Characterization of an autonomous subgenomic pestivirus RNA replicon

Abstract

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

FIG. 1

Schematic drawing of the different BVDV cRNAs used in this study. The individual viral polypeptides processed from the polyprotein are depicted with different kinds of shading. Ins, 27-base insertion characteristic of BVDV CP7 (55). The 3′-truncated RNAs are marked with the restriction cleavage sites used for linearization of the respective cDNA constructs (; see also Materials and Methods). The Ser 1752 mutation is indicated in DI9cpm1.

FIG. 2

Monitoring of BVDV RNA replication. (A) RNase protection assay performed as described in Materials and Methods at 24 h p.t. with MDBK cells transfected with RNA derived from BVDV CP7 cRNA or BVDV CP7Δ3′ cRNA. Lanes: p, input sense (−) and antisense (+) probes (280 nucleotides); c, protected products obtained with sense (−) and antisense (+) probes after RNase protection with BVDV CP7 cDNA; 1, RNase protection carried out with RNA from the nuclear fraction of BVDV CP7 cRNA-transfected MDBK cells (left lane, protection with sense probe; right lane, protection with antisense probe); 2, RNase protection carried out with RNA from the cytoplasmic fraction of BVDV CP7 cRNA-transfected MDBK cells (left lane, protection with sense probe without prior cycle of prehybridization-predigestion (pd); middle lane, protection with sense probe after prior cycle of hybridization-predigestion (pd); right lane, protection with antisense probe); 3, same experiments as in lanes 2 but carried out with cytoplasmic RNA from BVDV CP7Δ3′ cRNA-transfected MDBK cells. The figure is an autoradiogram of a 10% polyacrylamide–7 M urea gel. (B) IF analysis at 24 h p.t. of MDBK cells transfected with either BVDV CP7 cRNA or BVDV CP7Δ3′ cRNA. Anti-NS3 antibody was used. 1, MDBK cells transfected with BVDV CP7 cRNA (magnification, ×100); 2, same as 1 but at a magnification of ×400; 3, different image of MDBK cells transfected with BVDV CP7 cRNA (magnification, ×100); 4, same as 3 but at a magnification of ×400; 5, MDBK cells transfected with BVDV CP7Δ3′ cRNA (magnification, ×100); 6, MDBK cells transfected with BVDV CP7Δ3′ cRNA (magnification, ×400).

FIG. 3

Characterization of DI9c as an autonomous RNA replicon. (A) RNase protection analysis of MDBK and BHK-21 cells transfected with DI9c 24 h p.t. The protected fragments were analyzed on a 10% polyacrylamide–7 M urea gel. Lanes: p, input sense (−) and antisense (+) probes (1/10 of the experimental input was loaded); 1, RNase protection with cytoplasmic RNA obtained from MDBK cells transfected with DI9c using sense (−) and antisense (+) probes; 2, RNase protection with cytoplasmic RNA obtained from BHK-21 cells transfected with DI9c; 3, MDBK cells with DI9cΔ3′; 4, MDBK cells with DI9cpm1. (B) IF analysis of MDBK and BHK-21 cells transfected with DI9c, DI9cΔ3′, or DI9cpm1 24 h p.t. 1, MDBK cells with DI9c (magnification, ×100); 2 and 3, MDBK cells with DI9c (magnification, ×400); 4, BHK-21 cells with DI9c (magnification, ×100); 5, BHK-21 cells with DI9c (magnification, ×400); 6, MDBK cells with DI9cΔ3′ (magnification, ×100); 7, MDBK cells with DI9cpm1 (magnification, ×400); 8, BHK-21 cells with DI9cΔ3′ (magnification, ×100); 9, BHK-21 cells with DI9cpm1 (magnification, ×400).

FIG. 4

Determining the ratio of positive-strand RNA to negative-strand RNA in DI9c-transfected cells. BHK-21 cells were transfected in five independent experiments with DI9c, and the cytoplasmic RNA was extracted and subjected to an RNase protection assay 24 h p.t. To facilitate quantitation, positive and negative strands were both assayed with the same antisense probe, the latter by detection of positive strands which were protected by equimolar amounts of negative strands in a prehybridization-predigestion cycle (see Materials and Methods and Results). (A) The protected fragments were separated on a 10% polyacrylamide–7 M urea gel. p, amount of input probe that (with respect to the viral RNA) was added in excess into each protection assay mixture. (−), detected negative-strand RNA; (+), detected positive-strand RNA. (B) The major bands on the gel were extracted (indicated in panel A, lanes 5) and quantified by Cerenkov counting. Light grey columns represent the amounts of protected negative-strand RNA; dark grey columns represent the amounts of protected positive-strand RNA. (C) The ratio of positive-strand RNA to negative-strand RNA was calculated for each of the five experiments.

FIG. 5

The 5′-coding region of N^pro is essential for autonomous RNA replication of DI9c. (A) Top: schematic drawing of the DI9c derivatives used in this study (see the legend to Fig. 1A for details). As indicated (MG¹⁵⁹⁰) and as described in detail in Materials and Methods, DI9cNS3 RNA contains an additional AUG codon upstream of GGA coding for glycine 1590 of BVDV CP7 NS3. DI9cubi RNA also contains the ubiquitin gene (ubi), and DI9cΔN^proubi also contains the initial 42 codons of the N^pro gene (indicated by Δ). Bottom: RNA and primary amino acid sequence comparisons for the initial five codons of BVDV CP7 cRNA and the DI9cubi ORF. (B) RNase protection assay of BHK-21 cells 24 h p.t. with different DI9c derivatives. Lanes: 1, protection of RNA derived from cells transfected with DI9c (positive control); 2, protection of RNA p.t. with DI9cNS3; 3, protection of RNA p.t. with DI9cubi; 4, protection of RNA p.t. with DI9cΔN^proubi; p, sense probe (1/10 of the experimental input was loaded on the gel). (−), sense probe; (+), antisense probe.