Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter - PubMed (original) (raw)

Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter

U J Buchholz et al. J Virol. 1999 Jan.

Abstract

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

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Figures

FIG. 1

Schematic presentation of the BRSV ATue51908 genome (drawn to scale). The locations of transcripts (shaded bars) and protein-encoding frames (open bars) are shown relative to the viral RNA (vRNA) (solid bar). In the enlargements, the organizations of recombinant viruses and ATue51908 are compared. The 3′-terminal 45-nt BRSV leader regions are marked by horizontal stripes, and the 44-nt HRSV A2 leader regions are marked by vertical stripes. Genetic tags (_Not_I) and nucleotide deletions in the NS1 noncoding regions (shaded) of the recombinant viruses are indicated. The relative positions of corresponding nucleotides are given for the gene starts of NS1, NS2, and N and nucleotides flanking the NS2 deletion. The overall lengths of the vRNAs are shown on the right.

FIG. 2

Alignment of the BRSV ATue51908 3′ leader region (A) and 5′ trailer (B) sequences with those from other pneumoviruses. The DNA sequences are shown in 3′-to-5′ viral RNA sense. For HRSV strain A2 (28) and TRTV (33), only deviations from the ATue51908 sequence are indicated. Gaps are represented by dots, and the start signal of the first gene (NS1 in BRSV and HRSV, N in TRTV) and the L gene end signal are underlined. Numbering of the trailer sequences starts with the first nucleotide downstream of the L gene transcription stop signal. (C) Alignment of the NS1 noncoding region of the recombinant BRSVs containing the _Not_I tag (boxed) with the ATue51908 consensus sequence and the published A51908 sequence. The sequences are shown as DNA positive strands. Only nucleotides differing from the recombinant sequence are indicated. Gaps are indicated by dots, and the NS1 gene start signal and translation start codon are underlined.

FIG. 3

Demonstration of the _Not_I tag in the genome RNA of recombinant BRSVs. RT-PCR was performed on total RNA of BSR T7/5 cells infected with standard BRSV ATue51908 or with recombinant virus by using positive-sense primers annealing to the genome 3′ ends and a negative-sense primer binding in the NS1 gene. No PCR product was detected when the RT step was omitted. Digestion with _Not_I of the 324-bp (calculated size) RT-PCR product originating from recombinant viruses yielded two bands of calculated sizes of 82 and 242 bp, whereas the RT-PCR product from standard BRSV strain ATue51908 was not cleaved.

FIG. 4

Demonstration of NS2 gene deletion by RT-PCR. Total RNA of BSR T7/5 cells infected with standard BRSV ATue51908 or with recombinant virus was used for RT-PCR with a positive-sense primer hybridizing to the 3′ end of the BRSV genome RNA and a negative-sense primer binding in the N gene. Products from full-length recombinants and wild-type virus yielded a product of 1,179 bp (calculated size) spanning the NS2 gene, whereas deletion mutants gave rise to a 666-bp (calculated) fragment, reflecting the 513-nt deletion. No PCR product was detected when the RT step was omitted.

FIG. 5

Demonstration of virus transcripts by Northern hybridization. Total RNA of BSR T7/5 cells infected with rBRSV or standard ATue51908 was isolated 2 to 4 days after infection and separated on a 2% denaturing agarose gel. The blot was hybridized with PCR-derived probes specific for the BRSV N gene (nt 1429 to 2277) (A) and for the NS2 gene (nt 574 to 1026) (B). Transcripts corresponding to N mRNA (vRNA), bicistronic N/P readthrough transcripts, and NS2 mRNA are indicated.

FIG. 6

CPE of recombinant BRSVs in BSR T7/5 cells (A) and MDBK cells (B). (A) In BSR T7/5 cultures, all recombinants induced syncytia of cells detaching from the monolayer, which were indistinguishable from those of standard ATue51908, at 56 h postinfection at an MOI of 0.01. (B) In MDBK cells, both full-length recombinant viruses rBRSV and rH/BRSV and standard ATue51908 virus produced similar foci of enlarged cells at 5 days postinfection, whereas the NS2 deletion mutants produced only small foci of degenerating cells.

FIG. 6

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Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter - PubMed (original) (raw)