The 131-amino-acid repeat region of the essential 39-kilodalton core protein of fowlpox virus FP9, equivalent to vaccinia virus A4L protein, is nonessential and highly immunogenic

Abstract

The immunodominant, 39,000-molecular weight core protein (39K protein) of fowlpox virus (FP9 strain), equivalent to the vaccinia virus A4L gene product, contains highly charged domains at each end of the protein and multiple copies of a 12-amino-acid serine-rich repeat sequence in the middle of the protein. Similar repeats were also detected in other fowlpox virus strains, suggesting that they might confer a selective advantage to the virus. The molloscum contagiosum virus homolog (MC107L) also contains repeats, unlike the vaccinia virus protein. The number of repeats in the fowlpox virus protein does not seem to be crucial, since some strains have a different number of repeats, as shown by the difference in the size of the protein in these strains. The repeat region could be deleted, indicating that it is not essential for replication in vitro. It was not possible to delete the entire 39K protein, indicating that it was essential (transcriptional control signals for the flanking genes were left intact). The repeat region is partly responsible for the immunodominance of the protein, but the C-terminal part of the protein also contains highly antigenic linear epitopes. A role for the 39K protein in immune system modulation is discussed.

FIG. 1

(A) Schematic representation of the 39K protein homologs in FWPV, vaccinia virus, variola virus, and molluscum contagiosum virus. The terminus charged domains are hatched, whereas the shaded rectangles represent the repeats. The different intensity of shading represents the degree of conservation between the repeats. The symbol ∇ in the variola virus protein shows the position of the 10-aa deletion in the variola virus protein in comparison with the vaccinia virus protein. The protein sequences of the vaccinia virus WR strain and the Bangladesh-75 strain of variola virus are found in the SWISS-PROT database under accession no. p29191 and p33832, respectively. The amino acid sequence of the A4L homolog of molluscum contagiosum virus is found in the GenBank database under accession no. U60315. (B and C) Structure of the FWPV 39K protein (B) showing the positions of the primers used to amplify the different fragments (C). The black rectangles in panel C represent the FLAG C-terminal peptides.

FIG. 2

Western blot analysis of the fusion proteins expressed in E. coli. Extracts from bacteria (XL1 Blue), transformed with plasmids described in Fig. 1C, were transferred onto nitrocellulose paper and analyzed by Western blotting with the anti-FLAG peptide MAb (A); with three anti-39K protein MAbs, GB9 (B), 8F3 (C), and 3D9 (D); and with two hyperimmune chicken polyclonal sera, B63 (E) and B62 (F). In each panel, uninfected bacteria have been included as a negative control (lane XL) and purified FP9 has been included as a positive control (lane PV). The other lanes (N, NM, M, MC, C, and NMC) correspond to the fusion proteins in Fig. 1C.

FIG. 3

Fractionation of purified virus by detergent treatment. Purified virus was treated with 2% Triton X-100 (lanes 1 to 4) or 0.2% DOC (lanes 5 to 8) in the presence (lanes 1, 2, 5, and 6) or absence (lanes 3, 4, 7, and 8) of dithiothreitol and centrifuged for 30 min at 200,000 × g to separate the core fraction (lanes 2, 4, 6, and 8) from the solubilized envelope fraction (lanes 1, 3, 5, and 7). Western blots were revealed with the GB9 MAb.

FIG. 4

Immunolabelling of the 39K protein detected by the GB9 MAb. CEFs were infected with FP9 (A to D) or with 1-3 R1, the repeat deletion mutant (E and F), and fixed 66 h postinfection in 1% paraformaldehyde–0.5% glutaraldehyde. The viral factory is clearly labelled as well as the viroplasm of the immature particle in formation (A). In the intracellular mature virus, the intracellular enveloped virus, and the extracellular enveloped virus, the 39K protein appears on the periphery of the core (B, C, and D respectively). The deleted protein showed the same location as the wild-type protein in the immature particles (E), the intracellular mature particles (E), and the extracellular particles (F).

FIG. 5

(A) Variability of size of the 39K protein in different strains of FWPV determined by Western blotting with the 8F3 MAb: HP-1 (lane 1), HP-200 (lane 2), FP9 (lane 3), Poxine (lane 4), and Websters (lane 5). (B) Amplification of the repeat region by PCR with primers PR8 and PR9 in different strains of FWPV: HP-200 (lane 1), FP9 (lane 2), Poxine (lane 3), Websters (lane 4), and Chick-N-Pox (lane 5). (C) Determination of the number of conserved repeats in the 39K protein of different strains of FWPV determined by PCR with primers PR9 and PR19. The different strains of FWPV are as in panel B.

FIG. 6

(A) Structure of the PCR product (amplified with primers MASH48 and MASH49 and inserted into the pGNR plasmid), including the gene encoding the 39K protein (open areas) and 5′ termini of the adjacent genes, the equivalents of A5R and A3L (hatched areas). The repeats are indicated by arrowheads, and the positions of restriction sites used to generate the deletion mutants are shown. The late promoters of the 39K and 4b proteins (on the upper strand) are represented by narrow solid boxes, whereas the early/late promoter of the A5R equivalent (lower strand) is represented by a narrow shaded box. The cap sites of the three genes are represented by asterisks and are located 2 nucleotides upstream of the respective initiator codons. The genomic structures of the deleted clones are shown below the scale bar (in base pairs). (B) 39K protein deletion mutants. The gpt-negative clones were screened by PCR with primers MASH48 and MASH49. All the gpt-negative clones contained only a copy of the wild-type gene, like the clone represented in lane 1. One gpt⁺ intermediate clone was used as a control (lane 2), as well as the wild-type virus (lane 3). (C) Repeat-region deletion mutants. The gpt-negative clones were screened by PCR with primers PR8 and PR6. Two gpt-negative clones containing adapter A (1-3 R1 and 1-4 R1 clones) (lanes 1 and 2) and one gpt-negative clone containing adapter B (8-3 R1 clone) (lane 3) contained a deleted copy of the gene. Two gpt⁺ intermediate clones (8-2 R2 and 9-2 R2 clones) (lanes 4 and 5) and the wild-type virus (lane 6) were tested as controls. (D) Western blotting analysis, with MAb GB9, of successive plaque purifications of a gpt⁺ isolate (clone 1-3 R1) in nonselective medium (lanes 1 to 7). CEFs grown in 6-cm petri dishes were infected with the different isolates. When a cytopathic effect of approximately 80% was observed, the cells were washed, covered with 1 ml of PBS, and freeze-thawed once. One volume of electrophoresis buffer was added to 1 volume of cellular lysate, boiled, and separated by SDS-PAGE (15% polyacrylamide). In lanes 1 to 3, a wild-type protein is still expressed, but in lanes 4 to 7, only the deleted protein is observed when the isolate became gpt-negative. The wild-type virus was tested as a control (lane 8). The deletion mutant obtained after seven plaque purifications (lane 7) was passaged 12 times on CEFs and is shown in lane 10 alongside the second passage run as a control (lane 9). The wild-type virus (lane 11) and the 1-3 R1 deletion mutant (lane 12), purified on a sucrose gradient, were tested in parallel, showing that the deleted protein is present in the virion.

FIG. 7

Single-step growth curve experiment. CEFs were infected in triplicate with the repeat deletion mutant and with the wild-type virus. The supernatant was harvested, and the cells were freeze-thawed at various times postinfection. Intracellular virus (A) and extracellular viruses (B) were subjected to titer determination. The curves were drawn by using the mean of the triplicate sample values. The error bars show the standard deviation.