Combatting Against Cancer Using Virus: Oncolytic Virus M1 Reinvigorates T-cell immunity against glioblastoma

Glioblastoma multiforme (GBM), usually arising from glial precursor cells or neural stem cells, is the most common and lethal primary brain cancer in adults. It has very limited treatment options and a median life expectancy less than 2 years after diagnosis. Systemic and local immunosuppression induced by GBMs largely contributes to malignancy aggressiveness and resistance to multiple immunotherapies, such as immune checkpoint blockade (ICB) therapy, which is powerful for primary care of other types of solid tumors. 

Oncolytic virotherapy has attracted growing awareness as a potential transformative cancer therapy in recent years. It involves delivery of a genetically modified version of virus into the host and it would selectively destroy tumor cells while leaving neighboring healthy cells undamaged, overcoming a common limitation of chemotherapy. This targeted destruction is either achieved through direct oncolysis ("onco-" meaning cancer-related, "-lysis" meaning burst) or immune-mediated attack. Oncolytic therapy using alphacirus M1(OVM), which was originally isolated from Culex mosquitoes, recently emerged as a potential glioma therapeutic approach. The virus is delivered through intravenous administration and the main advantages are OVM's ability to selectively replicate in tumor cells overexpressing matrix remodeling-associated 8 (the marker allowing them to recognize and attach) and efficiency in penetrating through the blood brain barrier, which is a dense layer of blood vessels and cells that protect our brain from harmful substances but makes drug delivery difficult at the same time. Even though we might be worried that using virus as medium can be dangerous by itself at the first glance, the toxic genes are being taken out in advance and it's previously shown to be very safe: GBM patients showed high tolerance towards OVM in previous clinical trials and repeated intravenous administration of OVM is non-pathogenic for nonhuman primates (Zhang et al., 2016).

Despite of its huge therapeutic potential, the knowledge regarding how OVM initiates adaptive immunity against gliomas and how the interactions between OVM and our immune system would influence the therapeutic effects of ICB is still lacking. Hence the researchers are seeking to address these two knowledge gaps.

First, they confirmed the efficacy of OVM in reducing glioma progression. The immunocompromised mice were injected intracranially with GBM cell lines (GL261-Luciferase, GL261 and CT2A) to induce a glioma in the brain on day 0 and followed by daily tail intravenous injection of OVM or vehicle from day 5 to day 9. Vehicle includes the buffer solutions that researchers use to deliver OVM but containing no OVM, acting as the control condition. GL261-Luciferase is a cell line where it's infected with lentivirus whose genome was engineered to contain the luciferase gene so that wherever the tumor develops, it would glow. A stronger luminescence intensity (red) indicates the progression of glioma while bluish color indicates little tumor growth. It was found that OVM significantly inhibited the growth of glioma and prolonged the survival of glioma-bearing mice. 

Moving on to investigate the immune response, the researchers found that OVM successfully induced immunogenic cell death and reverse the GBM-induced immunosuppression both locally and systemically. Immunogenic cell death is characterized by the release of damage-associated molecular patterns (DAMPs) by tumor cells, which are molecules that normally stay inside the cell and becomes 'danger signals' recognized by immune cells when being released outside, driving adaptive-immunity elimination in tumor microenvironment. They examined the release of DAMPs, revealing significant CRT (externalized calreticulin, a multifunctional protein) exposure and elevated extracellular ATP (adenosine triphosphate, energy source for our cells) in the supernatant collected from glioma cell lines with OVM treatment. This indicates the occurrence of immunogenic cell death induced by OVM. 

Additionally, the number of CD4+ and CD8+ T cells increased significantly in peripheral blood of glioma-bearing mice following intravenous OVM treatment. Spleen atrophy and loss of splenic T-cell population were also recovered. Similarly, in the tumor local environment, they also found rapid increase in tumor-specific CD8+ T cells in the spleen. The measurement is achieved by orthotopical implantation of GL261 glioma cells expressing OVA (full-length ovalbumin, processed into SIINFEKL) and H-2Kb-SIINFEKL tetramer that binds to T cell receptors where H-2Kb is the mouse version of MHC class I protein. The recognition of glioma-derived-antigen-MHC complex by T cells allows the accurate tracking of tumor-specific T cells (Hsu et al., 2025). Hence, with OVM infection, both local and systemic immunity are boosted and recovered.

