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. 2020 May;101(5):533-541.
doi: 10.1099/jgv.0.001386. Epub 2020 Feb 24.

Leaky scanning translation generates a second A49 protein that contributes to vaccinia virus virulence

Sarah Neidel  1 , Alice A Torres  1 , Hongwei Ren  1   2 , Geoffrey L Smith  1
Affiliations

Leaky scanning translation generates a second A49 protein that contributes to vaccinia virus virulence

Sarah Neidel et al. J Gen Virol. 2020 May.

Abstract

Vaccinia virus (VACV) strain Western Reserve gene A49L encodes a small intracellular protein with a Bcl-2 fold that is expressed early during infection and has multiple functions. A49 co-precipitates with the E3 ubiquitin ligase β-TrCP and thereby prevents ubiquitylation and proteasomal degradation of IκBα, and consequently blocks activation of NF-κB. In a similar way, A49 stabilizes β-catenin, leading to activation of the wnt signalling pathway. However, a VACV strain expressing a mutant A49 that neither co-precipitates with β-TrCP nor inhibits NF-κB activation, is more virulent than a virus lacking A49, indicating that A49 has another function that also contributes to virulence. Here we demonstrate that gene A49L encodes a second, smaller polypeptide that is expressed via leaky scanning translation from methionine 20 and is unable to block NF-κB activation. Viruses engineered to express either only the large protein or only the small A49 protein both have lower virulence than wild-type virus and greater virulence than an A49L deletion mutant. This demonstrates that the small protein contributes to virulence by an unknown mechanism that is independent of NF-κB inhibition. Despite having a large genome with about 200 genes, this study illustrates how VACV makes efficient use of its coding potential and from gene A49L expresses a protein with multiple functions and multiple proteins with different functions.

Keywords: Bcl2-fold; NF-κB inhibitor; gene A49R; leaky scanning; multiple functions; vaccinia virus; virulence.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Two proteins are encoded from the A49L gene. HEK-293T cells were infected with VACV WR at 5 p.f.u./cell. Where indicated, 40 µg ml−1 cytosine arabinoside (AraC) was added. At 2, 4, 6 or 8 h p.i. cells were harvested and cell extracts were analysed by SDS-PAGE and immunoblotting (IB) with antibodies to the VACV proteins A49 and D8 and cellular α-tubulin. Red arrows mark the positions of the large (L) and small (S) A49 proteins and the star marks a nonspecific signal also present at 0 h.
Fig. 2.
Fig. 2.
The A49 S protein is expressed by leaky scanning translation. (a) Immunoblot. HEK-293T cells were transfected with plasmids encoding WT A49 or mutants [as shown in (b)]. The following day cell extracts were prepared and analysed by SDS-PAGE and immunoblotting with antibodies against A49 (top) and α-tubulin (bottom). Red arrows mark the large (L) and small (S) A49 proteins. (b) Nucleotide sequence of the WT and mutant A49 alleles used in (a) starting from the ATG codon 1 of the full-length open reading frame. Positions mutated from the WT sequence are shown in red. A49 stop contains an in-frame termination codon at nucleotides 13–15 and a T to C substitution at nucleotide 6 to remove an OOF ATG codon. A49 ΔM1 has the ATG codon (positions 1–3) changed to CGA, and the same T to C change at position 6 as for A49 stop. A49 OOF has an insertion of two nucleotides after nucleotide 6 to put the ATG codons at codon 1 and 20 in different reading frames.
Fig. 3.
Fig. 3.
Mutation of methionine 20 abrogates A49 L function. (a) Reporter gene assay. HEK-293T cells were transfected with empty vector (EV), or plasmids expressing WT A49 or mutant A49 proteins either lacking the N-terminal 19 aa (Δ19) or with M20 changed to L, A, I or F (M20L, M20A, M20I and M20F). The following day, cells were stimulated (black bars) with TNF-α (15 ng ml−1 for 8.5 h) and then lysates were prepared and the Renilla and firefly luciferase activity were measured. Data presented are the mean of the firefly luciferase activity normalized to the Renilla luciferase activity (each in triplicate), which were then normalized to unstimulated EV±sd. **P<0.01 compared to stimulated EV. (b). Immunoblot. Cell extracts, prepared as in (a), were analysed by SDS-PAGE and immunoblotting with antibodies against VACV protein A49 (top) or α-tubulin (bottom). (c) Virulence measurement of VACVs. Groups (n=5) of female BALB/c mice were infected intranasally with 5×103 p.f.u. of indicated VACVs or were mock-infected (mock) and the weight of each mouse was measured daily. The weight of each mouse was compared to its weight on day zero and the data are presented as the mean±sem for each group. Statistical analysis was by two-way ANOVA test. The data from A49M20L and vΔA49 were each significantly different from A49WR (P=0.0001 WR versus vΔA49, and P=0.001 WR versus vM20L), but were not significantly different from each other.
Fig. 4.
Fig. 4.
Analysis of A49 alleles making either only the large or small protein. (a) Reporter gene assay using plasmids described in (c) and in Figs 2 and 3, and conducted as described in Fig. 3. (b) Immunoblot (IB) showing expression of the different A49 proteins using extracts from cells prepared as in (a). (c) Nucleotide sequence of WT A49 and mutants. Changes from the WT sequence are shown in red. The top three rows are sequences present in plasmids expressing these A49 alleles and the bottom two rows show the sequence of WT or SoA (strong first ATG in oofATG) mutant virus. oofATG (out-of-frame ATG) is mutated to create an ATG codon at nucleotides 47–49, between the ATG codons at positions 1 and 20. V17G is like oofATG but also has nucleotide 50 changed from T to G to give the new ATG codon a stronger Kozak consensus sequence. SoA has the T at position −3 changed to A to give the codon 1 AUG a stronger Kozak consensus sequence.
Fig. 5.
Fig. 5.
Expression of A49 proteins in cells infected with VACVs. HEK-293T cells were infected at 5 p.f.u./cell with WT VACV (vWT) or viruses designed to make neither A49 protein (vΔA49), only the large (L) protein (vA49SoA), or only the small protein vA49Δ19. At 16 h p.i. cells were harvested and cell extracts were analysed by SDS-PAGE and immunoblotting with antibodies to VACV proteins A49 and D8 or α-tubulin. Red arrows indicate the positions of the large (L) or small (S) A49 proteins.
Fig. 6.
Fig. 6.
Virulence of VACVs. Groups of mice were infected with the indicated viruses as in Fig. 3c and were weighed daily. Data were analysed and are expressed as described in Fig. 3c. Statistical analysis was by two-way ANOVA test. Groups vA49Δ19 and vA49SoA were not significantly different from each other, but were each significantly different from both vΔA49 and A49WR (P=0.01 soA vs WR and soA versus vΔA49).
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