Study finds a new mammalian antiviral response pathway restricting poxviruses replication

By Neha MathurFeb 15 2023Reviewed by Aimee Molineux

In a recent study posted to the bioRxiv* preprint server, researchers identified a novel antiviral pathway different from the interferon (IFN) pathway that inhibited poxvirus(es) replication.

Study: A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins. Image Credit: Kateryna Kon / Shutterstock

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

The Facilitates Chromatin Transcription (FACT) complex is a chromatin remodeler that functions via coordinated interaction between the human suppressor of Ty 16 homolog 66 (hSpt16) and structure-specific recognition protein-1 (SSRP1) subunits. The FACT, an evolutionarily-conserved gene system, regulates messenger ribonucleic acid (mRNA) transcription of only specific cellular genes by regulating chromatin accessibility to transcriptional machinery. Thus, it is present in discrete genomic loci. Studies, however, have not deciphered the significance of FACT-mediated gene expression systems in human immunity.

Practically all mammalian viruses encode IFN antagonists because the type I IFN gene system triggers hundreds of antiviral genes upon encountering a viral antigen. Its activation is crucial during viral infection(s) in mammals, including humans. However, researchers have barely explored antiviral transcriptional processes that evolved before mammals-specific IFN responses.

Poxviruses, such as variola virus (VARV), caused smallpox, a fatal infectious disease that claimed millions of human lives over many decades it lasted. Despite its eradication in 1979, other poxviruses continue to pose prominent public health concerns; for instance, Mpox or Monekypox virus (MPXV) caused a deadly outbreak worldwide in 2022-2023. Nonetheless, poxvirus infections, in general, severely compromise host immunity. Thus, there is an urgent need to characterize host immune factors and pathways targeted by poxviruses.

A previous study showed that the vaccinia virus (VV) encoded A15R protein is critical for robust viral replication in mammalian cells. Its deficiency, however, did not alter the susceptibility of VV to IFN treatment in mammalian cell cultures. Strikingly, only A51R expression promoted viral RNA replication in non-permissive insect cells. Together, these observations point out that A51R inhibits undefined eukaryotic antiviral responses that evolved prior to the IFN responses.

About the study

In the present study, researchers first identified how VV A51R interacted with the hSpt16 subunit of FACT using a yeast two-hybrid screen and a human prey library. The hSpt16 subunit had nine overlapping prey clones, which implied A51R interaction domain in hSpt16 was nested within amino acid (AA) residues 758 to 893. The researchers confirmed the interaction between hSpt16 and Flag-A51R in A549 cells using the co-immunoprecipitation (Co-IP) methodology. Co-IP results confirmed an exclusive interaction between VV A51R and the hSpt16 subunit of FACT.

Next, the team used RNA interference (RNAi) to determine if FACT impacted VV replication. RNAi studies found that FACT depletion complemented the replication fault of A51R-depleted VV. Also, FACT broadly restricted cytoplasmic deoxyribonucleic acid (DNA) and RNA viruses.

For a more detailed probe of the role of FACT in antiviral immune responses, the researchers continued examining VV A51R-hSpt16 interactions. While on 10% acrylamide gels, hSpt16 resolved as one band, there were two hSpt16 bands of ~140 and 155 kiloDalton molecular weight (MW) on 6% gels. The (~15 to 20 kDa difference in MW of the two bands indicated that the upper band was a SUMOylated form of hSpt16. So, the team re-probed these distinct Co-IP membranes separately with small ubiquitin-like modifier 1 (SUMO-1) antibodies.

SUMOylation, a post-translational modification, involves the covalent attachment of SUMO protein(s) to lysine residues in target proteins via an enzymatic cascade that modifies their biological function.

Since Spt16 is conserved among eukaryotes, the researchers also evaluated Spt16 SUMOylation in several human cell lines and eukaryotes. Strikingly, SUMOylated Spt16 was present in all the tested human cell lines, including primary normal human dermal fibroblasts (NHDFs). Moreover, even monkey, mouse, hamster, and bat-derived cell lines had SUMOylated Spt16.

Study findings

The current study investigations revealed that early VV gene expression triggered FACT-dependent ETS proto-oncogene 1 (ETS-1) transcription factor (TF) expression termed the FACT-ETS-1 antiviral response (FEAR) pathway that restricted viral replication. Like FACT, ETS are primitive eukaryotic TFs, that originated nearly 600 million years ago in invertebrates. The hSpt16 SUMOylation regulated the antiviral FACT function and licensed FACT to bind transcriptionally active chromatin during viral infection.

The FEAR pathway has physiological relevance suggested by the finding that VV A51R countered this pathway by competing with SSRP1 to bind hSpt16^SUMO and tether it to microtubules (MTs) to block its accumulation in the nucleus. Moreover, the reduced pathogenicity of VV strains encoding A51R mutations that prevented hSpt16SUMOylation highlighted the contribution of this virus-host interaction to viral disease. Given that A51R proteins from multiple poxviruses (e.g., Ectromelia virus) bind hSpt16SUMO and associate with MTs suggests that it is a conserved poxvirus-host immune evasion mechanism. Other viruses harboring A51R are cowpox, Yaba-like disease viruses, and MPXV.

Both FACT and ETS-1 served crucial roles in activating the host transcriptional response to viral infection. However, ETS-1 restricted viral replication in an hSpt16^SUMO/FACT-dependent manner. The FEAR pathway appeared to predate and remain distinct from the IFN response. Likewise, ETS-1 likely plays an antimicrobial role in eukaryotes.

In addition, this study's results might aid probing the function of new host antiviral pathways and mechanisms of their regulation in other human etiologies, e.g., cancer. Studies have also shown a correlation between ETS-1 upregulation and poor survival in cancer patients. Thus, our finding that ETS-1 induction requires hSpt16SUMO function might yield insights into how overexpression of FACT promotes oncogenic gene expression and severe outcomes in cancer patients.

Furthermore, the study demonstrated that hSpt16^SUMO promoted its binding to monoubiquitinated H2B histone. This biomarker of active transcription sites in chromatin during viral infection also stimulates ETS-1 expression.

Conclusions

Overall, the current study demonstrated a broad role for FACT in antiviral immunity. Since FACT also restricted unrelated cytoplasmic RNA viruses, it proved to be a critical component of antiviral immunity. Future studies should investigate how RNA viruses activate and encounter FACT, including mechanisms governing its activation. Moreover, the study evidenced that the FEAR pathway of eukaryotes is a unique viral immune evasion mechanism. Thus, studies should also explore more genes regulated by ETS-1 during viral infection(s) and their role(s) in restricting them.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.