  

Before diving deeper into the mechanism, the researchers digressed a bit and addressed a main concern about the efficacy of OVM: Spleen is the major body 'filter' consisting of large number of immune cells that's found previously able to eliminate delivered oncolytic viruses and OVM is often 'trapped' inside the spleen of various host animals. Therefore, the researchers removed the spleen (splenectomy) from mice to find out the role of spleen in OVM treatment. To their surprise, opposite to the greater reduction in glioma progression that they have expected, the antitumor activity of OVM treatment was completely abrogated after removal. Splenectomy-OVM mice almost have the same low survival probability as vehicle control and no expansion of T-cell population in the peripheral blood and tumor microenvironment.

Consequently, the researchers tried to find out the specific cell population in the spleen that's mediating the reversal of immunosuppression and antitumor activity. They first employed a broad RNA sequencing and computational screening and found that B cells have the highest abundance and showed strongest predicted interactions with T cells after OVM treatment. To move beyond correlation, a series of validation experiment were conducted: In vitro co-culture experiments demonstrated that B cells from OVM-treated mice significantly enhanced activation of T cells but not other splenic immune populations and this activation is dose-dependent. Further, after blocking the B cells with anti-CD19 antibody, OVM administration failed to prolong the survival of glioma-bearing mice and increase the infiltration of T cell populations in the blood and tumor microenvironment. 

To elucidate the mechanism by which splenic B cells reverse immunosuppression, the researchers compared the interactions of B cells with CD8+ T cells in the OVM-treated group and the vehicle control group. They found that OVM treatment leads to much more frequent physical contacts between B and T cells, visualized through live cell imaging.

To determine the functional significance of this cell-to-cell contact, they created two conditions where the cells can directly interact with each other and where they are forced to be separated from each other by a membrane (transwell chamber). CD8+ T cell proliferation is measured by CFSE dilution (carboxyfluorescein succinimidyl ester,  a fluorescence dye that's split between new-born cells for every division) and CD8+ T cell activation is measured by GZMB+ (granzyme B, the 'weapon' T cells produce to kill target cancer cells). Using these two tracking tools, they discovered that T cell proliferation and activation is induced only when B cells and T cells that can freely contact with each other but not in transwell-separated groups. These results together suggests that the direct contact between B and T cells is essential for T-cell expansion and activation required for OVM-mediated immune enhancement.

In order to validate this finding in vivo, researchers targeted the Major Histocompatibility Complex class I (MHC-I), a critical component of the structural interface between B and T cells. B cells communicate and 'educate' T cells by presenting a fragment of tumor on MHC-I to activate them, which process is known as antigen presentation. Blocking MHC-I with a specific antibody completely abolished B-cell-induced CD8+ T cell proliferation and activation. Collectively, these results confirm that the direct physical interface is crucial for immunity restoration by OVM treatment. Later the researchers identified the specific subset of B cells that's responsible for this interaction to be Bst2+ B cells by computational screening and in-vivo evidence: Bst+B cells significantly prolonged the survival of B-cell-deficient, GL261-OVA-bearing mice after OVM stimulation while Bst2-B cells and B cells from vehicle control group did not.

The interface between B and T cells involves B cells 'presenting' a peptide derived from cancer cells or other sick cells on MHC-I protein, which is then recognized by T cell receptors (TCR). 

Finally, to bridge the above mechanistic findings with clinical application, the researchers tested if OVM treatment can help overcome immunosuppression that previously has prevent immune checkpoint blockade (ICB) therapy from functioning in glioblastoma patients. ICB therapy works by removing the "brake" on the T cells. Our bodies have these natural immune checkpoint proteins that's naturally used to prevent excessive inflammation from happening that would otherwise harm other healthy cells. But at the same time, this also grant tumor cells a tool to escape from immune responses. ICB drugs block these inhibitory signals and boost the working capacity of our immune system. 

PD-L1 and PD-1 are the immune checkpoint proteins that 'put a brake' on the T cell when bound. If either of them are blocked, which is what the researchers and ICB drugs do, T cells are free to attack the tumor cells. 

To test the synergy between ICB and OVM therapy, immunocompromised mice was injected intracranially on day 0 and from day 5 to day 9, the mice is divided into different groups and treated daily intravenously with different combinations of immune checkpoint protein antibodies, including isotype antibody as control, PD-1 and PD-L1 antibodies, with or without OVM. The researchers revealed that, comparing with other 5 combinations, OVM treatment and PD-1 inhibitor together yielded the greatest recruitment of CD8+ T cells into tumor microenvironment, suppression of intracranial glioma growth as well as restoration of splenic size and weight. Systematically, massive expansions of effector (activated) CD8+ and CD4+ T cells in peripheral bloodstream are also observed. Therefore, OVM synergizes with PD-1 inhibitors for glioma treatment by simultaneously reshaping systemic immunity and the local immune environment in the brain.

isotype= control isotype antibody; PD-1 and PD-L1 Ab= PD-1 and PD-L1 antibodies (ICB therapy); Vehicle= control for OVM treatment; OVM= oncolytic viral therapy. 

