatly facilitated by T cell activation, the ability of specific antigenic stimulation to augment HIV-1 replication in vivo has not been studied. We sought to determine whether vaccination of HIV-1-infected adults leads to activation of virus replication and the targeting of vaccine antigen-responsive T cells for virus infection and destruction. Should T cell activation resulting from exposure to environmental antigens prove to be an important determinant of the steady-state levels of HIV-1 replication in vivo and lead to the preferential loss of specific populations of CD4+ T lymphocytes, it would have significant implications for our understanding of and therapeutic strategies for HIV-1 disease. To begin to address these issues, HIV-1-infected individuals and uninfected controls were studied by measurement of immune responses to influenza antigens and quantitation of virion-associated plasma HIV-1 RNA levels at baseline and at intervals after immunization with the trivalent influenza vaccine. Influenza vaccination resulted in readily demonstrable but transient increases in plasma HIV-1 RNA levels, indicative of activation of viral replication, in HIV-1-infected individuals with preserved ability to immunologically respond to vaccine antigens. Activation of HIV-1 replication by vaccination was more often seen and of greater magnitude in individuals who displayed a T cell proliferative response to vaccine antigens at baseline and in those who mounted a significant serologic response after vaccination. The fold increase in viremia, as well as the rates of increase of HIV-1 in plasma after vaccination and rates of viral decline after peak viremia, were higher in individuals with higher CD4+ T cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Infecciones por VIH/inmunología , Vacunas contra la Influenza/inmunología , Linfocitos T/inmunología , Vacunación/efectos adversos , Replicación Viral , Adulto , Anticuerpos Antivirales/biosíntesis , Humanos , Inmunofenotipificación , Activación de Linfocitos , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Factores de TiempoRESUMEN
We analyzed the regulation of expression of a human cytomegalovirus (HCMV) early transcription unit encoded by EcoRI fragments R and d (map units, 0.682 to 0.713), located within the long unique segment of the genome. This region specified a 2.2-kilobase class of spliced transcripts which encode several related proteins. To define important upstream regulatory elements of this gene, we generated hybrid plasmids in which 5'-promoter sequences were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and tested these hybrid genes in transient expression assays in human fibroblast cells. The stimulation of CAT activity in HCMV-infected cells was found to reflect an induction of correctly initiated hybrid mRNA, which was dependent on the de novo synthesis of some virally induced factor(s). A time course experiment showed the correct early kinetics of CAT expression. Analysis of a series of 5'-promoter deletion plasmids, ending between -323 and -7 base pairs relative to the transcription start site, showed a stepwise reduction in inducible CAT activity, suggesting that this HCMV early promoter consists of multiple elements. One of these elements resembles the binding site of a previously identified cellular "transcription" factor. We also examined the role of specific virus-encoded factors in the transactivation of this promoter. Cotransfection of human fibroblasts with the 2.2-kilobase RNA promoter-CAT construct and plasmids containing different immediate-early genes showed that expression of CAT from this promoter was stimulated by the region of the HCMV genome encoding the immediate-early 1 and 2 gene products.
Asunto(s)
Antígenos Virales/genética , Citomegalovirus/genética , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces , Regiones Promotoras Genéticas , Transcripción Genética , Acetiltransferasas/genética , Secuencia de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferasa , Endonucleasas , Fibroblastos , Genes Virales , Humanos , Datos de Secuencia Molecular , Plásmidos , ARN Viral/análisis , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Factores de Transcripción/genética , TransfecciónRESUMEN
In this report, we identify the proteins encoded by the 2.2-kilobase class of early transcripts arising from a region of the strain AD169 human cytomegalovirus genome (map units 0.682 to 0.713) which contains cell-related sequences. These transcripts, encoded by adjacent EcoRI fragments R and d, have a complex spliced structure with 5' and 3' coterminal ends. Antiserum directed against a synthetic 11-amino-acid peptide corresponding to the predicted amino terminus of the proteins was generated and found to immunoprecipitate four infected-cell proteins of 84, 50, 43, and 34 kilodaltons. These proteins were phosphorylated and were associated predominantly with the nuclei of infected cells. The 43-kilodalton protein was the most abundant of the four proteins, and its level of expression remained relatively constant throughout the infection. Expression of the other proteins increased as the infection progressed. Pulse-chase analysis failed to show a precursor-product relationship between any of the proteins. A comparison of the [35S]methionine-labeled tryptic peptide maps of the four proteins from infected cells and an in vitro-generated polypeptide derived from the putative first exon showed that all four infected-cell proteins were of viral origin and contained a common amino-terminal region.
