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Updated: Jun 9, 2025
Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo
Published on: September 18, 2014
Poxin-deficient poxviruses are sensed by cGAS prior to genome replication
Sian Lant1, Alasdair J M Hood1, Joe A Holley1,2
1Department of Microbial Sciences, University of Surrey, Guildford, GU2 7XH, UK.
Abstract:
Poxviruses are dsDNA viruses infecting a wide range of cell types, where they need to contend with multiple host antiviral pathways, including DNA and RNA sensing. Accordingly, poxviruses encode a variety of immune antagonists, most of which are expressed early during infection from within virus cores before uncoating and genome release take place. Amongst these antagonists, the poxvirus immune nuclease (poxin) counteracts the cyclic 2'3'-GMP-AMP (2'3'-cGAMP) synthase (cGAS)/stimulator of interferon genes DNA sensing pathway by degrading the immunomodulatory cyclic dinucleotide 2'3'-cGAMP, the product of activated cGAS. Here, we use poxviruses engineered to lack poxin to investigate how virus infection triggers the activation of STING and its downstream transcription factor interferon-responsive factor 3 (IRF3). Our results demonstrate that poxin-deficient vaccinia virus (VACV) and ectromelia virus (ECTV) induce IRF3 activation in primary fibroblasts and differentiated macrophages, although to a lower extent in VACV compared to ECTV. In fibroblasts, IRF3 activation was detectable at 10 h post-infection (hpi) and was abolished by the DNA replication inhibitor cytosine arabinoside (AraC), indicating that the sensing was mediated by replicated genomes. In macrophages, IRF3 activation was detectable at 4 hpi, and this was not affected by AraC, suggesting that the sensing in this cell type was induced by genomes released from incoming virions. In agreement with this, macrophages expressing short hairpin RNA (shRNA) against the virus uncoating factor D5 showed reduced IRF3 activation upon infection. Collectively, our data show that the viral genome is sensed by cGAS prior to and during genome replication, but immune activation downstream of it is effectively suppressed by poxin. Our data also support the model where virus uncoating acts as an immune evasion strategy to simultaneously cloak the viral genome and allow the expression of early immune antagonists.
Insights
Poxviruses use poxin to suppress host antiviral responses. Removing poxin reveals viral genome sensing by cGAS and STING activation, indicating genome detection occurs before and during replication.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Poxviruses encode immune antagonists, like poxin, to evade host antiviral defenses.
- Poxin degrades 2'3'-cGAMP, a product of cGAS, thus inhibiting the STING-IRF3 pathway.
- Understanding poxin's role is crucial for deciphering poxvirus immune evasion strategies.
Purpose of the Study:
- To investigate how poxvirus infection triggers STING and IRF3 activation in the absence of poxin.
- To determine the timing and mechanism of viral genome sensing by host cells.
- To elucidate the role of virus uncoating in immune evasion.
Main Methods:
- Utilizing poxin-deficient vaccinia virus (VACV) and ectromelia virus (ECTV) infections in primary fibroblasts and macrophages.
- Employing DNA replication inhibitors (AraC) to differentiate sensing mechanisms.
- Using short hairpin RNA (shRNA) to target the virus uncoating factor D5.
Main Results:
- Poxin-deficient VACV and ECTV induced IRF3 activation in fibroblasts and macrophages.
- IRF3 activation in fibroblasts was DNA replication-dependent, while in macrophages, it was linked to incoming virions.
- Inhibition of virus uncoating reduced IRF3 activation in macrophages.
Conclusions:
- The viral genome is sensed by cGAS early in infection, prior to and during replication.
- Poxin effectively suppresses cGAS-STING pathway activation, highlighting its role in immune evasion.
- Virus uncoating is an immune evasion strategy that conceals the viral genome and facilitates the expression of early viral factors.
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