Skip to main content
. 2006 Dec 20;81(5):2391–2400. doi: 10.1128/JVI.01601-06

FIG. 5.

FIG. 5.

Mapping of ZAP target sequences in Zaire-EBOV and MARV Musoke genomes. (A) cDNA fragments of Zaire-EBOV- and MARV Musoke-specific genes were cloned into pGL3 or pGL3-B between the firefly luciferase gene and poly(A) site. Inserted sequences as well as the approximate size of each insert (in kilobases) are indicated below each bar. The L genes were divided into four fragments (L1 to L4). SIN corresponds to a SIN genome fragment known to be sensitive to ZAP. The plasmids were transfected into Rat2-Zeo or Rat2-NZAP-Zeo cells. Cotransfection of a plasmid not sensitive to ZAP encoding Renilla luciferase (pRL-TK) was used to normalize firefly luciferase values. Cells were lysed 48 h posttransfection, and luciferase activity was measured. The inhibition (n-fold) was calculated as the normalized luciferase activity in Rat2-Zeo cells divided by the normalized luciferase activity in Rat2-NZAP-Zeo cells. Data are means and ranges of duplicate transfection experiments. (B) The EBOV L4 fragment was further divided into three or four overlapping fragments. A schematic representation of the fragments is shown at the left. Transfection and calculation of inhibition was performed as described for panel A. Numbers in brackets depict the approximate size of the inserts in kilobases. Data represent means and ranges of duplicate transfection experiments. (C) Four overlapping sequences covering the genome of YFV (YF1 to YF4) were inserted into pGL-3. The size of each insert (in kilobases) is shown in brackets. Transfection and calculation of inhibition was performed as described for panel A. Data represent means and ranges of duplicate transfection experiments. (D) The pGL-3 plasmid containing the EBOV L4 fragment was used to analyze the activity of NZAP-Zeo zinc finger mutants in the luciferase reporter assay. Transfection was performed with Rat2 cells expressing NZAP-Zeo mutants (H86K, C88R, C168R, or H191R). Empty pGL3 was used as a control. Transfection and calculation of inhibition was performed as described for panel A. Data represent means and ranges of duplicate transfection experiments.