Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells

Abstract

We have developed a ribonuclease protection method suitable for sensitive detection of an RNA species in the presence of a large excess of its complementary strand, as for the detection of negative strands of positive-strand RNA viruses. By using this method to probe for poliovirus negative strands in virions, we found that positive strands are present in at least 40,000-fold excess over negative strands. Thus, we have confirmed that poliovirus encapsidation is highly specific for positive strands and have demonstrated that the genome-linked protein VPg, which is covalently attached to the 5' ends of both positive and negative strands, cannot be the sole determinant of RNA packaging. We tested the ratios of viral positive strands to negative strands in cells at different times during infection; this value ranged from approximately 40/1 to 70/1, being highest at 4 h and lower at 2 and 6 h postinfection.

OUP accepted manuscript

“…Thus, it is perhaps too early to dismiss the possibility of negative-strand ORFs in other (þ)ssRNA virus lineages. In most cases, however, any proteins encoded on the negative strand would be expected to be expressed at much lower levels than positive-strand encoded proteins simply because of the huge disparity in positive:negative RNA abundance during virus infection (typically of order 100:1; Novak and Kirkegaard 1991;Kopek et al 2007;Irigoyen et al 2016). Even for narnaviruses, less than 1-2 per cent of ScNV-20S viral RNA in infected cells is negativesense (Rodriguez-Cousiño, Esteban, and Esteban 1991;Fujimura, Soló rzano, and Esteban 2005), indicating that the rORF, where present, is likely to be expressed at a much lower level than the RdRp.…”

Enterovirus infections of the central nervous system

“…Since uridylylation is not observed during host cell transcription, this process may be a unique therapeutic target against EV infections, given that point mutations that block uridylylation may prevent viral growth (Paul et al, 1998). While VPg plays a role in both positive-and negative-strand RNA synthesis, it is still unknown how approximately 40 to 70 copies of positivestrand genome are produced for every negative-strand genome during an EV infection (Novak and Kirkegaard, 1991). Intriguingly, this ratio decreases upon the establishment of CVB3 persistence within the CNS suggesting that a double-stranded RNA genomic structure might assist in virus stability during persistence .…”

Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins

“…Positive-strand viral RNA accumulation in poliovirus-infected SK-OV-3 cells at 5 h post-infection was approximately 45% of the level observed in HeLa cells, suggesting a positivestrand RNA synthesis defect during poliovirus infection of the SK-OV-3 cell line. To further investigate the difference in RNA synthesis for poliovirus negative-strand RNAs (using the total cellular RNA prepared in the same manner described above), we carried out additional RNase protection assays for poliovirus negative-strand RNA using the two-cycle method that has been described previously (Novak and Kirkegaard, 1991). Interestingly, the maximum level of negative-strand RNA accumulation in poliovirus-infected SK-OV-3 cells was approximately 85% of the amount observed for infected HeLa cells ( Fig.…”

“…RNase protection assays to determine positive-strand and negativestrand RNA accumulation were performed as previously described (Novak and Kirkegaard, 1991;Brunner et al, 2005). A 100 ng aliquot of total RNA from infected cells was hybridized with 25 fmol of [ 32 P]UTPlabeled RNA probe, designated the (+) probe, containing nucleotides complementary to positive-sense sequences 5601 to 5809 of poliovirus viral RNA.…”