RESUMO
Although maximal synthesis of HSP70 is induced early (6-12 hr) after adenovirus type 5 (Ad5) infection of HEp-2 or HeLa cells, the total amount of HSP70 appears to be increased at late times of infection (18-24 hr). Since virion structural proteins also accumulate at these times, we investigated the possible interaction between Ad5 structural proteins and HSP70 by immunoprecipitation of infected cell extracts with antibodies to either ATP-affinity-purified HSP70 or to CsCl-gradient-purified Ad5 virions. We found that HSP70 and a 62-kDa Ad-specific protein coimmunoprecipitated from infected cell extracts. Antibody which recognizes one of these two proteins does not cross-react with the other. Thus, the association between HSP70 and the 62-kDa protein appears specific. Using different antisera to specific adenovirus structural proteins, we have identified the 62-kDa protein as the Ad5 fiber protein.
Assuntos
Adenovirus dos Símios/crescimento & desenvolvimento , Proteínas de Choque Térmico/metabolismo , Proteínas Estruturais Virais/metabolismo , Adenovirus dos Símios/genética , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células HeLa , Immunoblotting , Técnicas Imunológicas , Peso Molecular , Testes de Precipitina , Replicação ViralRESUMO
The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad pol). Ad pol and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad pol a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad pol genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.
Assuntos
Adenovirus dos Símios/genética , DNA Polimerase Dirigida por DNA/genética , Mutagênese Insercional , Proteínas Virais/genética , Replicação Viral , Adenovirus dos Símios/enzimologia , Adenovirus dos Símios/crescimento & desenvolvimento , Sequência de Aminoácidos , Sequência de Bases , Sobrevivência Celular , Clonagem Molecular , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos , Plasmídeos/genética , Precursores de Proteínas/genética , Terminologia como Assunto , Replicação Viral/genéticaRESUMO
Chronic infection with adenovirus types 5 and 6 was established in primary mononuclear leukocytes from human umbilical cord blood and in Epstein-Barr virus (EBV)-transformed B lymphocytes from human umbilical cord blood and from woolly monkey blood. Adenovirus could be recovered from cultures of primary leukocytes and of EBV-transformed lymphocytes for two and three months, respectively, without visible alteration of cell growth. Infection in cultures of EBV-transformed lymphocytes from woolly monkey blood was obliterated by exposure to antibody, but EBV-transformed lymphocytes from human umbilical cord blood contained small amounts of virus for prolonged periods that restored infection in the culture when antibody was removed. Thus, chronic infection of lymphoid cells by some adenoviruses is maintained by at least two mechanisms: cell-to-cell spread of virus in the absence of antibody and intracellular persistence of infectious virus in the presence of antibody.
Assuntos
Infecções por Adenoviridae/microbiologia , Infecções por Adenovirus Humanos/microbiologia , Linfócitos/microbiologia , Infecções por Adenoviridae/imunologia , Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/crescimento & desenvolvimento , Adenovírus Humanos/patogenicidade , Adenovirus dos Símios/crescimento & desenvolvimento , Adenovirus dos Símios/patogenicidade , Animais , Anticorpos Antivirais , Especificidade de Anticorpos , Cebidae , Transformação Celular Viral , Células Cultivadas , Doença Crônica , Herpesvirus Humano 4/metabolismo , Humanos , Linfócitos/imunologiaRESUMO
Simian adenovirus SA7 heated for 30 minutes at 70 degrees C retained part of its ability to induce tumors in newborn hamsters. Tumors were also induced by DNA extracted from the heated virus. However, neither residual replicating, i.e. cytopathogenic, nor transforming and T-antigen-inducing activities could be detected in vitro in CV-1 and hamster embryo cells inoculated with the heated virus. The results are discussed in terms of a possible release of viral nucleic acid at the high temperature. They also show that current tests for effective virus inactivation based exclusively on the recognition of replicating virus should be reevaluated.
