The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

Dhungel, Pragyesh; Cao, Shuai; Yang, Zhilong

doi:10.1371/journal.ppat.1006602

Cited by 47 publications

(78 citation statements)

References 62 publications

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“…In this biological context, mTOR suppression late in infection may contribute to host shutoff late in infection. Interestingly, postreplicative viral mRNAs harbor unusual 5= leader elements that enable both cap-dependent and -independent translation, and VacV also modifies host ribosomes in unusual ways to maximize translation (48,(104)(105)(106). While this unusual mode of initiation may help sustain their translation in specific cell types or under various stress conditions, our data show that maximal accumulation of late viral proteins nonetheless remains dependent on mTOR activity, which controls a wide range of translation factors beyond those that control initiation alone (48).…”

Section: Discussionmentioning

confidence: 80%

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

Meade

King

Munger

et al. 2019

J Virol

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Despite producing enormous amounts of cytoplasmic DNA, poxviruses continue to replicate efficiently by deploying an armory of proteins that counter host antiviral responses at multiple levels. Among these, poxvirus protein F17 dysregulates the host kinase mammalian target of rapamycin (mTOR) to prevent the activation of stimulator of interferon genes (STING) expression and impair the production of interferon-stimulated genes (ISGs). However, the host DNA sensor(s) involved and their impact on infection in the absence of F17 remain unknown. Here, we show that cyclic-di-GMP-AMP (cGAMP) synthase (cGAS) is the primary sensor that mediates interferon response factor (IRF) activation and ISG responses to vaccinia virus lacking F17 in both macrophages and lung fibroblasts, although additional sensors also operate in the latter cell type. Despite this, ablation of ISG responses through cGAS or STING knockout did not rescue defects in late-viral-protein production, and the experimental data pointed to other functions of mTOR in this regard. mTOR adjusts both autophagic and protein-synthetic processes to cellular demands. No significant differences in autophagic responses to wild-type or F17 mutant viruses could be detected, with autophagic activity differing across cell types or states and exhibiting no correlations with defects in viral-protein accumulation. In contrast, results using transformed cells or altered growth conditions suggested that late-stage defects in protein accumulation reflect failure of the F17 mutant to deregulate mTOR and stimulate protein production. Finally, rescue approaches suggest that phosphorylation may partition F17’s functions as a structural protein and mTOR regulator. Our findings reveal the complex multifunctionality of F17 during infection. IMPORTANCE Poxviruses are large, double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon-stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ∼100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses, we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.

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Section: Discussionmentioning

confidence: 80%

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

Meade

King

Munger

et al. 2019

J Virol

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“…This is due, at least in part, to the fact that poly(A) stretches ≥11 nucleotides induce sliding of both initiating 40S and elongating 80S ribosomes (Arthur et al, 2015;Koutmou et al, 2015;Shirokikh and Spirin, 2008), a phenomenon that contributes to the specific function of the 3′poly(A) tail in mRNA quality control (Joazeiro, 2017). It is interesting that, amongst reported functional studies of poly(A) leaders, these elements are generally absent and lack enhancer activity in human or yeast cells (Dhungel et al, 2017;Jha et al, 2017;Xia et al, 2011), both of which have no negative charge in their RACK1 loop and whose loopsour assays functionally validatedlack poly(A)-enhancer activity. By contrast, many 5′UTRs of the dicot plant A. thaliana harbor long poly(A) stretches (Guo et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity in a loop domain

Rollins

Jha

Bartom

et al. 2019

Journal of Cell Science

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Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved β-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plantbut not uncharged yeast or human loopenhances translation of mRNAs with adenosine-rich 5′ untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.

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“…Our TSS mapping uncovered a form of transcript slippage by the ASFV RNAP occurring on promoters that start with an A(+1)TA motif, where mRNAs are extended by one or two copies of the dinucleotide AU. This is reminiscent of VACV, where late gene transcripts containing a poly-A 5' UTR 21 are associated with improved translation efficiency and reduced reliance on cap-dependent translation initiation 47,48 ; likewise, distinct functional attributes of polyA leaders in translation have been documented in eukaryotes 49 . Whether the 5' AU-and AUAU-tailing is a peculiarity of the ASFV-RNAP initiation, or whether mRNA 5' leaders have any functional implications, remains to be investigated.…”

Section: Discussion [1014 Words]mentioning

confidence: 99%

Temporal Transcriptome and Promoter Architecture of the African Swine Fever Virus

Cackett

Matelska

Sýkora

et al. 2019

Preprint

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The African Swine Fever Virus causes haemorrhagic fever in domestic pigs and presents the biggest global threat to animal farming in recorded history. Despite its importance, very little is known the mechanisms and temporal regulation of transcription in ASFV. Here we report the first detailed viral transcriptome analysis of ASFV during early and late infection of Vero cells. In addition to total RNA sequencing, we have characterised the transcription start sites and transcription termination sites at nucleotide-resolution, revealing the distinct DNA consensus motifs of early and late promoters, as well as the sequence determinants for transcription termination. ASFV can utilise alternative promoters to generate distinct proteins from the same transcription unit that differ with respect to the polypeptide N-terminus. Finally, our results reveal that the ASFV-RNAP undergoes transcript slippage at the 5' end of transcription units that in a promoter sequence-specific manner results in the addition of 5'-AT and 5'-ATAT tails to mRNAs.

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The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

Cited by 47 publications

References 62 publications

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

mTOR Dysregulation by Vaccinia Virus F17 Controls Multiple Processes with Varying Roles in Infection

RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity in a loop domain

Temporal Transcriptome and Promoter Architecture of the African Swine Fever Virus

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