E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies

Abstract

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.

FIG. 1

(A) Schematic representation of SP6/wt and mutant plasmids which encode the BVDV E2 to NS2 proteins. The wt is shown at the top: the N-terminal domain of preprolactin (S) acts as a signal peptide (9) and is fused to E2. Amino acid sequences around the cleavage sites and the charged stretch of p7 are shown below. Amino acids which were substituted in this study are underlined. S*, signal peptide of the mouse immunoglobulin κ chain; aa, amino acids. The degree of cleavage at different sites after the introduction of mutations is indicated (see the box). (B) In vitro translation analysis of RNAs transcribed from SP6/wt and mutant plasmids. Microsomal membrane fractions of the translated products were analyzed by SDS-PAGE (9% polyacrylamide). The positions of BVDV proteins are indicated on the right. Sizes (in kilodaltons) of marker proteins are indicated on the left. The lanes combined in the figure are derived from the same gel.

FIG. 2

Processing at the p7-NS2 cleavage site of pestiviruses. (A) N terminus of BVDV NS2, strain CP7. [³H]leucine-labeled NS2 was synthesized from pRN653E2p7NS2 (9) by in vitro translation and used for N-terminal sequence analysis. The graph shows the radioactivity (in counts per minute [cpm]) released during each cycle. The N-terminal amino acid sequence of CP7 NS2 is shown at the top of the graph. (B) Alignment of the amino acid sequences in the region of the p7-NS2 cleavage site of representative strains from the four pestivirus species, BVDV-1 CP7, BVDV-1 NADL, BVDV-2 890, BDV X818, and CSFV Alfort.

FIG. 3

Pulse-chase analysis of the processing of E2-p7. MDBK cells infected with BVDV CP7 (CP7) or mock infected (m) are shown. At 19.5 h p.i., the cells were labeled with ³⁵S-protein-labeling mixture for 15 min and chased for the indicated times. Cell extracts were immunoprecipitated with a MAb directed against BVDV E2 and then incubated in the absence (−) or presence (+) of PNGase F. The precipitates were analyzed under reducing conditions by SDS-PAGE (11% polyacrylamide). The positions of E2-p7 and E2 before and after PNGase F digestion (indicated by *) are indicated by arrows on the right. Sizes (in kilodaltons) of marker proteins are indicated on the left.

FIG. 4

Characterization of viruses derived from CP7/wt and CP7/E2IRESp7. (A) Schematic representation of the RT-PCR analysis. Viral RNAs were transcribed from CP7/wt and CP7/E2IRESp7. UAG, translational stop codon; AUG, translational initiation codon; hatched box, signal sequence from the mouse immunoglobulin κ chain; arrows a and b, locations of primers used for the RT-PCR analysis. (B) RT-PCR analysis. cDNAs were synthesized from total RNAs of MDBK cells infected with viruses derived from CP7/wt (lane 2) and CP7/E2IRESp7 (lane 3). Mock-infected MDBK cells served as a control (lane 1). The amplified DNAs were separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. M, marker. (C) Growth analysis of viruses derived from CP7/E2IRESp7 and CP7/wt. MDBK cells were infected with the CP7/wt virus or CP7/E2IRESp7 virus at a multiplicity of infection of 0.05. Cell culture supernatants were collected at the indicated time points p.i.; virus titers were determined as log TCID₅₀ per milliliter.

FIG. 5

IF analysis of MDBK cells transfected with RNAs transcribed from CP7/wt, CP7/Δp7^15–51, CP7/p7SVV, CP7/p7II, and CP7/E2-p7. At 24 h p.t., NS3 expression was analyzed by IF (A, C, E, G, and I). At 72 h p.t., cell culture supernatants were collected and subsequently used for incubation of MDBK cells. At 48 h p.i., the replication of BVDV in the cells was monitored by IF analysis (B, D, F, H, and J). Only after transfection of CP7/wt RNA were infectious virions released to the supernatant.

FIG. 6

(A) Schematic representation of pcEF-p7neo and pcEF-E2IRESp7neo, which were used to establish the cell lines MDBK-p7 and MDBK-E2IRESp7. The expression vector is schematically shown at the top: the promoter region of human EF-1α, the polyadenylation signal from the simian virus 40 late region (PA), and a gene encoding aminoglycoside 3′-phosphotransferase (neo). The inserted fragments in the expression vector are shown below: the translational initiation codon (ATG); the IRES; genes encoding BVDV CP7 E2, p7, and the N-terminal half of NS2 (E2, p7, and NS2**); and the translational stop codon (TAG). The black box in pcEF-E2IRESp7neo indicates the signal peptide sequence derived from CSFV E^rns. The shaded boxes indicate the signal sequence of the mouse immunoglobulin κ chain. (B and C) Immunoprecipitation analysis with MAb directed against BVDV E2 (B) and Western blot analysis with rabbit serum directed against BVDV p7 (C) in MDBK, BVDV CP7-infected MDBK, MDBK-p7, and MDBK-E2IRESp7 cells.

FIG. 7

Schematic illustration of complementation experiments. Viral RNAs were transfected into MDBK, MDBK-p7, and MDBK-E2IRESp7 cells. Cell culture supernatants were used for incubation of MDBK cells. Replication of BVDV in the cells was monitored by IF analysis. The CP7-encoded polyprotein is shown at the top. Individual BVDV proteins encoded by the different viral RNAs are shown: E2-p7 (white box); E2 (shaded box); p7 (dotted box). BVDV proteins expressed in the MDBK-p7 and MDBK-E2IRESp7 cell lines are illustrated below their names in the same way. Rescue of viral RNA is indicated in columns at the right: +, positive; −, negative.