[Effect of deletion mutant IPS-1 on IFN-beta induction and its antiviral to HSV-1].
OBJECTIVE
The deletion mutant gene of IFN-beta promoter stimulator 1(DeltaIPS-1) was constructed by depleting the amino acid region from 300 to 444 loci and the recombinant plasmid pEF-BOS-FLAG/DeltaIPS-1 were constructed to study the effect of deletion mutant DeltaIPS-1 to IFN-beta induction and its antiviral to HSV-1.


METHODS
The mutant DeltaIPS-1 was obtained by overlap extension PCR. The depleting mutant DeltaIPS-1 recombinant plasmid pEF-BOS-FLAG/DeltaIPS-1 was constructed and identified by Xba I/Cla I digestion and DNA sequencing. HEK293T was Transiently transfected using the calcium phosphate precipitation method. The expression of pEF-BOS-FLAG/IPS-1 and pEF-BOS-FLAG/DeltaIPS-1 in HEK293T cell line was detected by Western blot. After transfected with different plasmids, HEK293T cells were infected by HSV-1 at different multiplicity of infection (MOI). The cytopathic effect (CPE) of the different plasmids in transfected cell groups was determined by amido black stainning. The viral titer of the supernatant of the cell culture medium in different plasmids of transfected groups at different time points was tested by plaque assay.


RESULTS
The eukaryotic expression vector of the depleting mutant IPS-1 gene was constructed successfully. The secretory volume of IFN-beta in pEF-BOS-FLAG/DeltaIPS-1 class was much lower compared with that in pEF-BOS-FLAG/IPS-1 class. The plaque assay showed the virus titre in pEF-BOS-FLAG/DeltaIPS-1 class was higher than that in pEF-BOS-FLAG/IPS-1 transfected class.


CONCLUSIONS
DeltaIPS-1 could decrease the secretory volume of IFN-beta in HEK293T cell and could not completely suppress the CPE of the cell infected by HSV-1. The antiviral function of DeltaIPS-1 to HSV-1 is weakened to some extent.
Ma
An
A
Institute of Pathogenic Biology, University of South China, Hengyang 421001, China. jhxiao223@163.com
Xiao
Jian-hua
JH
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D018259:Herpesvirus 1, Human / Q000254:growth & development*; D006801:Humans; D016899:Interferon-beta / Q000378:metabolism*; D009154:Mutation; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C505371:MAVS protein, human; D016899:Interferon-beta