Figure 1.
Organization of FCV genome and TURBS.
A) Schematic map of FCV genomic and subgenomic RNAs and location of the essential elements of the TURBS in the 3′ terminal sequence of ORF2. On top, the basic organization of the FCV genomic and subgenomic RNAs is illustrated (not drawn to scale). Shaded bars represent ORF2 and ORF3 coding for the capsid proteins and the so-called leader of the capsid protein (LC, light grey bar). The white bar symbolizes the nonstructural protein coding region (ORF1) with the different cleavage products named by size (within bar) and NS protein nomenclature (below bar). The nontranslated regions (NTR) at the 5′ and 3′ end are shown as black lines, the poly(A) tail at the 3′ end is indicated. VPg (virus protein, genome linked) present at the 5′ ends of both the genomic and subgenomic RNAs is symbolized by a black circle at the end of the line representing the 5′ NTR of the RNAs. The proteins encoded by the subgenomic RNA are indicated by abbreviations: LC: Leader of the capsid, VP1: major capsid protein, VP2: minor capsid protein. Red ‘T’ = TURBS. Below the scheme, the 3′ terminal 72 nucleotides of the ORF2 cDNA sequence are shown. Motif 1 (red), 2* and 2, determined by previous analyses [23], and the start/stop codons in the overlap-region of ORF2 and ORF3 are highlighted. B) Blow up of the cDNA region containing the TURBS with the proposed structure of the motif 2*, motif 1 (red) and motif 2 region. G-C and A-T pairing is symbolized by red and blue dots, respectively.
Figure 2.
Comparison of the 5′ terminal VP2-coding sequences of different FCV isolates.
Comparison of the cDNA sequences of the ORF3 5′ region for different FCV isolates. In the FCV 2024 sequence, the start/stop site and the two downstream stop codons in the ORF2 frame are highlighted. Residues conserved in all sequences are marked by asterisks.
Figure 3.
Effect of three nucleotide deletions downstream of the start/stop site on translation reinitiation.
A) cDNA sequences of wild type pCH1, a cDNA construct designed for expression of FCV ORFs 2 and 3 [23], and mutants thereof with deletions of 3 nucleotides within the 5′ part of ORF3. On the left, the name of the construct is given and on the right the VP2 expression level compared to the wt construct is given. In the wt sequence, the start/stop site and the downstream stop codons are highlighted. B) Autoradiographs show the proteins immunoprecipitated after transient expression of the constructs indicated on top. On the right site the name of the precipitated proteins is given. Slight variation in the electrophoretic mobility of the different VP2 proteins was observed which is especially obvious for the pMH49 product. According to nucleotide sequence analyses these differences are not due to unwanted second site changes and therefore reflect the differences in amino acid composition between the individual mutated proteins.C) The VP2 expression efficiency is given in a bar diagram with the standard deviation indicated by error bars. The presented data represent the mean values of three independent experiments given as the percentage of the value determined for pCH1 (data normalized relative to the levels of LC-VP1 expression).
Figure 4.
Influence of point mutations shortly downstream of the start/stop site on reinitiation frequency.
Results of transient expression studies with mutants of the sg mRNA expression construct pCH1 (wt) containing exchanges within the first 5 residues of ORF3. Panel (A) shows the names (left column) and sequences (middle column) of the different constructs with the start/stop site ATGA in bold face and the mutated residues underlined. For pCH1 the two stop codons in the ORF2 frame downstream of the start/stop site are also given in bold face. The right panel gives the VP2 expression rates as % of the wt (wt set to 100%) normalized to the VP1 expression rate. Panel (B) summarizes the expression rates of the different constructs determined via transient expression, in situ labelling with 35S amino acids, immunoprecipitation and phophorimager quantification in a bar diagram. The results given represent the mean values of at least three independent expression experiments and indicate the expression levels in % of the wt construct (wt set to 100%), normalized to the expression level of LC-VP1. Error bars give the standard deviation.
Figure 5.
Effect of number and location of stop codons downstream of the start/stop site on VP2 expression levels.
Results of transient expression studies with mutants of the sg mRNA expression construct pCH1 containing exchanges affecting the number and/or position of stop codons within the 5′ part of the ORF3 containing RNA. In (A) the sequences of the respective parts of the constructs are shown in the middle column. All stop codons in the given sequences are shown in bold face. Nucleotides differing from the wt sequence are underlined. For the wt pCH1, also the start codon of the start/stop site is given in bold face and the number of the stop codon is given above of the sequence. The names of the constructs and the determined VP2 expression levels are listed in the left and right columns, respectively. In (B) the results of transient expression studies conducted with the constructs are shown. The reinitiation frequency was determined via immunoprecipitation of the transiently expressed proteins, in situ labelled with 35S amino acids, and phophorimager quantification. The results given represent the mean values of at least three independent expression experiments and indicate the expression levels in % of the wt construct (wt set to 100%). The results were normalized to the amount of LC-VP1 to correct for variations in transfection rates. Error bars indicate standard deviation. Please note that stop codon #4 located closest to the 3′ end in the displayed part of the pCH1 sequence is in another reading frame than the first 3 termination codons.
Figure 6.
Analysis of putative translational readthrough at the start/stop site.
The results of transient expression studies aiming at detection of potential translational readthrough at the termination signal within the start/stop site are shown. The experiments were based on a synthetic expression construct, in which all Cys codons within the VP1-coding sequence were altered into Ser codons (WTΔCys). This construct and mutants thereof were analysed in transient expression studies. The mutants pMH168 and pMH170 were established by introducing Cys codons in the VP1 frame downstream of the termination signal of the start/stop site of WTΔCys as shown in the sequence in (A) (altered nucleotides underlined). Construct pMH169 served as a control. It was generated by changing the stop codon of the start/stop site in pMH168 into a Cys codon. For the wt construct pCH1, the start/stop sequence and the stop codons downstream thereof are given in bold face. In (B) the results of transient expression studies using either labelling with [35S] methionine (left part) or [35S] cysteine (right part) and immunoprecipitation. The names of the constructs are given on top of the gel. Vacc: negative control showing products precipitated from mock transfected vaccinia virus MVA-T7 infected cells.
