ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro
© Dai et al; licensee BioMed Central Ltd. 2011
Received: 30 August 2011
Accepted: 13 October 2011
Published: 13 October 2011
Dengue virus (DENV) and West Nile virus (WNV), close siblings of the Flaviviridae family, are the causative agents of Dengue hemorraghic shock or West Nile meningoencephalitis respectively. Vaccines against these two flaviviruses are currently unavailable. Interferon- Stimulated Gene 15 (ISG15), encoding an ubiquitin-like protein, is significantly induced by type I interferons or viral infections. Its roles in viral infections, however, vary with viruses, being either anti- or pro-viral. The exact roles of ISG15 in DENV and WNV infections remain unknown. In the current study, we evaluated the relevancies of ISG15 to DENV and WNV infection of a mouse macrophage cell line RAW264.7.
Quantitative PCR showed that mouse Isg15 was dramatically induced in DENV or WNV- infected RAW264.7 cells compared with non-infected cells. Isg15 and two other Jak-Stat related genes, Socs1 and Socs3, were silenced using siRNA mediated RNA interference. The intracellular DENV and WNV loads, as determined by quantitative PCR, were significantly higher in Isg15 silenced cells than control cells. The expression levels of interferon beta 1 (Ifnb1) were increased significantly in Isg15, Socs1 or Socs3 siRNA treated cells. Further investigation indicated that protein modification by ISG15, so called ISGylation, was significantly enhanced in DENV-infected cells compared to that in non-infected cells.
These findings suggest that ISG15 plays an anti-DENV/WNV function via protein ISGylation.
KeywordsISG15 Dengue Virus West Nile Virus ISGylation
Interferon Stimulate Gene 15
West Nile virus
- Ifnb1 :
Interferon beta 1
- Socs :
Suppressor of cytokine signaling
Dengue virus (DENV) and West Nile virus (WNV) are two major flaviviruses that can infect human via mosquito bites . Both viruses are (+) sense single strand RNA viruses and replicate in the cytoplasm of their host cells. DENV infection causes Dengue fever (DF) or Dengue hemorrhagic fever (DHF), and the latter may lead to death of the patient . WNV is a re-emerging zoonotic pathogen of medical importance. In humans, WNV infection may cause life-threatening meningoencephalitis or long-term neurologic sequelae . Vaccines against both DENV and WNV are currently not available, thus much efforts are needed to elucidate the mammalian host anti-viral mechanisms against these two flaviviruses in vitro and in vivo [1–3].
ISG15 is one of the first identified Interferon-Stimulated- Genes (ISGs). The expression of ISG15 can be induced by type I interferons (IFNs), viral infections and LPS, suggesting that ISG15 is a broad spectrum of stress response gene [4, 5]. The protein product of ISG15 shows a significant sequence homology to ubiqutin, and this protein can conjugate to numerous cellular proteins via isopeptide bonds. This kind of protein modification is called ISGylation, which also utilizes a three step reaction similar to protein ubiquitination. The specific enzymes for ISGylation are UBE1L (E1), UBCH8 (E2) and HERC5 (E3), respectively. Like ubquitination, ISGylation is reversible, mediated by deISGylation enzymes, such as UBP43. Notably, expression of UBE1L, UBCH8, HERC5, and UBP43 is also induced by IFN [4–7]. Till now, more than 200 cellular proteins were identified as the substrates of ISG15, while the functional consequences of reversible ISG15 modification of most target proteins are still largely unknown [8, 9].
A strong induction of ISG15 in response to IFN treatment or viral infections implies an antiviral role for ISG15, yet surprisingly initial studies using vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV) demonstrated that ISG15 or UBE1L are dispensable for anti-viral immune responses in mice [10, 11]. Nevertheless, more emerging work has shown that ISG15 plays an antiviral role against many viral pathogens in vitro and in vivo, including influenza A/B, herpes simplex virus type 1, murine gamma herpes virus, HIV and etc. [12–18]. ISG15 achieves its antiviral role by conjugating to target proteins including both host proteins and viral proteins and altering their functions. For example, ISG15 can conjugate to host anti-viral protein IRF3 and thus stabilize IRF3 by inhibiting its interaction with PIN1, a protein that promotes IRF3 ubiquitination and degradation . ISG15 was also found to modify the influenza A non-structural protein NS1A, and inhibit its immunosuppressive function . Little work has been done on the physiological relevancies of ISG15 to flavivirus infections [14, 20]. Surprisingly though, a recent study even showed that ISG15 is favorable of hepatitis C virus infection . It is though not clear why ISG15 "likes" some viruses, but "dislikes" some others, it is imperative to understand ISG15 function case-by-case. We hereby investigated the physiological role of ISG15 in DENV and WNV infection in a mouse macrophage cell line.
