RESUMO
The P38 promoter of the autonomous parvovirus minute virus of mice is strongly transactivated by the nonstructural protein NS1, a sequence-specific DNA-binding protein. In the context of the complete viral genome, the only unique cis-acting signals required for P38 transactivation by NS1 are the proximal Sp1 site and the TATA element. In the absence of additional upstream sequences, a dependence upon the NS1 binding site within the transactivation response region is observed. Addition of synthetic NS1 binding sites to transactivation response region deletion mutants can restore the ability of NS1 to transactivate P38, and NS1 transactivation has been directly correlated to its ability to bind upstream of the P38 promoter.
Assuntos
Vírus Miúdo do Camundongo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA Viral , Regulação Viral da Expressão Gênica , Genoma Viral , Camundongos , Vírus Miúdo do Camundongo/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Deleção de Sequência , Fator de Transcrição Sp1/metabolismo , TATA Box , Regulação para Cima , Proteínas não Estruturais Virais/químicaRESUMO
When the minute virus of mice NS2-specific exon was modified by either substitution or deletion, most P4-generated pre-mRNA was spliced from the large-intron donor at nucleotide 514 to the small-intron acceptor at nucleotide 2377. Improvement to consensus of large-intron splice sites in such mutants did not suppress exon skipping or restore large-intron excision. Therefore, sequences within the NS2-specific exon are required for inclusion of this exon into spliced, steady-state minute virus of mice RNA.
Assuntos
Éxons , Vírus Miúdo do Camundongo/genética , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Proteínas não Estruturais Virais/genética , Animais , Sequência de Bases , Linhagem Celular , Sequência Consenso , Cães , Íntrons , Rim , Vírus Miúdo do Camundongo/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Reação em Cadeia da Polimerase , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas Recombinantes/biossíntese , Deleção de Sequência , Transcrição Gênica , Transfecção , Proteínas não Estruturais Virais/biossínteseRESUMO
The two capsid proteins of minute virus of mice, VP1 and VP2, are generated from a single large open reading frame by alternate splicing of the capsid gene mRNA. Examination of the replication of a series of mutants that express only VP1, only VP2, or neither capsid protein demonstrates that VP2 is necessary for the accumulation and encapsidation of virus progeny single-stranded DNA. VP1 is dispensable for these functions but is required to produce an infectious virion. Virus that lacks VP1 binds to cells as efficiently as wild-type minute virus of mice but fails to initiate a productive infection. Because neither capsid protein is required for viral-DNA replication, these results suggest that virus lacking VP1 is blocked at a step during virus entry, subsequent to cell binding and prior to the initiation of DNA replication.
Assuntos
Capsídeo/genética , Vírus Miúdo do Camundongo/patogenicidade , Sequência de Aminoácidos , Animais , Transporte Biológico , Capsídeo/biossíntese , Capsídeo/ultraestrutura , Proteínas do Capsídeo , Linhagem Celular , Análise Mutacional de DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/crescimento & desenvolvimento , Dados de Sequência Molecular , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/patogenicidade , Virulência , Replicação ViralRESUMO
Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.
Assuntos
Capsídeo/metabolismo , Vírus Miúdo do Camundongo/química , Sequência de Aminoácidos , Sítios de Ligação , Capsídeo/química , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo , Células Cultivadas , Análise Mutacional de DNA , Vírus Miúdo do Camundongo/patogenicidade , Dados de Sequência Molecular , Mutagênese , Processamento de Proteína Pós-Traducional , Relação Estrutura-Atividade , Tripsina/farmacologia , VirulênciaRESUMO
cDNAs corresponding to RNA from the autonomous parvovirus minute virus of mice were cloned into constitutive and inducible expression vectors. These clones generate viral NS2, VP1, and VP2 proteins individually. Initial examination of these clones by transient expression analysis and analysis of stably transformed murine cell lines inducibly expressing these constructs indicated that they will be useful tools for characterizing the function of individual minute virus of mice gene products.
