RESUMO
We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused "squelching" of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1's transactivation activity that can enhance viral gene expression and help in virus replication.
Assuntos
Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/metabolismo , Penaeidae/virologia , Proteína de Ligação a TATA-Box/metabolismo , Transativadores/metabolismo , Vírus da Síndrome da Mancha Branca 1/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Penaeidae/genética , Penaeidae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , TATA Box , Proteína de Ligação a TATA-Box/genética , Transativadores/genética , Ativação Transcricional , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/metabolismoRESUMO
Voltage-dependent anion channel (VDAC) proteins abound in the outer membrane of mitochondria. They play an important role in mitochondrial membrane permeabilization (MMP), which can lead to stress-induced cellular apoptosis and necrosis. Several pathogens regulate this MMP in their host cells to benefit their replication cycle, while in other cases, the host can use the same mechanism to combat pathogenesis. In this study, the first shrimp VDAC gene was identified and characterized from Marsupenaeus japonicus (MjVDAC). Its open reading frame (ORF) contained 849 bp encoding 282 amino acids. The deduced MjVDAC protein includes the 4-element eukaryotic porin signature motif, the conserved ATP binding motif (the GLK motif) and a VKAKV-like sequence known in other organisms to be involved in the protein's incorporation in the mitochondrial outer membrane. Tissue tropism analysis indicated that MjVDAC is abundant in the heart, muscle, stomach and pleopod. MjVDAC proteins colocalized with mitochondria in transiently transfected Sf9 cells and in shrimp hemocytes. dsRNA silencing of shrimp VDAC delayed white spot syndrome virus (WSSV) infection by 1 day in different shrimp organs. Taken together, these findings suggest that MjVDAC is likely to be involved in WSSV pathogenesis.
Assuntos
Penaeidae/virologia , Filogenia , Canais de Ânion Dependentes de Voltagem/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Potencial da Membrana Mitocondrial/imunologia , Dados de Sequência Molecular , Penaeidae/genética , Penaeidae/imunologia , RNA/química , RNA/genética , Interferência de RNA/imunologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de Sequência , Análise de Sequência de DNA , Canais de Ânion Dependentes de Voltagem/genéticaRESUMO
It has recently been suggested that Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in the alternative adaptive immune system of invertebrates. Here, we isolated and characterized the first shrimp Dscam from Litopenaeus vannamei. The LvDscam protein had an extracellular domain but lacked the expected transmembrane domain and cytoplasmic tail, both of which are found in all other members of the Dscam family (and may also be found in other L. vannamei Dscams that have not yet been isolated). In nervous tissue, expression levels of LvDscam were unexpectedly low. Phylogenetic analysis suggests that LvDscam is far from the Dscams found in other invertebrates. Nevertheless, the domain architecture of the extracellular region of LvDscam is similar to other invertebrate Dscams, and it exhibits the typical configuration of 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD). Cloning and characterization of a total of 62 cDNAs from hemocytes collected from WSSV-free, WSSV-persistent and WSSV-acute-infected shrimp revealed 23 alternative amino acid sequences in the N-terminal of Ig2, 30 in the N-terminal of Ig3 and 13 in the Ig7 domain. This implies that LvDscam can potentially encode at least 8970 unique isoforms. Further analysis suggested that the LvDscam Ig2 and Ig3 regions are more functionally important than Ig7 in the shrimp's specific immune response against WSSV. We discuss how this tail-less, soluble Dscam can still play an active role in alternative adaptive immune response even while its axonal guidance functionality may be impaired.
