Figure 1.
Determination of coronaviral poly(A) tail length in virions used for inoculation.
(A) Strategy for determining coronaviral poly(A) tail length in virus used for inoculation. RNA extracted from BCoV harvested at 48 hpi was decapped and head-to-tail ligated. BCoV 5’ UTR-positive-strand-specific primer 2 (for RT) and BCoV 3’ UTR-negative-strand specific primer 1 were used for RT-PCR with the ligated RNA as template. Note that the head-to-tail ligation could be inter-molecular or intra-molecular. Inter-molecular-ligated RNA is represented here and in the following figures. The amplified RT-PCR product was sequenced to determine poly(A) tail length. (B) Sequence of the amplified RT-PCR product. The poly(A) tail length in virus used for inoculation was ~45 nt.
Figure 2.
Poly(A) tail length on intra-cytoplasmic coronaviral positive-strand RNA at different times post-infection.
(A) RT-PCR product synthesized from ligated RNA by the method described in Figure 1A for cytoplasmic RNA extracted from infected cells at the indicated times post-infection. RT-PCR products ranging in length from ~250 bp to ~300 bp were observed. (B) Plot of the poly(A) tail lengths as determined by sequencing RT-PCR products of ligated ends described in (A). M, ds DNA size markers in nt pairs. Values in (B) represent the mean±SD of three individual experiments.
Figure 3.
Poly(U) tract length on intra-cytoplasmic coronaviral negative-strand RNA at different times post-infection.
(A) Strategy for determining coronaviral poly(U) tract length. The strategy used was the same as that described in Figure 1A except the primer used for RT was primer 1. The RT-PCR products were used to determine coronaviral poly(U) tract length. (B) Determination of RT-PCR product sizes. Varying lengths of RT-PCR products ranging from ~250 bp to ~300 bp were observed at different times post-infection. (C) RT-PCR control reactions. Left panel: RT-PCR reveals a ~300 bp product with RNA from BCoV-infected cells extracted at 9 hpi (lane 1), but not with a mixture of RNA from mock-infected cells (800 ng), from in vitro synthesized subgenomic mRNA 7 transcripts (100 ng), and from in vitro synthesized transcripts representing full-length BCoV genomic RNA (100 ng) (lane 2). Right panel: RT-PCR reaction mixture containing RNA from BCoV-infected cells at 9 hpi along with no exogenous primers (lane 1). RT-PCR reaction mixture containing RNA from BCoV-infected cells at 9 hpi along with a single primer that binds β actin mRNA (lane 2). Samples from a complete reaction with RNA from infected cells at 9 hpi served as a size reference (Figure 3C, left panel, lane 1, and Figure 3C, right panel, lane 3). (D) The length of poly(U) tracts at different times post-infection as determined by sequencing RT-PCR products obtained from samples used for panel (B). M, ds DNA size markers in nt pairs. Values in (D) represent the mean±SD of three individual experiments.
Figure 4.
The length of poly(A) and poly(U) on subgenomic mRNA 7 positive strand and negative strand, respectively.
(A) Strategy for determining poly(A) tail length on coronaviral subgenomic mRNA 7 positive strand. Total cellular RNA extracted from BCoV-infected HRT cells was decapped and head-to-tail ligated. To ensure the poly(A) tail sequence is specifically derived from subgenomic mRNA 7 rather than genomic RNA, BCoV leader positive-strand-specific primer 4 (for RT) and BCoV leader (-)-strand-specific primer 3 were used for RT-PCR with ligated positive-strand coronaviral RNA as a template. The expected length of RT-PCR product is near 1.8 kb containing subgenomic mRNA 7-specific poly(A) tail. The RT-PCR product was sequenced to determine subgenomic mRNA 7 poly(A) tail length. (B) Strategy for determining poly(U) tract length on coronaviral subgenomic mRNA 7 negative strand. The method used to determine the length of poly(U) tail on negative-strand subgenomic RNA is the same as that for poly(A) tail length on positive-strand subgenomic mRNA 7 except the primer used for RT was primer 3. The expected length of RT-PCR product which contains subgenomic mRNA 7-specific poly(U) tract is ~1.8 Kb. The RT-PCR product was sequenced to determine subgenomic mRNA 7 poly(U) tract length. (C) RT-PCR product synthesized with the methods described in panels (A) and (B). The length of subgenomic mRNA 7 RT-PCR products containing poly(A) (left panel) or poly(U) (right panel) was near 1.8 kb at the different times postinfection. (D) RT-PCR control reactions. Same as described for Figure 3C, except for the use of primers 3 and 4 (not 1 and 2) in lane 1, left panel, and lane 3, right panel. (E) The length of coronaviral subgenomic RNA 7 poly(A) tail (left panel) and subgenomic RNA 7 poly(U) tract (right panel) at different time points postinfection as determined by sequencing RT-PCR products obtained in Figure 4C. M: ds DNA size markers in nt pairs. Values (E) represents the mean±SD of three individual experiments.
