Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus

Abstract

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.

FIG. 1

Northern blot analysis of total RNA from MDBK cells infected with BVDV strain CP Rit or CP7 and noninfected cells (n.i.). Before transfer and hybridization, RNA was separated on a 1.0% agarose gel under denaturing conditions. The blot was hybridized with a 2.5-kb NotI-NsiI fragment from the cDNA clone pA/BVDV (33). RNA ladder sizes (in kilobases) are indicated. Migration positions of the viral genomic RNAs are marked with arrows. The additional band visible below the viral RNA of CP7 represents a gel artifact resulting from large amounts of rRNA.

FIG. 2

(A) Genome organization of a noncp pestivirus (NCP) and RT-PCR strategy for identification of a duplication of the NS3 gene. The positions and orientations of primers Ol NS3R and Ol BVDV7100 are indicated by arrowheads below the bars. RT-PCR with this primer pair allows a specific amplification only for a duplicated NS3 gene. The bull’s-eye icon indicates a putative insertion. (B) RT-PCR product obtained from RNA of BVDV CP Rit-infected cells (lane 1). RNA from cells infected with noncp BVDV strain 519-NCP (2a) served as negative control (lane 2). The cDNA fragment was separated on a 1.0% agarose gel and stained with ethidium bromide. M, size standard; KB, kilobases.

FIG. 3

(A) Schematic representation of the genome organization of a noncp pestivirus (NCP) and CP Rit. For CP Rit, the genomic region encoding NS3, NS4A, and the N-terminal 132 aa of NS4B (NS4B*) is duplicated. In addition, two cellular insertions are located directly upstream of the NS3 gene. These cellular sequences encode part of ribosomal protein S27a (S27a*, shaded box) and an N-terminally truncated ubiquitin (ubi*, solid box), respectively. (B) Deduced amino acid sequence of part of the CP Rit sequence. The positions of NS4B*, S27a*, ubiquitin*, and NS3 are indicated. The two cellular insertions are underlined.

FIG. 4

Identification of the bovine mRNA encoding ubiquitin and S27a. (A) RT-PCR product obtained from RNA of noninfected MDBK cells with primers Ol UeR and Ol Ue (lane 1). The cDNA fragment was separated on a 1.0% agarose gel and stained with ethidium bromide. Negative control, double-distilled H₂O (lane 2); M, size standard; KB, kilobases. (B) Nucleotide and deduced amino acid sequence of the coding region of the bovine mRNA. The positions of ubiquitin and S27a are indicated. Regions which correspond to the two cellular insertions found within the genome of CP Rit are underlined. (C) Comparison of the structure of the bovine mRNA sequence and the respective part of the CP Rit genome encompassing the two cellular insertions, S27a* and ubi*, respectively. Nucleotide sequence identities between the bovine mRNA and the two cellular insertions of CP Rit are indicated.

FIG. 5

(A) Schematic representation of part of the CP Rit genome organization and the fusion proteins encoded by constructs pRit-A, pRit-B, pRit-C, and pRit-D. For initiation of translation, each construct starts with an additional methionine. The positions of primers Ol Rit4B1 (•), Ol S27a* (■), Ol Rit-ubi4 (★), Ol RitNS3R (⧫), and Ol Rit4AR (◣), used for cloning of the respective constructs, are indicated below the bar on the top. (B and C) Immunoblot analysis of CP Rit nonstructural proteins. After infection with vaccinia virus MVA-T7pol, BHK-21 cells were transfected with pRit-C (lanes 1), pRit-A (lanes 2), pRit-B (lanes 3), and pRit-D (lanes 4). Cells were lysed 16 h posttransfection or postinfection, and the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8% polyacrylamide) under reducing conditions, transferred to nitrocellulose, and incubated with anti-NS3 monoclonal antibody (B) or anti-P1 serum directed against NS4A and NS4B (C). +, MDBK cells infected with CP Rit. −, Negative control (BHK-21 cells infected with MVA-T7pol but not transfected). The sizes (in kilodaltons) of marker proteins are indicated on the left. The positions of NS3 (B) and NS4B-specific fusion proteins (C) are marked with arrows. Additional bands visible in panel C represent nonspecific reactions due to the background level of the rabbit antiserum. With respect to pRit-D, it has actually not been demonstrated that NS4A is cleaved off.