Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles
Figure Lengend Snippet: ORF2 and ORF3 are required for releasing viral particles to infect naïve HepG2C3A cells. ( A ) Schematic representation of the transcomplementation system for packaging HEV virions. ( B ) Representative flow cytometry plots demonstrating efficient ORF2 and ORF3 expression. HepG2C3A cells were transduced with pLVX-ORF2-IRES-zsGreen and/or pLEX-ORF3-IRES-mCherry or not transduced. Flow cytometric analysis was performed 4 d following transduction to quantify the frequencies of ORF2 and/or ORF3 expressing cells. ( C ) Infection kinetics of transcomplemented HEV in HepG2C3A cells. Cell culture supernatants from naïve HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, were collected 5 d posttransfection with rHEVΔORF2/3[Gluc] RNA. Naïve HepG2C3A cells were incubated with these supernatants. After 12 h, cells were washed and Gaussia luciferase activity quantified in the cell culture supernatants at the indicated time points. ( D ) Five days following transfection of rHEVΔORF2/3[Gluc] RNA into HepG2C3A, or HepG2C3A cells transduced with HEV ORF2 or/and ORF3, lysates were used to infect naïve HepG2C3A cells. Gluc activity was measured in the supernatants 4 d postinfection. Shown are averages and SDs of triplicate measurements of three independent experiments. * P
Article Snippet: To construct a lentiviral construct that encodes Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3, the Kernow C1/p6 ORF2 or Kernow C1/p6 ORF3 cDNA was amplified by PCR from the HEV Kernow C1/p6 construct (a kind gift from Suzanne Emerson, NIH, Bethesda, MD) and then cloned into pLVX-IRES-zsGreen1 or pLEX-IRES-mCherry vectors using the In-Fusion HD Cloning Kit (Clontech).
Techniques: Flow Cytometry, Cytometry, Expressing, Transduction, Infection, Cell Culture, Incubation, Luciferase, Activity Assay, Transfection