Mutational Disruption of cis-Acting Replication Element 2C in Coxsackievirus B3 Leads to 5'-Terminal Genomic Deletions.
While the wt genome with the mutated CRE(2C) displays suppressed replication levels similar to those observed in a CVB3-TD strain, mutation of the CRE(2C) function in a CVB3-TD strain does not further decrease replication.In this report, we demonstrate that while CVB can replicate without a uridylylating CRE(2C), the replication rate suffers significantly.This demonstrates that VPg can prime without being specifically uridylylated and that this priming is error prone, resulting in the loss of sequence information from the 5' terminus.
Affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
- Enterovirus B, Human/genetics*
- Gene Deletion*
- Replication Protein C/genetics*
- Virus Replication/genetics*
- Base Pairing
- Base Sequence
- DNA Primers/genetics
- Gene Knockout Techniques
- HeLa Cells
- Molecular Sequence Data
- Reverse Transcriptase Polymerase Chain Reaction
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Figure 5: T7 RNA transcripts from CVB3 and CVB3-CKO clones can be transfected into mice, and virus replicates posttransfection. Nested RT-PCR could detect viral RNA using total RNA from the hearts and spleens of mice transfected with wt CVB3 or CVB3-CKO T7 RNA transcripts. (A) A representative gel demonstrating the necessary 10-fold serial dilutions of total extracted tissue RNA (in this case, from heart) to detect the low level of replication of CVB3-CKO in mouse tissue at 20 days posttransfection (lanes 1 and 8, molecular size marker; lane 2, no-RNA RT-PCR control; lane 3, untransfected mouse RNA; lane 4, 10-fold dilution of RNA from a CVB3-CKO-transfected mouse; lane 5, 100-fold dilution; lane 6, 1,000-fold dilution; lane 7, 10,000-fold dilution). CVB3-CKO RNA was detectable only at a 1,000-fold dilution of RNA (arrow, lane 6). (B) Detection of viral RNA in the spleens of transfected mice (lanes 1 and 9, molecular size marker; lane 2, no-cDNA PCR control; lane 3, no-RNA RT-PCR control; lane 4, HeLa cell RNA control; lane 5, 1,000 copies of CVB3 cDNA from T7 transcripts; lane 6, untransfected mouse RNA; lane 7, 1,000-fold dilution of RNA obtained from a CVB3-CKO-transfected mouse at day 20 posttransfection; lane 8, 100,000-fold dilution of RNA obtained from a wt CVB3-transfected mouse at day 8 posttransfection). (C) Detection of viral RNA in the hearts of transfected mice (lanes 1 and 8, molecular size marker; lane 2, no-cDNA PCR control; lane 3, no-RNA RT-PCR control; lane 4, untransfected mouse RNA; lane 5, 1,000 copies of CVB3 cDNA from T7 transcripts; lane 6, 100-fold dilution of RNA obtained from a CVB3-CKO-transfected mouse at day 20 posttransfection; lane 7, 10,000-fold dilution of RNA obtained from a wt CVB3-transfected mouse at day 4 posttransfection). (D to K) RNase-treated homogenates of spleens were passaged onto HeLa cells at approximately 90% confluence and incubated overnight with (D to G) or without (H to K) neutralizing antibodies (D and H, no homogenate; E and I, control mouse homogenate; F and J, homogenate obtained from CVB3-CKO-transfected mice at day 20 posttransfection; G and K, homogenate obtained from CVB3-transfected mice at day 8 posttransfection). Magnifications, ×100. CPE was observed only in cultures inoculated with homogenates from mice transfected with wt CVB3 RNA (K). (L) Following 5 days of incubation, total RNA was tested by nested RT-PCR and analyzed on 2% agarose gels (lanes 1 and 14, molecular size marker; lanes 2 and 3, CVB3-CKO plus neutralizing antibody; lanes 4 and 5, CVB3-CKO minus neutralizing antibody; lanes 6 and 7, wt CVB3 with neutralizing antibody; lanes 8 and 9, wt CVB3 without neutralizing antibody; lane 10, no-cDNA control; lane 11, no-RNA RT control; lane 12, uninfected control with neutralizing antibody; lane 13, uninfected control without neutralizing antibody), demonstrating that CVB3-CKO (arrows, lanes 4 and 5) and wt CVB3 (arrows, lanes 8 and 9) were detected in tissue homogenates following transfection of mice. The absence of amplimers in lanes 2 and 3 (CVB3-CKO) and lanes 6 and 7 (wt CVB3) demonstrates that virus is neutralized by anti-CVB3 neutralizing antibody.
Our observations that CVB3-CKO produced infectious virus and can be passaged in cell culture led us to ask if this was reproducible in vivo. A/J mice were chosen for use in this experiment, as long-term CVB3 persistence has been demonstrated in this strain of mouse and this strain has regularly been used in our studies of CVB3-TD replication (25, 28, 55). Mice were inoculated i.p. with lipid-conjugated CVB3-CKO or wt CVB3 T7-transcribed RNA and killed 8 days (wt CVB3) or 20 days (CVB3-CKO) postinoculation. Spleen and heart RNA preparations were assayed for the presence of viral RNA by RT-PCR. It was necessary to perform serial dilutions of RNA extracted at day 20 posttransfection from tje tissues of mice that had been transfected with CVB3-CKO in order to detect the low-level replication of this mutant virus against the high background level of tissue RNA. Viral RNA was detectable only at higher dilutions (Fig. 5A; compare lanes 4 [10-fold dilution], 5 [100-fold dilution], and 7 [10,000-fold dilution] to lane 6 [arrow, 1,000-fold dilution], though the required dilution varied slightly among the different samples. Both CVB3-CKO RNA and wt CVB3 RNA were detectable in spleen (Fig. 5B, lanes 7 and 8, respectively) and heart (Fig. 5C, lanes 6 and 7, respectively), demonstrating the infectivity of T7 transcripts of CVB3 genomes in mice.