This study provides profound mechanistic details for how emerging OVM treatment reverses the immunosuppressive environment of GBM, which is incurable and has grim prognosis. Most significantly, it establishes OVM as a transformative catalyst for ICB therapy, which is historically ineffective for GBM. The findings also illuminate a remarkable capability of OVM bypassing the 'toxic', immunosuppressed tumor microenvironment in the brain, and re-directing the site of immune cell communication to the 'clean sanctuary', the spleen. Within this protected environment, the recovery of B- and T-cell physical interface structures allows for robust antigen presentation, subsequent T cell activation, and eventually elimination of the tumor cells. The study has a strong advantage in its comprehensive validation of each step. For instance, when assessing the efficacy of the therapies, they would use several measures like the size of the spleen, tumor growth and immune cells activation level. Also, when confirming the determining role of splenic B cells, they combined both computational tools and biological checks, including knockout and correlation experiments. Moving forward, these discoveries open a new frontier for translational medicine. While mouse models provide an essential foundation, future research should evaluate OVM in non-human primates and eventually human clinical trials to characterize its safety profile in more complex in vivo environment. Long-term studies are also preferable because so far only the decrease in size of tumor is. reported but future relapse is still possible, especially given the ability of tumors to develop resistance mutations. Furthermore, the mechanism behind OVM-directed B-cell migration to the spleen and T-cell migration to the brain can be elucidated to identify potential signaling molecule targets.

Citations:

For images:

https://www.researchgate.net/figure/The-origin-of-glioblastoma-During-normal-embryonic-development-and-in-the-adult-brain_fig2_371833392

https://vyriad.com/science/oncolytic-virus-platforms/
https://www.cancerresearch.org/immunotherapy-by-treatment-types/oncolytic-virus-therapy
https://www.nature.com/articles/s41592-020-01031-0
https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/checkpoint-inhibitors

https://rcastoragev2.blob.core.windows.net/13974d8439d3384abb2302e11f2da069/PMC9563749.pdf

For academic literature:
the primary article:
‌Han, Yu, et al. “Oncolytic Virus M1 Reinvigorates CD8+ T-Cell Immunity against Glioblastoma through B-Cell-Dependent Antigen Cross-Presentation in the Spleen.” Cellular & Molecular Immunology, 4 Mar. 2026, www.nature.com/articles/s41423-026-01396-w/figures/8, https://doi.org/10.1038/s41423-026-01396-w. Accessed 26 Mar. 2026.

Others:

‌National Cancer Institute. “Https://Www.cancer.gov/Publications/Dictionaries/Cancer-Terms/Def/Blood-Brain-Barrier.” Www.cancer.gov, 2 Feb. 2011, www.cancer.gov/publications/dictionaries/cancer-terms/def/blood-brain-barrier.

‌Hsu, Chung-Yao, et al. “Polymerised Superparamagnetic Antigen Presenting Cell Lymphocyte Capture for Enriching Tumour Reactive T-Cells and Neoantigen Identification.” Nature Communications, vol. 16, no. 1, 2 June 2025, www.nature.com/articles/s41467-025-60321-3, https://doi.org/10.1038/s41467-025-60321-3. Accessed 26 Mar. 2026.

“Up Close: How Immune Checkpoint Inhibitors Revolutionize Cancer Treatment | What’s up at Upstate | SUNY Upstate.” Www.upstate.edu, www.upstate.edu/whatsup/2023/100523-up-close-how-immune.php.

Zhang, Haipeng, et al. Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates after Multiple Rounds of Repeated Intravenous Injections. Vol. 27, no. 9, 1 Sept. 2016, pp. 700–711, https://doi.org/10.1089/hum.2016.038. Accessed 1 Aug. 2023.

‌“Defined Immune Response Tracking in Mice: OVA MHC Tetramers – Caltag Medsystems.” Caltagmedsystems.co.uk, Mar. 2024, www.caltagmedsystems.co.uk/information/defined-immune-response-tracking-in-mice-ova-mhc-tetramers/. Accessed 29 Apr. 2026.

‌“Oncolytic Virus Therapy and Its Side Effects.” Cancer.org, 2023, www.cancer.org/cancer/managing-cancer/treatment-types/immunotherapy/oncolytic-virus-therapy.html.

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Hepatitis B Virus Genome Packaging and Reverse Transcription

Introduction

Hepatitis B Virus (HBV) is an infectious liver virus that contains a genome consisting of circular, double-stranded DNA. HBV is a member of a family of viruses that takes RNA genetic information generated during infection, and transcribes it into DNA to package into new viral particles (1). The process of reverse transcription is unique to viruses, as there is no need to generate DNA from RNA anywhere else in biological life. 