Isg15 is upregulated upon DENV or WNV infection
Isg15 activities repress DENV and WNV infection in RAW264.7 cells
siRNA and oligo-primer sequences for this study:
siRNA seq for mouse gene Isg15
siRNA seq for mouse gene Socs1
siRNA seq for mouse gene Socs3
Forward primer for Quantitative-PCR of mouse beta-actin
Reverse primer for Quantitative-PCR of mouse beta-actin
Forward primer for Quantitative-PCR of DENV E gene
Reverse primer for Quantitative-PCR of DENV E gene
Forward primer for Quantitative-PCR of WNV E gene
Reverse primer for Quantitative-PCR of WNV E gene
Forward primer for Quantitative-PCR of mouse Isg15 gene
Reverse primer for Quantitative-PCR of mouse Isg15 gene
Forward primer for Quantitative-PCR of mouse Socs1 gene
Reverse primer for Quantitative-PCR of mouse Socs1 gene
Forward primer for Quantitative-PCR of mouse Socs3 gene
Reverse primer for Quantitative-PCR of mouse Socs3 gene
Ifnb1 expression is upregulated when Isg15, Socs1 or Socs3 is silenced
Protein ISGylation is enhanced in DENV infected cells
Protein modification by ubiquitin and ubiquitin-like proteins, including ISG15, SUMO and Nedd8, Fat 10, has been shown to be important for many cellular processes, such as cell cycle, stress response and immune response . ISG15 has been shown to be upregulated upon type I interferon stimulation, microbial infections and tumor growth [4, 5, 22, 23]. Expression of ISG15 is reported to be controlled by transcriptional factors Stat, NFkb, p53 and JNK . These studies suggest that ISG15 is generally a stress-responsive gene.
The role of ISG15 and its conjugation activity in viral infections has been intensively studied recently, though controversial results have been reported with different viruses [4, 7]. One study shows that Isg15 is largely dispensable for the type I IFN-induced antiviral response against VSV and LCMV in mice . While some other work indicate that Isg15 -/- or Ube1L -/- (ISG15 E1) mice are more susceptible to many viral infections, such as influenza A/B, herpes simplex virus and SNV, than WT mice . ISG15 can modify a number of important antiviral proteins, including RIG-1, IRF3, MDA-5, Mx1, PKR, STAT1 and JAK1, and potentially influence their functions [4, 19, 24]. However, Functional consequences of this reversible modification are still mysterious. For examples, ISGylation of RIG-I negatively regulates RIG-I signaling and leads to a reduction of IFN production ; while ISGylation of IRF3 prevents the ubiquination and degradation of IRF3 during DNV infection . ISG15 can inhibit the ubiquitination of Gag and Tsg101 of HIV-1, disrupts their interaction, and prevents the virus assembles , while ISG15 also promotes HCV infection in an in vitro model . Generally speaking, ISGylation does not lead to degradation of target proteins, in stead, ISGylation stabilizes the target proteins by inhibiting their ubiquitination and degradation .
On the other hand, viruses may also block the antiviral activity of ISG15 . For example, nario-viruses and arterviruses encode ovarian-tumor (OTU)-domain containing proteases, which can deconjugate ISG15. Overexpression of OUT proteases antagonizes the antiviral effect of ISG15 .
Since ISG15 plays distinct role in different viral infection model, we use DENV and WNV to explore the function of ISG15 in these two vector-borne RNA viruses. Our results suggest that ISG15 has an antiviral activity against these two flaviviruses. And the antiviral activity of ISG15 likely relies on its protein-modifying activity. Our results also confirmed the inhibitory effect of Socs1 and Socs3, on antiviral mechanisms such as the Jak-Stat pathway. Future directions would be characterizing the target proteins of ISGylation during DENV or WNV infection, and indentifying novel anti-flavivirus molecules and/or new mechanisms mediated by ISGylation. These studies may significantly increase our knowledge on the pathogenesis of flavivirus infection and advance the development of novel therapeutics or drugs against these life-threatening viruses.
This work was supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT1075), and research grants Q413400211 from Soochow University, China. P. W. is supported by a Career Development Award from Northeast Biodefense Center (U54-AI057158-Lipkin).
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