Cytoplasmic Viral RNA-Dependent RNA Polymerase Disrupts the Intracellular Splicing Machinery by Entering the Nucleus and Interfering with Prp8

Liu, Yen Chin; Kuo, Rei Lin; Lin, Jing; Huang, Peng; Huang, Yi; Liu, Hsuan; Arnold, Jamine J.; Chen, Shu Jen; Wang, Chia Hui; Cameron, Craig Ε.; Shih, Shin Ru

doi:10.1371/journal.ppat.1004199

Cited by 52 publications

(73 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An NLS-like sequence within HRV16 3D (PXKTKLXPS 27 ) similar to sequences from yeast ribosomal proteins was previously predicted by Amineva et al (6) based on in silico analysis/sequence comparison (and which is similar to the NLS motif of foot-and-mouth disease virus [FMDV] 3D pol [32]), but mutational analysis had not been previously applied to test NLS functionality and the possibility that HRV protease activity might contribute to nuclear localization had not been assessed. Our data here clearly indicate that mutagenesis of this sequence does not appear to impact nuclear localization of HRV16 3CD under the conditions tested, while GFP-3D itself, although localizing to a slightly greater extent in the nucleus than GFP-3CD (P ϭ 0.03), is predominantly cytoplasmic, completely in line with previous studies for enterovirus 71 (where overexpression can lead to weak nuclear localization in transfected cells) (33) and poliovirus 3D pol (29). Enterovirus 71 and poliovirus 3D…”

Section: Discussionsupporting

confidence: 91%

“…pol are known to associate with splicing factor Prp8 in the nucleus, leading to inhibition of pre-mRNA splicing, resulting in a decrease in mRNA and accumulation of pre-mRNA intermediates (33). Results for FMDV also suggest an important role for 3D pol nuclear localization in virus infection, whereby NLS mutations that inhibit it result in attenuated FMDV infection (28), although a link to splicing factor interaction has not been assessed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Walker

Jensen

Croft

et al. 2016

J Virol

View full text Add to dashboard Cite

The human rhinovirus (HRV) 3C and 2A proteases (3C pro and 2A pro , respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C pro is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A pro activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C pro , 3D, and mutant derivatives thereof, we show that 3C pro is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C pro activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A pro fusion proteins, we demonstrate formally that 2A pro activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C pro is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A pro activity and not 3C pro activity, which is observed only later in infection. The results thus define the temporal activities of 2A pro and 3CD/3C pro activities in HRV serotype16 infection. IMPORTANCEThe human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.

show abstract

Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Walker

Jensen

Croft

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…S8 and Supplementary Table S2). PRPF8 is a splicing factor known to have an anti-apoptotic effect in neurons and Picornaviruses infection2627. ZIKV infection also results in downregulation of a splicing factor, SFPQ, which might result in impairment of neuronal development.…”

Section: Discussionmentioning

confidence: 99%

Zika virus disrupts molecular fingerprinting of human neurospheres

Garcez

Nascimento

Vasconcelos

et al. 2017

Sci Rep

109

View full text Add to dashboard Cite

Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

show abstract

“…It is 500 therefore possible that HRV does not actively target mRNA transcription in the same 501 way as poliovirus. Consistent with the view point that HRV might not depend on 502 rapid shut-down of mRNA transcription is the recent observation that unlike 503 poliovirus, HRV fails to use 3D to suppress mRNA splicing [44]. A second possible 504 explanation relates to the observation that while most gene transcription is quickly 505 suppressed by enteroviruses, a minority of transcripts -some with antiviral activity -506 are both upregulated and translated [45,46].…”

Section: Comparing the Ability Of A16 B4 And C2 Proteases To Shutdowmentioning

confidence: 86%

Rhinovirus 2A is the predominant protease responsible for instigating the early block to gene expression encountered in infected cells

Smart

Filippi

Smalley

et al. 2019

Preprint

View full text Add to dashboard Cite

1Human rhinoviruses express 2 cysteine proteases, 2A and 3C, that are responsible for 2 viral polyprotein processing. Both proteases also suppress host gene expression by 3 inhibiting mRNA transcription, nuclear export and cap-dependent translation. 4However, the relative contribution that each makes in achieving this goal remains 5 Methods 83Cell lines and reagents 84 293T cells were maintained at 37°C in a 5% CO2 incubator with DMEM (Invitrogen, 85Fisher Scientific UK Ltd, Loughborough, UK) supplemented with 10% foetal bovine 86 serum, 100 U/ml penicillin, 100 µg/ml streptomycin. Experimental treatment of cells 87 included incubation in medium supplemented with 200 µg/ml cycloheximide (Sigma, 88 Gillingham, UK) and/or 5 µg/ml actinomycin D (Cayman Chemical Cambridge 89Bioscience Ltd, Cambridge, UK). 90 91 DNA constructs 92Generation of some constructs relied on gene synthesis (Invitrogen, Fisher Scientific 93 UK Ltd, Loughborough, UK). In instances where this was the case, sequences ordered 94 were codon optimized for mammalian expression and then manually adjusted to 95 minimize the existence of unwanted splice donor and splice acceptor sites using the 96 online programmes HSF3[25] and the NetGene2 server [26]. The initial plasmid 97 expressing GFP, VSV-tagged 2A and HA-tagged 3C from HRV16 was generated by 98 cloning a single synthetic DNA fragment into an in-house dual promoter plasmid via 99EcoRI and SalI restriction sites to generate pCIPEP-A16 TAG (+/+) (see Fig. S1 for 100 sequence). Synthesized DNAs encoding for HRV-B4 and HRV-C2 proteases (Table 101 S1) were exchanged with their respective counterparts in pCIPEP-A16 TAG (+/+) using 102ClaI and SbfI (2A) and BsiWI and SalI (3C) restriction sites. Inactivation of the 103 proteases involved a two-step PCR approach, which introduced a Cys>Ala mutation 104 in the active site of both 2A and 3C. A similar PCR-based strategy was used to 105 generate constructs expressing tag-free proteases and a 3C protease with a c-Myc 106 nuclear localization signal (PAAKRVKLD) fused directly to its C-terminus. Primer 107 sequences are available on request. 108The synthetic DNA encoding for the Thioredoxin-Nanoluciferase(Nluc)PEST 109 fusion protein linked by a peptide derived from translating the hepatitis delta virus 110 (HdV) ribozyme sequence (HdV WT Nluc; Fig. S2) was initially cloned into pCDNA3.

show abstract

Cytoplasmic Viral RNA-Dependent RNA Polymerase Disrupts the Intracellular Splicing Machinery by Entering the Nucleus and Interfering with Prp8

Cited by 52 publications

References 56 publications

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Rhinovirus 16 2A Protease Affects Nuclear Localization of 3CD during Infection

Zika virus disrupts molecular fingerprinting of human neurospheres

Rhinovirus 2A is the predominant protease responsible for instigating the early block to gene expression encountered in infected cells

Contact Info

Product

Resources

About