Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence
Figure 2
Mutant viruses produce equivalent levels of Gag protein but the virus N26 is transmitted better.
(A) The schematic diagram of the HTLV-1 molecular clones indicates the amino acid change in each clone. The initiation codon for Orf-I is mutated in p12KO such that no Orf-I protein is made. The changes did not affect the sequence and/or function of the overlapping pX region genes. Infectious molecular clones or control DNA were co-transfected with an HTLV-1-LTR-luciferase construct and the renilla-luciferase transfection efficiency control into 293T-cells and culture supernatants or protein lysates prepared 48 hours after transfection. (B) The HTLV-1 promoter activity induced by the HTLV-1 mutant was measured by assaying luciferase activity from transfected cell lysates. Luciferase activity for each clone (indicated on the x-axis) from three independent transfection experiments was graphed (n = 3). LTR-luciferase activity was normalized using the transfection efficiency control renilla-luciferase activity. Error bars indicate the standard deviation. (C) Western blot analysis of protein lysates from transfected cells was assayed for intracellular p24Gag expression (top panel) or the loading control, tubulin (bottom panel). (D) Culture supernatants from transfected 293T-cells were collected, spun to remove debris and assayed for p19Gag levels using an HTLV-1 ELISA kit. The values graphed are from three independent experiments (n = 3). (E) Stable producer 729.6 B-cell lines were cloned and used to quantify the transmission of the viral mutants. The 729-HTLV-1-producing cells or parental control cells were co-cultured with BHK1E6 cells and 48 hours later, adherent cells were stained for -galactosidase activity. Graphed is the number of blue cells per well for the indicated clone from three independent wells (n = 3). Error bars indicate standard deviation. By ANOVA and t- test, transmission of WT, D26N and G29S was significantly different than control (p<0.0001). Transmission of D26N was significantly different than WT, G29S and p12KO (p = 0.0007). There was no significant difference among transmission of WT, G29S and p12KO. Western blot analysis for HTLV-1 p24Gag was performed on whole cell extracts from 729-HTLV-1 producing cell lines. The housekeeping gene tubulin is shown for a loading control (lower panels).