Figure 5.
The poly(A) and poly(U) lengths on BCoV DI RNA positive strand and negative strand, respectively.
(A) Diagram of BCoV DI RNA-M used to determine poly(A) and poly(U) lengths on BCoV DI RNA during coronavirus infection. To distinguish the DI RNA-specific poly(A) tail and poly(U) tract from those on BCoV helper virus RNAs, the BCoV DI RNA 3’ UTR was replaced with the MHV 3’ UTR to synthesize a previously documented replication-competent, packagable BCoV DI RNA-M [6]. The methods used to determine the DI RNA-M poly(A) and poly(U) lengths are similar to those described in Figures 2 and 3 except that primer 5 is used. This method enables discrimination between helper virus RNA and DI RNA-M as the origin of the poly(A) and poly(U). (B) Sequence of the poly(A) tail on BCoV DI RNA-M in the inoculum. The experiment described in panel (A) shows that the poly(A) tail length of BCoV DI RNA-M in the virus used for inoculation was ~26 nt. (C) RT-PCR product synthesized with the method described in panel (A). Varying lengths of RT-PCR products which contained DI RNA-M poly(A) tail (left panel) and poly(U) tract (right panel) ranged from ~200 bp to ~250 bp at different times following infection with virus passage 1 (VP1) or 2 (VP2). (D) RT-PCR control reactions. Left panel: RT-PCR with primers 5 and 2 reveals a ~200-bp product with RNA extracted from total cell RNA collected at 24 hpi with DI RNA-M-containing BCoV (lane 1), but not from a mixture of RNA extracted from BCoV-infected cells at 24 h of VP1 (100 ng) and from positive-strand DI RNA-M (100 nt) (lane 2). Right panel: A ~200-bp product was not obtained from RT-PCR reactions with RNA from cells infected with DI RNA-M-containing BCoV and no exogenous primers (lane 1), or from the same RNA with a primer that binds β actin mRNA (lane 2). (E) The length of DI RNA-M poly(A) tail (left panel) and poly(U) tract (right panel) at different time points of VP1 and at 48 h of VP2 as determined by sequencing RT-PCR products obtained in panel (C). M, ds DNA size markers in nt pairs. Values (E) represents the mean±SD of three individual experiments.
Figure 6.
Effect of coronaviral poly(A) tail length on translation of non-replicating coronaviral DI RNA in virus infected cells.
(A) DI RNA constructs used for replication and translation assay. The open box represents a single, 2.2 kb open reading which is followed by a stippled box representing an in-frame 18-nt His-tag coding region. (B) Replication of DI RNAs. A Northern blot of RNAs made at 48 hpi following infection with VP1 to determine the replication level of mutated DI RNAs. Lanes 1 through 4 show the accumulation of DI RNA. (C) Abundance of non-replicated DI RNA and DI RNA-expressed proteins. Expression of His-tagged DI RNA protein was measured by Western blot analysis with antibody specific to the Histidine-tag. BCoV-infected HRT cells were transfected with the named DI RNA at 2 hpi and at the indicated times proteins or RNAs were extracted for analysis. Protein samples from cell lysates were harvested at 4, 8, and 21 hpt and the His-tagged protein (row 1) and cellular β actin (row 2) were measured by Western blotting. For RNA measurements, RNA was extracted at the indicated times and Northern probing was done with the TGEV reporter probe to measure DI RNA levels (row 3), with intra-N subgenomic mRNA probe to measure subgenomic N mRNA levels (row 4), subgenomic M mRNA and DI RNA levels (row 6), with 18S rRNA-specific probe to measure 18 S rRNA levels (row 5), and with an oligo-histidine-specific probe to detect His-tag coding sequence in viral subgenomic RNA and His-tagged DI RNA (row 7). (D) Quantification of translated protein from individual DI RNA constructs at different time points. Values (D) represent the mean±SD of three individual experiments.