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. 2001 Jan;75(1):125-33.
doi: 10.1128/JVI.75.1.125-133.2001.

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging

K Dalton  1 , R Casais, K Shaw, K Stirrups, S Evans, P Britton, T D Brown, D Cavanagh
Affiliations

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging

K Dalton et al. J Virol. 2001 Jan.

Abstract

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

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Figures

FIG. 1
FIG. 1
D-RNAs made to investigate the sequences derived from the 5′ end of the IBV genome necessary for replication and rescue (rescue indicates replication and packaging of the RNA into virus particles). The top diagram shows the genome of IBV (not to the same scale as that of the other diagrams), with the six genes marked, and shows the parts which have been retained in D-RNA CD-61. Thin horizontal lines indicate deletions relative to CD-61. Numbers under the diagrams are restriction site positions relative to the IBV Beaudette genome (27,607 nt; data bank accession no. M95169). Replication and rescue were determined by quantification of CAT protein by ELISA or, for D-RNAs without a CAT gene, by Northern blot analysis. A “+” for replication and for rescue indicates that the amount of CAT protein expressed from a D-RNA was very similar to the amount of CAT protein expressed by D-RNA CD-61CATPmaCI in passage P0 (Vero cells; replication) and after serial passage in CK cells (rescue), respectively. The “±” for CD-12 indicates that it was detected only by RT-PCR. The “±” for CD-12CAT indicates that only very low levels of CAT were expressed during passage. Restriction sites are indicated by their positions in the IBV Beaudette genome. An, poly(A).
FIG. 2
FIG. 2
D-RNAs made to investigate the sequences derived from the 3′ end of the IBV genome necessary for replication and rescue. The 507-nt 3′ UTR has been expanded to show the variable and conserved regions I and II, respectively. The open boxes on the right-hand side indicate regions I and II of the 3′ UTR and the poly(A) tail, and the numbers indicate how many of these nucleotides remain. Other details are explained in the legend to Fig. 1.
FIG. 3
FIG. 3
Schematic representation of the predicted stem-loop structure present in region II of the 3′ UTR of IBV; 19 IBV and 3 TCoV isolates were compared. The sequence of the predicted structure corresponds to nt 27312 to 27353 of the genome of the Beaudette strain. The arrows show the positions of nucleotide differences of the strains indicated alongside. Those nucleotides not marked by arrows did not vary between the strains. Deletions are indicated by “▵,” and an insertion is indicated by a black arrowhead. The 3′ UTR sequences were established by Boursnell et al. (1) (IBV Beaudette and M41), Williams et al. (34) (IBV Gray, Arkansas 99, and Holland 52), Sutou et al. (30) (IBV KB8523), Sapats et al. (27) (IBV Vic S, V5/90, N1/62, N9/74, N2/75, N1/88, Q3/88, and V18/91), and Breslin et al. (3) (TCoV isolates Minnesota, Indiana, and NC95) and by us for the remaining isolates. Strains M41, D207, HV10, HV140, KB8523, and N9-74 had the same sequence and potential structure as IBV Beaudette.
FIG. 4
FIG. 4
D-RNAs made to investigate sequences necessary for packaging of the RNA into viruslike particles. nk, not known. Any D-RNA without a CAT reporter gene could be detected by Northern blot analysis only after one or more passages (P1 onwards)—replication could not be assessed if rescue had not occurred. Other details are explained in the legend to Fig. 1.
FIG. 5
FIG. 5
Detection of D-RNAs by Northern blot analysis. D-RNAs CD-61, CD-36, CD-39, and CD-40ΔXbaI without a CAT reporter gene were electroporated into helper virus-infected CK cells (A) or Vero cells (B) at 8 h p.i. Resultant particles were serially passaged in CK cells (P1 to P6). Total cellular RNA was extracted, electrophoresed in an agarose gel, and blotted onto nitrocellulose filters that were then probed with a 5′ genomic probe. The arrows show the position of the expected D-RNA band. Lane 51 shows CD-51 at P0, and lane M is a marker RNA that contains CD-61 P4 RNA. Blank lanes were a consequence of unsuccessful recovery of total RNA. The replication and rescue of these D-RNAs are shown in Fig. 4. g, IBV genomic RNA.
FIG. 6
FIG. 6
D-RNAs used to study the deletion of replicase 1b sequence and its replacement by structural protein genes. In these constructs, the CAT reporter gene was inserted at an XcmI site, 804 nt from the 5′ end.
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References

    1. Boursnell M E G, Binns M M, Foulds I J, Brown T D K. Sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus. J Gen Virol. 1985;66:573–580. - PubMed
    1. Breslin J J, Smith L G, Fuller F J, Guy J S. Sequence analysis of the matrix/nucleocapsid gene region of turkey coronavirus. Intervirology. 1999;42:22–29. - PMC - PubMed
    1. Breslin J J, Smith L G, Fuller F J, Guy J S. Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3′ untranslated region identifies the virus as a close relative of infectious bronchitis virus. Virus Res. 1999;65:187–193. - PMC - PubMed
    1. Brian D A, Chang R-Y, Hofmann M A, Sethna P B. Role of subgenomic minus-strand RNA in coronavirus replication. Arch Virol. 1994;9(Suppl.):173–180. - PubMed
    1. Cavanagh D, Brian D A, Brinton M A, Enjuanes L, Holmes K V, Horzinek M C, Lai M M C, Laude H, Plagemann P G W, Siddell S G, Spaan W, Taguchi F, Talbot P J. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol. 1997;142:629–633. - PubMed

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