Asunto(s)
Citomegalovirus/genética , Genes Virales , Fosfoproteínas/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Compartimento Celular , Glicosilación , Técnicas Inmunológicas , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Fosforilación , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , TripsinaRESUMEN
In this report we describe the kinetics of appearance and fine mapping of a 2.2-kilobase (kb) class of transcripts arising from a region of the human cytomegalovirus genome which contains cell-related sequences. These transcripts are encoded by adjacent EcoRI fragments R and d (map units 0.682 to 0.713), located within the long unique segment of the genome. The 2.2-kb RNAs were first detected at 8h postinfection and appeared at comparable or slightly lower levels at 28 and 72 h postinfection. At late times (72 h) additional transcripts were detected with probes from this region. RNase, S1 nuclease, and exonuclease VII protection analyses of 8- and 28-h RNA indicated that the 2.2-kb RNAs had a complex spliced structure consisting of invariable 5' and internal exons and a heterogeneous 3' exon. The position of the 5' end of the RNA was determined with respect to the nucleotide sequence. Analysis of this sequence showed that the cell-related sequences were contained within a long open reading frame in the 5' exon.
Asunto(s)
Citomegalovirus/genética , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Endonucleasas , Regulación de la Expresión Génica , Genes Virales , Peso Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Factores de TiempoRESUMEN
Human cytomegalovirus (HCMV) cloned EcoRI fragments R and b hybridized strongly, under standard high-stringency conditions, to uninfected cellular DNA of human, murine, or sea urchin origin. Less hybridization was detected with fragments, A, C, E, WL(F), WN(H), I, M, O, P, Q, V, c, d, and e. Southern blot analysis of the HCMV-related human DNA localized the major sites of hybridization of HCMV EcoRI fragments R, b, and d to defined regions of the 28S rRNA gene.
Asunto(s)
Citomegalovirus/genética , ADN Viral/análisis , Animales , Humanos , Ratones , Hibridación de Ácido Nucleico , Erizos de MarRESUMEN
The degree of relatedness between previously identified cross-hybridizing regions within human cytomegalovirus strain AD169 and the avian retrovirus oncogene v-myc were investigated by nucleotide sequence comparison. We found that the homologous regions between the human cytomegalovirus genome and v-myc are limited to short G + C-rich regions in each genome and that the human cytomegalovirus genome shares little or no homology with the human c-myc gene.
Asunto(s)
Virus del Sarcoma Aviar/genética , Citomegalovirus/genética , Genes Virales , Oncogenes , Secuencia de Bases , ADN Viral/genética , Humanos , Hibridación de Ácido Nucleico , Polidesoxirribonucleótidos/genéticaRESUMEN
In this report, we describe the size and kinetics of appearance of RNAs from the long repeat of human cytomegalovirus. The most abundant RNA from this region was a 2.7-kilobase (kb) species that was detected throughout the infection and was most abundant at 27 and 72 h after infection. The 2.7-kb RNA was the only major species detected with a probe that included the terminus of the long repeat and the heterogeneous L-S junction region. Other transcripts were detected with probes from the internal portion of the long repeat, including an immediate-early RNA of 1.3 kb, early and late RNAs of 1.2 kb, and minor late transcripts of 4.4, 3.6, 3.3, and 1.8 kb. S1 nuclease and exonuclease VII protection analyses of RNA from immediate-early, early, midpoint, and late times in the infection indicated that the major 2.7-kb RNA was not spliced and that the RNA mapped within the long repeat, 1.6 kb from the heterogeneous region. No evidence for temporally regulated changes in transcription initiation, splicing, or choice of 3' end of this RNA was observed. Nuclease protection analysis also demonstrated that the second most abundant late RNA from this region, the 1.2-kb species, was not spliced and had the same polarity as the 2.7-kb RNA. The 1.2-kb also mapped entirely within the long repeat, with its 3' terminus 1.7 kb upstream from the 5' terminus of the 2.7-kb RNA.
Asunto(s)
Citomegalovirus/genética , ARN Viral/análisis , Transcripción Genética , Secuencia de Bases , Humanos , Cinética , Hibridación de Ácido NucleicoRESUMEN
The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.