Assuntos
Diabetes Mellitus Experimental/terapia , Genes Reporter , Terapia Genética , Animais , Dependovirus/genética , Cães , Expressão Gênica , Engenharia Genética , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Camundongos , Músculo Esquelético/metabolismo , Plasmídeos , Transformação GenéticaRESUMO
Herpes simplex virus (HSV) has been shown to induce DNA amplification in the host cell genome, which can be suppressed by the adeno-associated virus type 2 (AAV-2) rep gene (Heilbronn et al., 1990, J. Virol. 64, 3012-3018). In an attempt to define domains of Rep which are required for this effect a set of expression constructs was generated for Rep mutants with either N-terminal and/or C-terminal truncations, with small internal deletions, or with point mutations. In transient cotransfection assays these mutants were tested for the inhibition of HSV-induced DNA amplification and in parallel for DNA replication of a rep-defective AAV genome. Our data show that the C-terminal region of Rep where spliced and unspliced proteins differ is dispensable for both AAV DNA replication and inhibition of HSV-induced DNA amplification. The N-terminus of Rep is required for AAV DNA replication, whereas the first 174 amino acids can be deleted without loss of function for the inhibition of DNA amplification. Rep52 which starts at methionine 225 is neither sufficient, nor required for this effect. We further analyzed the region between amino acids 174 and 225: A stretch of 16 highly hydrophilic amino acids is dispensable for the inhibition of DNA amplification, but it is required for AAV DNA replication. Deletion of two short motifs spanning putative protein kinase C phosphorylation sites each strongly reduce both AAV DNA replication and inhibition of DNA amplification, whereas a single amino acid substitution of one of these sites abolished AAV DNA replication with no effect on the inhibition of DNA amplification. Our data show that most, but not all, of the sequence elements within the N-terminus of Rep78 required for AAV DNA replication coincide with those required for the inhibition of HSV-induced DNA amplification. A replication-negative version of Rep78 comprising the internal 60% of the protein still carry the entire inhibitory function for HSV-induced DNA amplification.
Assuntos
Proteínas de Ligação a DNA/genética , Dependovirus/genética , Amplificação de Genes , Simplexvirus/fisiologia , Proteínas Virais/genética , Animais , Linhagem Celular , Cricetinae , Cricetulus , DNA Viral/biossíntese , Células HeLa , Humanos , Vírus 40 dos Símios/genéticaRESUMO
We have previously described the apparent acquisition by human herpesvirus 6 (HHV-6) of the multifunctional rep gene of the helper-dependent human parvovirus adeno-associated virus type 2 (AAV-2). We report here that HHV-6 is a full helper virus for AAV-2 replication, suggesting a mechanism for transfer of the rep gene between the two viruses by recombination of replicative intermediates. The HHV-6 rep gene cloned under control of the human cytomegalovirus immediate early promoter complemented replication of a rep-deficient AAV-2 genome. In cotransfection experiments with heterologous promoters linked to the CAT reporter gene, HHV-6 rep activated the human immunodeficiency virus (HIV) long terminal repeat (LTR) in fibroblast cell lines but not in T-cells. In contrast, AAV-2 rep inhibited HIV LTR activity in both fibroblast and T-cell lines. The effect of HHV-6 and AAV-2 rep genes on the HIV LTR was independent of the NF-kappa B, Sp1, and TATA box elements. These results suggest that HHV-6 Rep is a multifunctional regulatory protein with properties related to, but distinct from, those of AAV-2 Rep.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , Dependovirus/crescimento & desenvolvimento , Regulação Viral da Expressão Gênica/genética , Vírus Auxiliares/fisiologia , Herpesvirus Humano 6/fisiologia , Proteínas Virais/genética , Replicação Viral/genética , Linhagem Celular , DNA Viral/biossíntese , Proteínas de Ligação a DNA/fisiologia , Genes Reguladores/fisiologia , Genes Virais/fisiologia , Teste de Complementação Genética , Repetição Terminal Longa de HIV/genética , Humanos , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência/fisiologia , Homologia de Sequência do Ácido Nucleico , Ativação Transcricional , Transfecção , Proteínas Virais/fisiologiaRESUMO
Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.
