Vaccinia virus A12L protein and its AG/A proteolysis play an important role in viral morphogenic transition

Abstract

Like the major vaccinia virus (VV) core protein precursors, p4b and p25K, the 25 kDa VV A12L late gene product (p17K) is proteolytically maturated at the conserved Ala-Gly-Ala motif. However, the association of the precursor and its cleavage product with the core of mature virion suggests that both of the A12L proteins may be required for virus assembly. Here, in order to test the requirement of the A12L protein and its proteolysis in viral replication, a conditional lethal mutant virus (vvtetOA12L) was constructed to regulate A12L expression by the presence or absence of an inducer, tetracycline. In the absence of tetracycline, replication of vvtetOA12L was inhibited by 80% and this inhibition could be overcome by transient expression of the wild-type copy of the A12L gene. In contrast, mutation of the AG/A site abrogated the ability of the transfected A12L gene to rescue, indicating that A12L proteolysis plays an important role in viral replication. Electron microscopy analysis of the A12L deficient virus demonstrated the aberrant virus particles, which were displayed by the AG/A site mutation. Thus, we concluded that the not only A12L protein but also its cleavage processing plays an essential role in virus morphogenic transition.

Figure 1

Tet-dependent replication of vvtetOA12L and one-step growth curve. a. Tet-dependent replication of vvtetOA12L. T-REx 293 cells were infected with vvtetOA12L at an MOI of 1 PFU/cell in the presence of tetracycline (Tet) at various concentrations of 0, 10, 20, 30, and 40 μg/mL. The infected cell extracts harvested at 24 hpi were titered on BSC 40 cells to determine the virus yields. b. One-step growth curve. T-REx 293 cells were infected with vvtetOA12L in the presence and absence of Tet (30 μg/mL) and harvested at 3, 5, 8, 12, and 24 hpi. Each virus titer (PFU/ml) was scaled in log phase.

Figure 2

Essentiality of A12L protein in VV replication. In order to determine the essentiality of A12L protein in virus replication, T-REx 293 cells were infected with vvtetOA12L in the presence/absence of Tet (Tet+/-). The lack of A12L was complemented by the transient expression of plasmid born A12L under the control of an early/late synthetic promoter (pA12L) or the native promoter (233 nucleotide upstream of A12L ORF, p233-A12L). In addition, the N-terminal AG/A site mutated A12L was constructed to rescue the absence of A12L (AG/A). pA12L: A12L ORF under the control of the early/late synthetic promoter; p233-A12L: plasmid born A12L under the native promoter; pRB21: vector plasmid alone; AG/A: plasmid born A12L with N-terminal AG/A site mutation into ID/I. Each virus titer (PFU/ml) was scaled in log phase.

Figure 3

Morphology defects in the absence of A12L expression. To investigate a role of A12L protein in virus assembly, T-REx 293 cells were infected by vvtetOA12L in the presence (a, b) and the absence of Tet (c, d). In the presence of Tet, spherical IV particles were demonstrated, which evolved into the biconcave IMV particles. The inner layer of the core is localized along with the outer membrane (panel b). In the absence of Tet (c and d), mostly IV-like particles (IV*) were observed with accumulated viroplasms (V). IV-like particles contained little of viral dense materials in the membranes, which formed irregular-shape. Some of IV particles were developed into IMV-like particles, of which cores showed abrogated condensation along with abnormal-shaped layer as demonstrated in box at the panel d.

Figure 4

Morphology defects by abrogated AG/A cleavage of A12L. In order to examine VV morphology by rescuing the absence of A12L, we transfected plasmid born A12L under the control of an early/late synthetic promoter (pA12L) and AG/A mutant plasmid of A12L (AG/A), and infected with vvtetOA12L in the absence of Tet. The transient expression of A12L induced regular IV and IMV particles (panel a) while the AG/A mutation into ID/I displayed defective phenotypes (panel b through d). Arrows in panel a indicate center-concaved inner layer of the core. Panel b and c show IV particles with little or almost empty viral materials while panel d demonstrates the aberrant layers of the cores.