RESUMO
In order to establish criteria for the serodiagnosis of foamy virus infections we investigated the extent to which sera from infected individuals of human and primate origin react with structural and non-structural virus proteins in immunoblot assays. Using lysates from infected cells as the source of virus antigen, antibodies were preferentially detected against the Gag proteins and the non-structural Bet protein. Both the Gag precursor molecules of 70 and 74K apparent M(r) and the cytoplasmic 60K M(r) Bet protein were found to be phosphorylated, the latter being synthesized in large amounts in infected cells. Rabbit antiserum raised against recombinant human foamy virus (HFV) Gag major capsid protein cross-reacted with foamy viruses of chimpanzee, gorilla, orang-utan, rhesus monkey and African green monkey origin. This was reflected by a broad cross-reactivity of the respective monkey sera to the Gag proteins of the various foamy virus isolates. Cross-reactivity of antisera against the Bet protein was restricted to viruses from man and the great apes. Recombinant Gag and Bet proteins expressed in prokaryotes or in insect cells were readily recognized by foamy virus-positive primate sera. Screening serum samples from chimpanzees with HFV Gag and Bet proteins expressed by recombinant baculoviruses revealed that 18 out of 35 (52%) were positive for Gag antibodies. Of these, 13 (72%) showed antibodies against the Bet protein, indicating that Bet antigen is of value in serological screening for foamy virus infections.
Assuntos
Produtos do Gene gag/imunologia , Primatas/sangue , Proteínas dos Retroviridae/imunologia , Spumavirus/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Reações Cruzadas , Produtos do Gene gag/análise , Genes gag , Gorilla gorilla , Humanos , Insetos , Rim , Pulmão , Macaca mulatta , Dados de Sequência Molecular , Peso Molecular , Pan troglodytes , Fosforilação , Pongo pygmaeus , Primatas/virologia , Proteínas Recombinantes/análise , Proteínas dos Retroviridae/análiseRESUMO
Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Virais , Fases de Leitura Aberta , Proteínas dos Retroviridae/genética , Spumavirus/genética , Transativadores/genética , Sequência de Bases , Western Blotting , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Imunofluorescência , Genoma Viral , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Mapeamento por Restrição , Proteínas dos Retroviridae/análise , Proteínas dos Retroviridae/metabolismo , Vírus 40 dos Símios/genética , Spumavirus/fisiologia , Transativadores/análise , Transativadores/metabolismo , Replicação ViralRESUMO
An infectious molecular clone (pHSRV) of the human Spumaretrovirus (HSRV) was constructed using viral DNA and cDNA clones. The infectivity of pHSRV was proven by transfection of cell cultures and subsequent infection of susceptible cultures with cell free transfection derived virus. pHSRV derived virus produced foamy virus typical cytopathic effects in susceptible cultures. Infected cells could be stained specifically with foamy virus antisera by means of indirect immunofluorescence. Radioimmunoprecipitation revealed the presence of characteristic HSRV structural proteins in pHSRV infected cultures. By cotransfection of pHSRV and an indicator plasmid it was found that pHSRV is able to transactivate the viral LTR. Viral transcripts were found to be approximately 200 bases longer in pHSRV infected cultures compared to wildtype infected cultures. This difference is most likely due to an insertion of DNA of non-viral origin in the U3 region of the 3'LTR of the infectious clone.