Involvement of a bovine viral diarrhea virus NS5B locus in virion assembly

Abstract

A novel mutant of bovine viral diarrhea virus (BVDV) was found with a virion assembly phenotype attributable to an insertion into the NS5B polymerase locus. This mutant, termed 5B-741, was engineered by reverse genetics to express NS5B with a C-terminal peptide tag of 22 amino acids. Electroporation of bovine cells with genomic RNA from this mutant showed levels RNA synthesis which were regarded as sufficient for infectivity, yet infectious virions were not produced. Pseudorevertants of mutant 5B-741 that released infectious virions and formed plaques revealed a single nucleotide change (T12369C). This change resulted in a leucine-to-proline substitution within the NS5B tag (L726P). Genetic analysis revealed that indeed a single nucleotide change encoding proline at NS5B position 726 in the pseudorevertant polyprotein mediated recovery of virion assembly function without improving genomic RNA accumulation levels. A subgenomic BVDV reporter replicon (rNS3-5B) was used to analyze the consequences of alterations of the genomic region encoding the NS5B C terminus on replication and assembly. Interestingly, rNS3-5B-L726P (revertant) replicated with the same efficiency as the rNS3-5B-741 mutant but produced 10 times more virions in a trans-packaging assay. These results indicated that impairment of assembly function in 5B-741 was independent of RNA accumulation levels and agreed with the observations from the full-length mutant and revertant genomes. Finally, we recapitulated the packaging defect of 5B-741 with a vaccinia virus expression system to eliminate possible unwanted interactions between the helper virus and the packaged replicon. Taken together, these studies revealed an unexpected role of NS5B in infectious virion assembly.

FIG. 1.

Primary structure of the BVDV NS5B peptide tag mutants. The C terminus of NS5B is expanded to show nucleotide and amino acid sequences of mutant 5B-741, engineered with a C-terminal extension of 22 amino acids (underlined), and the representative sequence of three independent pseudorevertants, 5B-741rev, indicating the single-nucleotide transition, T12369C (nomenclature based on the WT base, position, and the mutant base), which converted the leucine 726 codon to proline (NS5B residue numbering, derived from GenBank accession no. M31182).

FIG. 2.

The NS5B C terminus conditions the production of infectious viral progeny. (A) Infectivity of in vitro-transcribed RNA from WT BVDV and NS5B mutant genomes determined by infectious center assay. BHK-21 cells were electroporated with RNA transcripts from pNADLp15 or from p5Bgdd, p5B-741, or p5B-L726P serially diluted and plated with additional fresh MDBK cells with agar overlay. (B) Immunofluorescence assay of cells electroporated with in vitro-transcribed RNA. RNA transcripts (2 μg) from 5Bgdd (1), mutant 5B-741 (3), 5B-L726P (5), N-ΔINS ncBVDV (7), and WT BVDV NADLp15 (9) were electroporated into RD420 cells and RNA replication was monitored by IIF using anti-E2 antibodies. Release of infectious virus was assessed by harvesting the culture medium (panels 1, 3, 5, 7, and 9) at 24 hpe, inoculating into fresh RD420 cells, and monitoring by IIF using anti-E2 antibodies (panels 2, 4, 6, 8, and 10, respectively). (C) Northern blot analysis of total RNA from cells electroporated with RNA from NS5B mutants. (a) Total RNA from BHK-21 cells electroporated with 5Bgdd (1), 5B-741 (2), 5B-L726P (3), N-ΔINS (4), and N-p15 (5) was analyzed, and 28S rRNA is indicated as a control. (b) The transferred RNA was hybridized to a ³²P-labeled single-stranded DNA probe specific to BVDV. The migrations of the viral RNA and rRNA are indicated; labels below the figure indicate theradioactive probe hybridization signal as measured by phosphorimager analysis for each of the lanes, relative to that of the ncpBVDV N-ΔINS (lane 4) (59). (D) Western blot analysis to demonstrate expression of structural and NS proteins in electroporated cells with mutant NS5B RNA. Panels: 1, 5Bgdd; 2, 5B741; 3, L726P; 4, N-ΔINS; 5, NADLp15. The total protein was resolved by SDS-polyacrylamide gel electrophoresis and probed with anti-E2 and anti-NS3 MAbs, and signal was developed with a chemiluminescence detection system. Molecular mass markers are indicated (in kilodaltons). (E) Plaque phenotype of NS5B mutants. Plaques formed by revertant i-5B-741rev (a), the virus recovered from the engineered mutant i-5B-L726P (b), or WT BVDV strain NADL (c) on bovine cells infected for 60 h. The average diameters of the plaques are indicated.

