Inhibition of hepatitis C virus RNA replication by ISG15 does not require its conjugation to protein substrates by the HERC5 E3 ligase

Domingues, Patricia; Bamford, Connor; Boutell, Chris; McLauchlan, John

doi:10.1099/jgv.0.000283

Cited by 28 publications

(30 citation statements)

References 26 publications

(34 reference statements)

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“…Functional characterisation of A549 ISG15 knockout cell lines. (a) CRSIPR/Cas9 genome editing was used to knockout ISG15 expression in A549 cells followed by single-cell cloning (following previously reported procedures (18)). Four independent clones were treated with 1000 IU ml-1 IFN-α for 24 and 48 h, or left untreated, and protein expression was tested by immunoblot analysis of ISG15, MxA and β-Actin.…”

Section: Resultsmentioning

confidence: 99%

“…A549-ISG15 -/- cells were generated by CRISPR/Cas9n system that utilises the D10A dual ‘nickase’ mutant of Cas9 (Cas9n) that ostensibly limits off-target effects. Briefly, to disrupt exon 2 of the ISG15 gene, single guide RNA (sgRNA) sequences were cloned using the pPX460 and transfected into A549 cells as previously described (18). Transfectants were enriched after 48 h by treating cells with puromycin (1 μg/ml) for 2 d and then diluted to single cells in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

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Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Holthaus

Vasou

Bamford

et al. 2019

Preprint

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18Interferons (IFNs), produced during viral infections, induce the expression of hundreds of stimulated genes (ISGs). Some ISGs have specific antiviral activity while others regulate the cellular 20 response. In addition to functioning as an antiviral effector, IFN-stimulated gene 15 (ISG15) is a 21 negative regulator of IFN signalling and inherited ISG15-deficiency leads to autoinflammatory 22 interferonopathies where individuals exhibit elevated ISG expression in the absence of infection. We 23 have recapitulated these effects in cultured human A549-ISG15 -/cells and (using A549-UBA7 -/cells) 24 confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of 25 the type-I IFN response. ISG15-deficient cells pre-treated with IFN-α were resistant to paramyxovirus 26 infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition 27 of global protein synthesis leading us to ask whether resistance was due to the direct antiviral 28 activity of ISGs or whether cells were non-permissive due to translation defects. We took advantage 29 of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal 30 antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in IFN-α-31 pre-treated ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of 32 the IFN response. However, resistance could be induced if cells were pre-treated with IFN-α for 33 longer times, presumably due to inhibition of protein synthesis. These data show that the cause of 34 virus resistance is two-fold; ISG15-deficiency leads to the 'early' over-expression of specific antiviral 35ISGs, but the later response is dominated by an unanticipated, ISG15-dependent, loss of 36 translational control. 37 FBS (1:1000). Following PBS washes, cells were incubated for 1 h with goat anti-mouse IgG 131 antibodies conjugated to alkaline phosphatase (Abcam Cat# ab97020) diluted 1:1000 in PBS, 3%FBS. 132 Cells were washed in PBS and signals were detected using SIGMAFAST BCIP/NBT (Sigma). 133 Reverse transcription quantitative PCR. 134Total cellular RNA was purified from cells that had been treated with 1000 IU/ml IFN-α2b (Intron A) 135 for 18 h, or left untreated, using TRIzol reagent (ThermoFisher Scientific) and Direct-zol RNA 136

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Holthaus

Vasou

Bamford

et al. 2019

Preprint

Self Cite

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“…Several cellular proteins involved in antiviral signaling, including RIG-I, MDA-5, STAT1, JAK1, IRF3, PKR, Mx1, and RNase L, were also identified or suggested as substrates for ISGylation [16][17][18][19][20][21]. ISGylation suppressed replication of diverse viruses, such as influenza virus (type A and B) [22][23][24][25], human immune deficiency virus (HIV) [26,27], hepatitis C virus (HCV) [28][29][30], Japanese encephalitis virus [31], Sindbis virus [23,32,33], Ebola VP40 virus-like particle [34,35], herpes simplex virus type-1 [23], murine γ-herpesvirus 68 [23], vaccinia virus [36], dengue and West Nile viruses [37], porcine reproductive and respiratory syndrome virus [38], Kaposi's sarcoma-associated herpesvirus (KSHV) [39], and respiratory syncytial virus [40]. However, the antiviral mechanism of ISGylation against specific viruses is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators

Kim

et al. 2016

PLoS Pathog

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Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.

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“…For example, ISG15 is a critical ISG with antiviral activity against DNA and RNA viruses [Lenschow et al, 2007]. While silencing of ISG15 has been shown to increase HCV replication [Domingues et al, 2015], high hepatic ISG15 expression levels are also associated with high viral load and poor response to interferon therapy, suggesting that HCV might exploit ISG15 as part of an immune evasion strategy [Broering et al, 2010;Chen et al, 2010].…”

Section: Interferon Stimulated Genesmentioning

confidence: 99%

Interferon stimulated genes and innate immune activation following infection with hepatitis B and C viruses

Hayes

Chayama

2016

Journal of Medical Virology

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Although not directly cytopathic, hepatitis B and C viruses (HBV and HCV) are responsible for millions of deaths per year, and the aging patient population presents a severe public health challenge. Most cases of acute HCV become chronic, but treatment has become increasingly successful following the development of direct acting antiviral agents. Conversely, most cases of acute HBV are cleared in the pre-symptomatic stage and do not become chronic, but treatment options for chronic HBV are limited and rarely result in clearance. Despite these differences, interferon is partially effective against both viruses and has long been used in therapy. Newer treatments have largely moved away from interferon in favor of more targeted approaches, but interferon signaling remains the body's first line of defense against viruses. The recent discovery of type III interferon and IFNL4 has yielded new insights into the mechanism of ISG activation and revealed potential new therapeutic targets. J. Med. Virol. 89:388-396, 2017. © 2016 Wiley Periodicals, Inc.

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Inhibition of hepatitis C virus RNA replication by ISG15 does not require its conjugation to protein substrates by the HERC5 E3 ligase

Cited by 28 publications

References 26 publications

Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Direct antiviral activity of interferon stimulated genes is responsible for resistance to paramyxoviruses in ISG15-deficient cells

Consecutive Inhibition of ISG15 Expression and ISGylation by Cytomegalovirus Regulators

Interferon stimulated genes and innate immune activation following infection with hepatitis B and C viruses

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