The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.
Keywords: PRRSV; RNA sequencing; arterivirus; chromosomes; gene expression; gene expression regulation; infectious disease; microbiology; nidovirus; porcine reproductive; programmed ribosomal frameshifting; respiratory syndrome virus; ribosome profiling; subgenomic mRNAs; transcriptome; translatome.
Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells’ ribosomes – the machines in the cells that make proteins – to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a ‘frameshift’, and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.
© 2022, Cook et al.