FIG. 3.

Structure and function of a BVDV reporter replicon in bovine cells. (A) Diagram of the structure of the rNS3-5B and rNS3-5Bgdd BVDV reporter replicons (see Materials and Methods for details). (B) Integrity of in vitro-synthesized RNA analyzed by electrophoresis in MOPS-formaldehyde denaturing gel followed by staining with ethidium bromide. (C) Kinetics of luciferase reporter expression in RD420 cells. In vitro-synthesized RNA (2 μg) from the WT BVDV reporter replicon rNS3-5B (grey bars) or the rNS3-5Bgdd (open bars) was electroporated into 2 × 10⁶ RD420 cells, and cells were divided into aliquots and incubated for the indicated times to determine luciferase activity from 10⁵ cells. Values shown are means of three experiments; error bars show the standard deviations of the means. (D) Correlation between luciferase production and intracellular viral RNA levels. RD420 cells were electroporated as for panel C and at the indicated times harvested for Northern blotting and quantification by phosphorimaging. Total cellular RNA samples were electroporated with rNS3-5Bgdd and harvested at 24 h, or with rNS3-5B and harvested at 24-h intervals after electroporation (lanes 2 to 6, respectively). The ethidium bromide-stained gel (a) demonstrates equal loading of the RNA blotted and hybridized to the ³²P-labeled BVDV probes (b). The rNS3-5B radioactive hybridization signal intensity values shown below each lane have been normalized to that of the 96-hpe sample.

FIG. 4.

trans-encapsidation of rNS3-5B by complementing BVDV structural proteins. (A) RD420 cells were infected with BVDV2 or mock infected and cultured for 72 h (step 1) prior to electroporation with rNS3-5B. After electroporation with replicons (step 2), the cells were harvested for luciferase assay (step 3) to determine the packageable replicon levels, whereas the culture medium was divided into two aliquots, which were incubated with BVDV2-neutralizing or nonneutralizing antibodies prior to inoculation onto fresh bovine cells (step 4). After 24 h, the cells were harvested (step 5) to determine luciferase activity generated by the transduced replicon. (B) Reporter replicon packaging assay. Luciferase activity in RD420 cells (RLU/10⁵ cells) that were mock infected (open bars) or infected with BVDV2 (solid bars) and harvested at 48 hpe with the replicon RNAs, representing the size of the packageable replicon pool. Luciferase activity from RD420 cells (RLU/10⁵ cells) harvested 24 h after inoculation with culture fluid filtrates from mock-infected cells (hatched clear bars) or from BVDV2-infected cells (hatched bars on grey background) after incubation with either 10 μg of neutralizing antibody/ml (ascending hatch) or with 10 μg of nonneutralizing antibody/ml (descending hatch). The reporter activity in these cells is indicative of the activity of replicons transduced by functional virions.

FIG. 5.

The NS5B-741 locus modulates the encapsidation of a BVDV replicon. (A) Luciferase activity is shown for mock-infected or BVDV2 virus-infected cells (clear bars) harvested at 48 hpe with the WT or mutant replicons (as indicated). The cell culture fluid harvested at this time from BVDV2-infected cells was filtered and inoculated onto a naïve cell monolayer. Cells were harvested at 24 h after inoculation, and their luciferase expression levels were determined (shaded bars). (B) RPE for mutant replicons compared to the WT replicon, which was considered 100%.

FIG. 6.

Transduction of BVDV NS5B mutant replicons packaged in cells infected with a helper vaccinia virus-T7 expressing the BVDV polyprotein with a catalytically inactive NS5B. Luciferase activity of 10⁵ cells transfected with rNS3-5Bgdd, rNS3-5B-741, rNS3-5B-L726P, or WT replicon RNA and infected with vaccinia virus recombinant vT7-ncpNADLgdd, as indicated, at 24 hpe, when cell culture medium was harvested to assess replicon packaging efficiency. Firefly luciferase activities of cells inoculated with culture medium filtrates from the packaging cell monolayers and lysed at 24 h after inoculation are shown. Values are averages from two experiments.