Functional advantages of triplication of the 3B coding region of the FMDV genome

Abstract

For gene duplication to be maintained, particularly in the small genomes of RNA viruses, this should offer some advantages. We have investigated the functions of a small protein termed VPg or 3B, which acts as a primer in the replication of foot-and-mouth disease virus (FMDV). Many related picornaviruses encode a single copy but uniquely the FMDV genome includes three (nonidentical) copies of the 3B coding region. Using sub-genomic replicons incorporating nonfunctional 3Bs and 3B fusion products in competition and complementation assays, we investigated the contributions of individual 3Bs to replication and the structural requirements for functionality. We showed that a free N-terminus is required for 3B to function as a primer and although a single 3B can support genome replication, additional copies provide a competitive advantage. However, a fourth copy confers no further advantage. Furthermore, we find that a minimum of two 3Bs is necessary for trans replication of FMDV replicons, which is unlike other picornaviruses where a single 3B can be used for both cis and trans replication. Our data are consistent with a model in which 3B copy number expansion within the FMDV genome has allowed evolution of separate cis and trans acting functions, providing selective pressure to maintain multiple copies of 3B.

Keywords: 3B; FMDV; evolution; gene duplication; picornavirus; trans-complementation.

FIGURE 1

FMDV genome replication can occur with fewer than three functional copies of 3B. A, Annotated cartoon of FMDV replicon showing the capsid‐coding region replaced with a reporter gene. B, BHK‐21 cells in duplicate wells of a 6‐well plate were pretreated for 1 hour with 10 µg/mL of actinomycin D at 37°C. Cells were then transfected with T7 RNA transcripts of respective replicon constructs and radio‐labeled with [³H]‐U. These include WT, a polymerase active site mutant (3D‐GNN) and replicons where one, two, or all the 3Bs have been inactivated by Y3F substitution (Table 1). At 8 h.p.t., total RNA extract was harvested and quantified spectrophotometrically. RNA synthesis was quantified as scintillation counts per microgram of RNA (n = 3 ± S.D., *P < .05, **P < .01, ***P < .001)

FIGURE 2

Competitive advantage of multiple copies of FMDV 3B. A, FMDV replicon, highlighting the P3 region. Constructs were designed (listed in Tables 1 and S1) with one or two copies of 3B but maintaining the 3A‐3B1 and 3B3‐3C junctions. B, BHK‐21 cells were transfected with T7 RNA transcripts of GFP replicon constructs. Expression was monitored hourly and is shown as fluorescence positive cells at 8 h.p.t. Each data set represents an average of two wells (n = 3 ± S.D.). C, Co‐transfection of BHK‐21 cells with equimolar concentrations of T7 transcripts of FMDV mCherry replicon 3B3 and ptGFP replicon constructs with various copies of 3B. Expression of mCherry and ptGFP was monitored hourly and is shown as fluorescence positive cells at 8 h.p.t. Each replicate represents an average of two wells (n = 3 ± S.D., *P < .05, **P < .01)

FIGURE 3

Replication of constructs incorporating gene fusions. A, Schematic of the FMDV replicon. Several fused constructs were designed (Tables 1 and S1) with each 3B fused to its neighbor, including fusions between 3A and 3B1 or 3B3 and 3C. Chimeric constructs were designed to maintain the 3A‐3B1 and 3B3‐3C junctions. The P3 region is expanded for clarity. Fused regions are indicated with asterisks. Replicon constructs were transcribed, and RNA transfected into BHK‐21 cells. Replication was monitored using an IncuCyte. B, Replication of the fused constructs. C, Replication of fusion constructs that incorporate inactivating Y3F substitutions. D, Replication of fusion constructs incorporating both inactivating Y3F substitution and 3B deletions. Figure shows total GFP fluorescence at 8 h.p.t. Baseline represents input translation. Each replicate represents an average of two wells (n = 2 ± S.D., **P < .01, ***P < .001)

FIGURE 4

FMDV can tolerate an additional copy of 3B. BHK‐21 cells in duplicate wells of a 6‐well plate were pretreated for 1 hour with actinomycin D at 37°C. Cells were transfected with T7 replicon RNA transcripts, which include a novel 3B (termed 3Bx) as either an additional 3B or as the only functional 3B. Constructs used previously (Figure 1) were also included as controls. At 1 h.p.t., [³H]‐U was added, and replication monitored as mCherry expression using an IncuCyte for 8 hours. A, Schematic representation of FMDV WT and 3Bx replicon constructs. B, Total mCherry fluorescence at 8 h.p.t. Each replicate represents an average of two wells (n = 3 ± S.D. *P < .05, **P < .01, ***P < .001). C, At 8 h.p.t., total RNA was extracted and quantified spectrophotometrically. [³H]‐U incorporation was measured as scintillation counts per microgram of RNA (n= 3 ± S.D. **P < .01)

FIGURE 5

Trans‐complementation by 3B in FMDV differs from enteroviruses. A, Cartoon illustrating trans‐complementation experiment with two replication‐incompetent replicon constructs. Replication‐competent constructs were included as controls. Figures B, C, and D show average fluorescence of duplicate wells at 8 h.p.t. of two replicons co‐transfected into BHK‐21 cells. B, PV‐GNN‐mCherry and PV‐3B^Y3F‐GFP with controls. Each replicate represents an average of two wells (n = 3 ± S.D., **P < .01, ***P < .001). C, EV71‐GNN‐mCherry and EV71‐3B^Y3F‐GFP with controls. Each replicate represents an average of two wells (n = 2 ± S.D. *P < .05). D, FMDV‐GNN‐mCherry with GFP replicons encoding one, two, or three active 3Bs. Each replicate represents an average of two wells (n = 3 ± S.D., *P < .05, **P < .01)