Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4

Abstract

Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid.

Keywords: A3; Core wall; Nucleocapsid; Vaccinia virus.

Fig. 1

Initial characterization of vA3i. (A) Illustration of the final genome structure in the region between A3L and A4L genes in vA3i. The viral A3L and A4L genes are represented by black arrows. pT7 and pE/L represent the bacteriophage T7 and synthetic vaccinia virus early/late promoters, respectively. GPT = guanine phosphoribosyl transferase. EMC = the encephalomyocarditis virus independent ribosome entry site. SLO = modified (stem-loop) lac operator. (B) Viral plaques phenotype. Cells were infected with WR or vA3i, incubated at 37°C in the presence or absence of IPTG and stained with crystal violet after 7 days. (C) One-step growth curve. Cells were infected with WR or vA3i at an MOI of 10 and incubated at 37°C in the presence or absence of IPTG. After varying times of infection, cells were harvested and the virus titer was determined by plaque assay in the presence of IPTG. (D) Accumulation of A3 during infection. Cells were infected at an MOI of 10 with WR or vA3i and incubated in the presence or absence of IPTG at 37°C. At varying times post-infection, cells were harvested and samples were analyzed by SDS-PAGE and western blot with antibodies against A3. The numbers above each lane indicate hours post-infection.

Fig. 2

Analysis of protein synthesis. Cells were infected with WR or vA3i at an MOI of 10 and incubated in the presence or absence of IPTG at 37°C. At varying times post-infection, infected cells were incubated with medium containing ³⁵S-methionine. After 30 minutes, cells were harvested in sample buffer and analyzed by SDS-PAGE and autoradiography. The numbers on the top indicate hours post-infection. The star on the right marks the unidentified band observed during infections with vA3i in the absence of IPTG. The molecular weight in kDa is on the left.

Fig. 3

Analysis of protein processing. (A) Cells were infected at an MOI of 10 with WR or vA3i and incubated in the presence or absence of IPTG at 37°C. After 8 h of infection, cells were incubated with ³⁵S-methionine for 30 minutes. Some cells were harvested after the pulse (P) while others were incubated in the presence or absence of IPTG for varying times (chase). The numbers on the top represent hours after the pulse. The molecular weight in kDa is on the right. (B) Cells were infected as described previously and harvested in sample buffer. Samples were analyzed by SDS-PAGE and western blot using antibodies against L4 or A10 proteins. The numbers on the top represent hours of infection.

Fig. 4

Analysis of morphogenesis. Cells infected with WR or vA3i at an MOI of 10 were processed for electron microscopy after 24 hours of infection. (A) and (B) WR; (C) and (D) vA3i in the presence of IPTG; (E) and (F) vA3i in the absence of IPTG. Normal immature virions are represented in (A) and (C). Normal mature virions are represented in (B) and (D). (E) and (F) represents abnormal IVs and MVs produced under non-permissive conditions. Arrows point to half-empty immature virions. Bars = 500 nm.

Fig. 5

Immunogold labeling against structural proteins. Cells infected with WR or vA3i at an MOI of 10 were processed for electron microscopy after 24 hours of infection. Ultrathin sections were probed with anti-D13 (10 nm gold) and anti-A27 (15 nm gold) (A) to (C) or with anti-A10 (D) to (I). (A), (D) and (E) WR; (B), (F) and (G) vA3i in the presence of IPTG; (C), (H) and (I) vA3i in the absence of IPTG. Bars = 250 nm.

Fig. 6

Immunogold labeling against L4. Cells infected with WR or vA3i at an MOI of 10 were processed for electron microscopy after 24 hours of infection. Ultrathin sections were probed with anti-L4 (10 nm gold) and anti-A14 (15 nm gold). (A) and (B) WR; (C) and (D) vA3i in the presence of IPTG; (E) and (F) vA3i in the absence of IPTG. Bars = 250 nm.

Fig. 7

Quantification of L4 labeling. Several electron micrographs were analyzed and the number of gold nanoparticles labeling individual viral particles was quantified and plotted in a histogram.

Fig. 8

Analysis of viral proteins localization. Cells were infected at an MOI of 2 for 9 hours with WR or vA3i in the absence or presence of IPTG. Infected cells were prepared for analysis by confocal microscopy, using antibodies against several viral proteins. The images represent the collapsed Z-stack.