Figure 7.
Ability for hairpin structure formation in the start/stop region of wt and mutant sequences.
A) Sequences of the 5′ terminal region of the VP2 coding RNA derived from the wt construct pCH1 or mutants thereof displaying the indicated changes. Mutated nucleotides are underlined and deleted residues are indicated as horizontal lines. In the wt sequence the start/stop residues as well as termination codons downstream thereof are given in bold face. On the left site, the name of the cDNA constructs is given whereas on the right the free energy of the secondary structure (determined by Mfold, given as ΔG in kCal/mol) and the VP2 expression efficiency as percent of the wt level is given. B) Mfold calculated structures of a selected set of the RNAs given in (A). The start codon of the start/stop site is given in bold face, the stop codon is highlighted by a line. Nucleotides differing from the wt sequence are given in red and circled. A-U or G-U pairing is indicated by blue dots, G-C pairing by red dots.
Figure 8.
Test for a correlation of hairpin stability with reinitiation frequency.
A) Sequences of the 5′ terminal region of the VP2 coding RNA derived from the wt sg mRNA encoding construct pCH1 or mutants thereof displaying changes affecting stability of putative hairpin structures closely downstream of the start/stop region. Mutated nucleotides are underlined. Differences in mutants pMH163 and pMH165 with regard to mutants pMH74 and pMH75, respectively, are given in lower case. For the wt construct pCH1 the start/stop region and termination codons downstream thereof are given in bold face. Left of the sequences, the names of the constructs are given, whereas on the right side the stability of the secondary structure (determined by MFold and given as ΔG in kCal/mol) and the VP2 expression rate (given as mean value of at least three independent experiments in % of the wt level which was set to 100%) are shown. B) Mfold calculated structures of the RNAs derived from the constructs given in (A). The start codon of the start/stop site is given in bold face, the stop codon is highlighted by a line. Nucleotides differing from the wt sequence are circled or marked by a box when a stretch of several nucleotides is affected. A-U or G-U pairing is indicated by blue dots, G-C pairing by red dots.
Figure 9.
Recovery and analysis of viruses with mutations altering VP2 expression rates.
In the upper part, the cDNA sequence of part of the TURBS region is shown for the constructs specified on the left with the encoded amino acid sequence of wt given in the bottom line. Since the introduced mutations are concentrated in different regions only these parts of the sequence harbouring exchanges are shown resulting in two different blocks. Nucleotide exchanges are highlighted in red. In the amino acid sequence, the two residues that are changed in mutants pCH83/pCH84 or pCH12/pCH13 are shown in red. In the lower part, the sequences coding for the aminoterminal part of VP2 of a third group of constructs with exchanges in this area are shown. Below each nucleotide sequence, the encoded amino acid sequence is given. Changes with regard to the wt sequence are given in red. The Leu (L) at position 7 of VP2 is shown in green since this position was shown before to be critical for virus viability [16]. On the left, the names of the constructs are given whereas on right site the VP2 expression efficiencies in per cent of the wt level are indicated (given as mean value of at least three independent experiments).
Table 1.
Results of virus mutant recovery experiments.
Figure 10.
Growth curves of viruses with TURBS mutations.
Growth curves of viruses recovered from the full length constructs containing the mutations displayed by pCH2, pCH3 and pCH83 (VpCH2, VpCH3 and VpCH83, respectively) in comparison to the virus recovered from the wt infectious cDNA clone. The curves show the results of a representative experiment in titer (TCID50) at a given time point (hours post infection – hpi). The infection was done at an m.o.i. of 0.0003.
Figure 11.
Construction, and analysis of viruses expressing VP2 via a separate sg mRNA.
Establishment and characterization of viruses containing in their genomes duplicated sg mRNA promoters. (A) Genome organization of the different viruses is given as a scheme (not drawn to scale). Grey bars represent ORF2 and ORF3 coding for the capsid proteins and the leader of the capsid protein (LC, light grey). The polyprotein containing the nonstructural proteins (encoded by ORF1) is symbolized by a white bar with the designation of the different cleavage products. Black lines represent the nontranslated regions (NTR) at the 5′ and 3′ end, the 3′ poly(A) tail is indicated. VPg (virus protein, genome linked) present at the 5′ ends of both the genomic and subgenomic RNAs is symbolized by a black circle at the end of a line representing the 5′ NTR of the RNAs. The proteins encoded by the subgenomic RNA are indicated by abbreviations: LC: Leader of the capsid, VP1: major capsid protein, VP2: minor capsid protein. The red ‘T’ stands for the TURBS. The sg promotor region and duplicated fragments thereof are shown in light blue, a nonrelated sequence used as a spacer in sgad249 is represented by a black bar. (B) Northern blot with RNA isolated from cells infected with the viruses indicated above of the gel. Hybridization with a probe covering the VP2-coding sequence was done as described before [27] (C) The VP2 expression rate of the recovered viruses was determined by in situ labelling with 35S amino acids, immunoprecipitation and phosphorimager analysis. The results are shown as percent of the wt virus expression rate (set to 100%). The values were normalized to the VP1 expression rate. Error bars represent standard deviation determined from at least 3 independent experiments (D) Growth curves of the recovered viruses inoculated at a starting m.o.i. of 0.0003. The graph shows the mean values of 2 to 3 experiments with the standard deviation indicated by error bars.