Immunoprecipitated proteins were separated on SDS-10%PAGE, followed by Coomassie Brilliant Blue staining. At 2 days post transfection, cell lysates were immunoprecipitated with an anti-Flag (NP1 Flag, lane 2) or control IgG (lane 1). HEK293 cells were transfected with pIHBoV1 ΔNP1 and pCI-NP1 Flag. (B) Co-immunoprecipitation (IP) and SDS-(10%) PAGE. dRF, mRF, and ssDNA represent double, monomer replicative form DNA and single stranded DNA, respectively. HBoV1 duplex DNA excised from pIHBoV1 was used as a size marker (M) of ~5.5 kb (Lane 1). At 2 days post transfection, Hirt DNA were extracted in each transfection group and analyzed by Southern blotting using a full-length HBoV1 genome as a probe. #Imagequant tl iqtl software ge healthcare education plusHEK293 cells were transfected with pIHBoV1, pIHBoV1 ΔNP1, and pIHBoV1 ΔNP1 plus pCI-NP1 Flag, respectively. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.Īffinity purification of NP1-interatcing cellular proteins. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (K D ) of 95 nM and 122 nM, respectively. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. NP1 plays an important role in viral DNA replication and pre-mRNA processing. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children.
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