The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

Wang, Tong-Yun; Fang, Qiongqiong; Feng, Cong; Liu, Yonggang; Wang, Haiming; Zhang, Hongliang; Tian, Zhen; Tang, Yan-Dong; Cai, Xuehui

doi:10.1080/22221751.2019.1679010

Cited by 20 publications

(13 citation statements)

References 42 publications

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“…This highlights the possibility that the composition of the PRRSV RTC could change over time, for example if extra copies of nsp10, 11 or 12 are supplied from ORF1b sgRNA translation (as well as the potential contribution of increased ORF1ab frameshift efficiency). This provides a potential mechanism of regulating viral replication, for example by altering the ratio of gRNA to sgRNA production (as observed in Figure 2 and Figure 8), as both nsp10 (the helicase) and nsp12 are thought to be involved in promoting sgRNA transcription [129][130][131][132][133][134] . Nsp11 (NendoU) is an endoribonuclease found in many nidoviruses, which has broad substrate specificity in vitro 135 , and is also an innate immune antagonist [136][137][138][139][140] .…”

Section: Discussionmentioning

confidence: 99%

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Cook

Brown

Shang

et al. 2021

Preprint

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Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and analyse the host response to infection. We quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), −2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF- stimulatory viral protein (nsp1β) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we also found PRF efficiency at the canonical ORF1ab frameshift site increases over time, in apparent 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 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) 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 temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

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Section: Discussionmentioning

confidence: 99%

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Cook

Brown

Shang

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…This highlights the possibility that the composition of the PRRSV RTC could change over time, for example if extra copies of nsp10, 11 or 12 are supplied from ORF1b sgRNA translation (as well as the potential contribution of increased ORF1ab frameshift efficiency). This provides a potential mechanism of regulating viral replication, for example by altering the ratio of gRNA to sgRNA production (as observed in Figures 2 and 8), as both nsp10 (the helicase) and nsp12 are thought to be involved in promoting sgRNA transcription [130][131][132][133][134][135] . Nsp11…”

Section: Non-canonical Orf1b Sgrnas May Modulate Expression Of Orf1bmentioning

confidence: 99%

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Cook

Brown

Shang

et al. 2022

eLife

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Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, we use 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 quantified viral gene expression over a timecourse of infection, and calculated the efficiency of programmed ribosomal frameshifting (PRF) at both sites on the viral genome. At the nsp2 frameshift site (a rare example of protein-stimulated frameshifting), −2 PRF efficiency increases over time, likely facilitated by accumulation of the PRF-stimulatory viral protein (nsp1β) during infection. This marks arteriviruses as the second example of temporally regulated PRF. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in apparent 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 125-codon ORF overlapping nsp12, which is expressed as highly as nsp12 itself at late stages of replication, and is likely translated from novel subgenomic (sg) 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 temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, we identified a highly translated, short upstream ORF (uORF) in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This is the first application of RiboSeq to arterivirus-infected cells, and reveals new features which add to the complexity of gene expression programmes in this important family of nidoviruses.

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“…In contrast, mutations that inactivate PCPβ activity of nsp1β completely block viral genomic and sgRNA synthesis [11]. Similar to nsp1α, nsp12 is not required for viral genomic RNA synthesis but is indispensable for viral sg RNA synthesis [94]. Two Cys residues at position 35 and 79 in nsp12 are cooperatively required for sg mRNA synthesis [12].…”

Section: Understand the Regulation Of Viral Rna Synthesismentioning

confidence: 99%

Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics and the Major Applications

Chaudhari

2020

Viruses

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Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive sense, single-stranded RNA virus that is known to infect only pigs. The virus emerged in the late 1980s and became endemic in most swine producing countries, causing substantial economic losses to the swine industry. The first reverse genetics system for PRRSV was reported in 1998. Since then, several infectious cDNA clones for PRRSV have been constructed. The availability of these infectious cDNA clones has facilitated the genetic modifications of the viral genome at precise locations. Common approaches to manipulate the viral genome include site-directed mutagenesis, deletion of viral genes or gene fragments, insertion of foreign genes, and swapping genes between PRRSV strains or between PRRSV and other members of the Arteriviridae family. In this review, we describe the approaches to construct an infectious cDNA for PRRSV and the ten major applications of these infectious clones to study virus biology and virus–host interaction, and to design a new generation of vaccines with improved levels of safety and efficacy.

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The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis

Cited by 20 publications

References 42 publications

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Ribosome profiling of porcine reproductive and respiratory syndrome virus reveals novel features of viral gene expression

Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics and the Major Applications

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