RESUMEN
Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology.
Asunto(s)
Proteínas de la Cápside/química , Cápside/química , Virus de la Leucemia Bovina/química , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Bovinos , Cristalografía por Rayos X , Virus de la Leucemia Bovina/genética , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
It is widely accepted that the majority of cancers result from multiple cellular events leading to malignancy after a prolonged period of clinical latency, and that the immune system plays a critical role in the control of cancer progression. Bovine leukemia virus (BLV) is an oncogenic member of the Retroviridae family. Complete genomic sequences of BLV strains isolated from peripheral blood mononuclear cells (PBMC) from cattle have been previously reported. However, a detailed characterization of the complete genome of BLV strains directly isolated from bovine tumors is much needed in order to contribute to the understanding of the mechanisms of leukemogenesis induced by BLV in cattle. In this study, we performed a molecular characterization of BLV complete genomes from bovine B-cell lymphosarcoma isolates. A nucleotide substitution was found in the glucocorticoid response element (GRE) site of the 5' long terminal repeat (5'LTR) of the BLV isolates. All amino acid substitutions in Tax previously found to be related to stimulate high transcriptional activity of 5'LTR were not found in these studies. Amino acid substitutions were found in the nucleocapsid, gp51 and G4 proteins. Premature stop-codons in R3 were observed. Few mutations or amino acid substitutions may be needed to allow BLV provirus to achieve silencing. Substitutions that favor suppression of viral expression in malignant B cells might be a strategy to circumvent effective immune attack.
Asunto(s)
Leucosis Bovina Enzoótica/virología , Genoma Viral , Virus de la Leucemia Bovina/genética , Linfoma de Células B/veterinaria , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Virus de la Leucemia Bovina/química , Virus de la Leucemia Bovina/metabolismo , Linfoma de Células B/virología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinaria , Alineación de Secuencia/veterinariaRESUMEN
Bovine leukemia virus (BLV)-infected cattle were classified by their proviral load into low and high proviral load profiles (LPL and HPL, respectively). Blood from these animals was used to infect sheep to obtain multiple identical copies of integrated provirus. An env fragment of BLV was amplified from all infected sheep and sequenced. The sequences that were obtained were compared to already published BLV genome sequence, resulting in three clusters. Mutations could not be attributed to the passage of provirus from cattle to sheep and subsequent amplification and sequencing. The description of two different proviral load profiles, the association of the BoLA-DRB3.2 0902 allele with the LPL profile, the availability of complete BLV sequences, and the comparison of a variable region of the env gene from carefully characterized cattle are still not enough to explain the presence of animals in every herd that are resistant to BLV dissemination.
Asunto(s)
Leucosis Bovina Enzoótica/virología , Virus de la Leucemia Bovina/genética , Provirus/genética , Enfermedades de las Ovejas/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Virus de la Leucemia Bovina/química , Virus de la Leucemia Bovina/clasificación , Virus de la Leucemia Bovina/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Provirus/clasificación , Provirus/aislamiento & purificación , Alineación de Secuencia , Ovinos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.
Asunto(s)
Leucosis Bovina Enzoótica/virología , Variación Genética , Virus de la Leucemia Bovina/genética , Provirus/genética , Secuencia de Aminoácidos , Animales , Argentina , Secuencia de Bases , Bovinos , Análisis por Conglomerados , ADN Viral/química , Genes env/genética , Genotipo , Japón , Virus de la Leucemia Bovina/química , Virus de la Leucemia Bovina/clasificación , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Provirus/química , Provirus/clasificaciónRESUMEN
OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.