Figure 1.
diagram of MDV vTR secondary structure, location of the CR1 and CR4-5 domains, and incorporated mutations.
A) The pseudoknot (core), containing the template sequence, and the CR4-CR5 domains containing the P6.1 stem loop, are indicated with boxes. B) The pseudoknot domain including the sequence of wild-type vTR template and AU5 template mutant (AU5). Nucleotide changes in the template sequence are shown in red. C) The CR4-CR5 domain showing detailed representations of the P6.1 stem-loop and the structures of wild-type P6.1 (left) and mutant P6.1 stem-loop (P6.1mut) (right) are shown. Nucleotide changes of the wt P6.1 stem-loop (blue) are shown in red and have been previously published [22].
Figure 2.
Expression of vTR harboring the mutant template (AU5) decreases cell proliferation of an avian cancer cell line.
A) Analysis of telomerase activity in primary CEC cultures, the chicken fibroblast cell line DF-1 with or without TERT expression, and the quail QT35 cancer cell line using TRAP assays. TRAP products representing telomere elongation and internal control (IC) are indicated. B) RT-qPCR of vTR copies in polyclonal empty vector, vTR or AU5 QT35 cell lines induced with 1 µg/ml doxycycline (Dox) for 3 and 5 d or left uninduced. Data is shown as relative quantitation (RQ) of vTR copies relative to quail GAPDH RNA copies that served as an endogenous control. C) Percent (%) confluency of vector, vTR, and AU5 cell lines over the course of 31 d. Results are shown as means and standard errors of three independent experiments. P values were determined between each group using Student's t tests.
Figure 3.
MDV harboring a template mutant vTR replicate comparable to parental and revertant viruses in vitro.
A–B) Plaque areas were determined for 35 (A) or 100 (B) randomly selected plaques for indicated viruses. Results are shown as mean plaque areas in percent of the parental vRB-1B with standard deviations (error bars). C–D) Multi-step growth kinetics of indicated viruses were performed in triplicates and are shown as means with standard deviations (error bars).
Figure 4.
Tumor induction and in vivo replication of MDV harboring mutant template sequence (AU5) vTR.
MD-susceptible chickens were inoculated with 1,000 PFU of either vRB-1B (n = 12) or vAU5 (n = 11) in experiment 1 (A and B) and 2,000 PFU of vRB-1B (n = 17), vAU5 (n = 19), or vAU5rev (n = 17) in experiment 2 (C and D). A and C) Necropsies were performed on chickens following onset of clinical signs of MD during both experiments and the percent of infected chickens developing tumors over 13 wk was determined. B and D) DNA was obtained from peripheral blood of chickens infected with each respective virus and viral genome copies were determined using qPCR assays. MDV ICP4 copies were normalized to the chicken iNOS gene and are shown as MDV genome copies per 1×106 cells with standard error of mean bars. Viremia induced by vAU5 was significantly reduced when compared to vRB-1B (in experiment B) 14dpi, p = 0.007; 21dpi, p = 0.003; 28dpi, p = 0.014; in experiment D) 14dpi, p = 0.013; 21dpi, p = 0.004; 28dpi, p = 0.049) and vAU5rev (in experiment D) 10dpi, p = 0.022; 14dpi, p = 0,003) at the time points indicated by asterisks (*) using Student's t tests.
Figure 5.
In vitro replication of parental, mutant, and revertant viruses.
Plaque areas were determined for 100 randomly selected plaques for the indicated viruses. Results are shown as mean plaque areas in percent of the parental vRB-1B with standard deviations (error bars).
Figure 6.
Secondary mutation of the vTR-TERT interaction domain, P6.1, rescues MDV replication and lymphomagenesis.
MD-susceptible chickens were infected with vRB-1B (n = 17), vP6.1mut (n = 16), vAU5 + P6.1mut (n = 18), vAU5 + P6.1rev (n = 19), or vAU5rev + P6.1rev (n = 18). A) DNA was obtained from blood of infected chickens and MDV genome copies are shown per 1×106 cells as in Fig. 3. Significant differences in genome copies between vAU5 + P6.1rev and vRB-1B (14dpi, p = 0.013; 21dpi, p = 0.004; 28dpi, p = 0.049) and vAU5rev + P6.1rev (14dpi, p = 0.002) are indicated with an asterisk (*) using Student's t test. B) Tumor incidences for each group infected with viruses contained only the AU5 mutation (empty boxes), additional/exclusively the P6.1 mutation (grey symbols) or parental and complete revertants (black symbols) were measured for 13 weeks.
Figure 7.
Immunization with the vAU5 mutant viruses protects chickens from lethal MDV infection.
A) MD-incidence in N2a (A; n = 14) and B) P2a (B; n = 14) chickens vaccinated with either vAU5, CVI988 or media alone (Mock) before challenge-infection with RB-1B. Precent protection from the onset of disease or being tumor-positive at termination of the experiment is shown in % of the animals.
Figure 8.
Proposed model for abrogation of tumor induction by mutant template sequence vTR through incorporation of mutant telomere sequences in transformed T cells.
Expression of vTR AU5 leads to telomere instabilities, aberrant chromosome separation and segregation, and finally apoptosis induction in the presence of TERT (upper panel). Without vTR interaction with TERT by mutation of the P6.1 stem loop, mutant template sequences (AU5) are not incorporated in the telomeres of transformed cells and thus proliferation of transformed cells continues, leading to lymphomas.
Table 1.
Primers used for cloning and generation of mutant and revertant AU5 constructs.