Replication of MDV in vivo.

<p><b>(A)</b> Blood samples of chickens infected with the indicated viruses were taken at 4, 7, 10, 14, and 28 days post infection. <b>(B)</b> Contact chickens were sampled 14, 21, 28, 35 and 42 days p.i. Viral titers in the blood are shown as MDV genome copy numbers per 1×10⁶ cells of eight infected chickens per group. The detected viral loads are not statistically different among the groups, Kruskal-Wallis H test.</p

Characteristics of parental and recoded UL30 genes.

<p>Characteristics of parental and recoded UL30 genes.</p

Distribution of codon pair bias (CPB) scores and recoding of the MDV gene UL30.

<p><b>(A)</b> Distribution of calculated codon pair bias (CPB) scores of 15,762 predicted chicken, 112 MDV, and recoded MDV UL30 genes. Each light blue circle represents a calculated CPB score of a single chicken protein coding gene plotted against its protein length (amino acids). The arithmetic mean of all 15,762 CPB scores is 0.0755. The blue diamonds represent 112 predicted MDV protein coding genes. The pink circle represents original, wild type, labeled WWW, MDV UL30 gene. RWW, RRW and RRR (yellow crosses), which overlap each other and the parental UL30 represent calculated CPB scores of recoded MDV UL30 genes that have one, two or three segments of the ORF codon pairs-randomized. The red diamonds and green squares represent recoded UL30 genes that have either first, first two or all three segments of UL30 ORF codon pair-deoptimized (DWW, DDW, DDD) or optimized (OWW, OOW, OOO). <b>(B)</b> Structure of the MDV UL30 genomic region. UL30 encodes the catalytic subunit of the DNA polymerase, UL31 encodes a nuclear egress protein. UL30 and UL31 overlap at their 3’ ends by 98 nucleotides. UL30 is 3,663 nucleotide long and was divided into three equally long subsequences (1,221 nucleotides each), and each sequence was individually codon pair-optimized, -deoptimized, or -randomized. By recoding the individual MDV UL30 parts separately it is possible to generate different MDV UL30 mutants where only one (DWW, WDW, WWD), two (DDW, DWD, WDD), or all three UL30 segments (DDD) are recoded. The last 201 nucleotides of UL30 (blue triangles), which contain a polyadenylation signal and the overlapping coding sequences of UL31 were not altered by recoding.</p

Quantification of RNA expression and protein production from the recoded UL30 genes.

<p>HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P<0.05.</p

Codon pair deoptimization of UL30 impairs disease development and tumor formation in vivo.

<p><b>(A)</b> MD incidence in chickens infected with the parental (vWWW), mutant (vOOO, vDWW, vDDW) and revertant (vOOO-Rev, vDWW-Rev, vDDW-Rev) viruses. Animals infected with the vDDW mutant showed lower MD incidence than those that were infected with the parental virus. Comparison of survival curves via Log-rank (Mantel-Cox) test did not identify statistical differences between the groups. Tumor incidence in infected chickens (<b>B</b>) and contact sentinel chickens that were housed together with the infected chickens (<b>C</b>). Chickens that were infected with three revertant viruses were housed together in one room and shared one group of sentinel chickens. Tumor incidence is shown as the percentage of animals per group. Differences in tumor incidence among the groups of infected chickens are statistically significant. Tumor formation was impaired in contact chickens that were housed together with vDWW or vDDW infected chickens. Statistical analysis was done by Chi-square test, P<0.0001.</p

Multi-step growth kinetics of indicated viruses shown as mean and SEM.

<p>1×10⁶ CECs were infected with 100 PFU and viral progeny was titered 1–6 days post infection. <b>(A)</b> Comparison of growth curves of the parental (vWWW) and mutant viruses (vRRR, vOOO, vDWW and vDDW); n = 6, Kruskal-Wallis H test, * indicates P<0.05. <b>(B)</b> Comparison of growth curves of the parental (vWWW) and revertant viruses (vRRR-Rev, vOOO-Rev, vDWW-Rev and vDDW-Rev); n = 6, Kruskal-Wallis H test, P<0.05.</p

Model of chTR and vTR during MDV infection with vP6.1mut.

<p>chTR (gray) and wt vTR are able to interact with TERT (blue) and mediate telomerase activity, which is crucial for the survival of early MDV-transformed cells during initial crisis. P6.1mut vTR (Rose) is not able to interact with TERT and can, therefore, not contribute to telomerase activity. P6.1mut, as well as wt vTR, is able to interact with RPL22 (Red) and potentially also other factors (green) which mainly contributes to transformation and tumor dissemination.</p

P6.1 stem-loop mutation does not affect lytic replication <i>in vivo</i>, but delays MD incidence.

<p>A) qPCR analysis of the viral <i>ICP4</i> gene and the host <i>iNOS</i> gene. Blood samples were taken at 4, 7, 10, 14, 21, and 28 dpi and total DNA was extracted. Mean MDV genome copies/10⁶ blood cells of eight infected chickens per group as determined by qPCR analysis are shown with standard deviations (error bars). B) 1^st animal experiment: MD incidence in percent in chickens infected with vRB-1B (n = 5), vP6.1mut (n = 10) and vP6.1rev (n = 8) during the indicated time period C) 2^nd animal experiment: MD incidence in percent of vP6.1mut (n = 22) and vP6.1rev (n = 20) during the indicated time period. The time to develop MD in 50% of the inoculated animals (MD₅₀) is indicated (dashed line) and was significantly increased in the P6.1mut group (p = 0.0012).</p

Effect of the P6.1 mutation on telomerase activity.

<p>A) Schematic of the CR4–CR5 domain including a detailed representation of the P6.1 stem-loop. Structure of wild-type P6.1 (left) and mutant P6.1 stem-loop (P6.1 mut) (right) is shown. Nucleotide changes of the wt P6.1 stem-loop (blue) are shown in red. B) <i>In vitro</i> transcribed β-actin control RNA, wt vTR RNA, vTR containing the P6.1 mutation (P6.1) or a mutation in the template sequence (AU5) was analyzed on a 2% denaturing agarose-formaldehyde gel. Expected vTR size is indicated by the black arrow. C) Chicken TERT-His was translated <i>in vitro</i> using rabbit reticulocyte lysates and subsequently analyzed via western blotting using an anti-5x-His antibody. The expected size of TERT-His is indicated with the black arrow. D) Telomerase activity of the <i>in vitro</i> transcribed vTR variants was analyzed using gel based TRAP-assays. TRAP products and the internal control (IC) are indicated. The results shown are representative for three independent experiments showing similar results.</p

Primers used for cloning and mutagenesis.

<p>Underlined sequences indicate restriction enzyme sites. Bold indicates mutated sequences.</p