For liver cells infected with HBV to form infectious viral particles, the viral DNA produced during this infection must fit inside new viral particles to generate more viruses to infect more cells (1). This process is called packaging, and is required in all viral infections, regardless of the types of cells they infect, or the type of genome that the virus packages its cells with. Viral genomes are not very large compared to more complex life, such as in human cells, but the size of an average viral particle is also much smaller than an animal cell, and fitting the entire genome of a virus into its particle is a major hurdle that any virus must overcome, HBV included, and understanding the mechanisms behind this process may prove valuable in understanding viral replication (1). This paper describes their process of uncovering how HBV packages its DNA genome into the mature viral particle (2).

Viral Particle Imaging

The paper by Gibes et al. 2026 describes their process of imaging Duck HBV (DHBV) and human HBV viral particles to uncover how the DNA inside these viruses is organized in a manner that allows them to fit inside such a small particle. DHBV was generally easy to use, which is why some of their experiments started using it rather than HBV (2). They did this by purifying DHBV and later HBV viral particles and imaging them using cryo-electron microscopy, which is used to take pictures of small particles such as viruses that would be too small for normal microscopes (Figure 1).

Figure 1: Cryo-em image of HBV particle. a. Fully developed HBV protein capsid. White shapes represent the geometric points of symmetry that pattern the capsid's outer layer. b. First layer of DNA (lavender) showing 11 concentric rings of DNA. c. Second layer of DNA (purple) showing 7 concentric rings of DNA. d. The third and final layer of DNA (blue) showing less-defined concentric rings of DNA (2).

Structural Analysis

They saw that the viruses contained their DNA in layers wrapped around protein, forming concentric DNA rings inside the viral particle (Figure 1 b-d). To generate this structure, the precursor RNA associates with the capsid, or viral shell, proteins. The RNA is then reverse transcribed into the DNA, which replaces the RNA. The structures they derived from their experiments hint that the process of copying the RNA into DNA may cause the rings of DNA to slide around the proteins, and as more layers are added, the DNA would become more rigid in its position in the viral particle (2). One of the ends of the capsid protein monomers that the DNA associates with is rich in arginine, a positively charged amino acid, which can interact with the negatively charged backbone of DNA to stabilize the negative charges that would end up being so tightly packed into the viral particle (Figure 2). 

Figure 2: Structures of charged molecules relevant to capsid stability a. Structure of L-arginine b. Structure of a portion of the DNA backbone, “Base” represents A, T, C, or G (2)

They speculate that the tightly packed DNA can also act as a spring inside the viral particle, so that when it infects a new liver cell, the force from the tightly wound DNA can cause the particle to burst open and facilitate genome entry into the cell (2). 

Conclusions

To better understand how HBV packages its entire genome into its viral particle, the authors imaged mature HBV and DHBV particles to visualize the organization of the DNA inside. They revealed that the DNA of HBV and DHBV is contained in layers of circular concentric loops. The authors speculate that the process of packaging the particle takes place during reverse transcription of the RNA segments that HBV creates following infection, and that the positively charged capsid proteins facilitate stabilizing the DNA inside the virus (2). 

Works Cited

1. Gibes N, Culhane K, Liu H, Xi J, Pionek K, Nair S, Loeb DD, Hu J, Zlotnick A, Wang JC. DNA synthesis inside the hepatitis B virus creates a high-energy spool. bioRxiv [Preprint]. 2026 Jan 23:2026.01.22.701187. doi: 10.64898/2026.01.22.701187. PMID: 41648622; PMCID: PMC12871785.

2. Watson AG, Mulay AS, Gill US. Chronic hepatitis B in 2025: diagnosis, treatment and future directions. Clin Med (Lond). 2025 Nov;25(6):100527. doi: 10.1016/j.clinme.2025.100527. Epub 2025 Nov 4. PMID: 41192690; PMCID: PMC12681808.

Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

 Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

Influenza A virus (IAV) is a member of the influenza family, which contains Influenza A-D. Influenza A and B are the subtypes primarily responsible for the seasonal influenza cases.  Influenza subtypes have further genetic diversity due to their hemagglutinin (HA) protein, which has 18 identified variants, and the neuraminidase (NA) protein that has 11 identified variants. The variants of these proteins present on the viral surface determine the nomenclature for a specific subtype of virus; for example, the influenza A H1N1 virus (Spanish Flu) would present variant 1 for both the HA and NA proteins (1).

Although vaccinations are available each year for the emerging strains, ~5 million people each year develop severe symptoms, with 650,000 yearly deaths. In addition to its well-known symptoms of fever, tiredness, dizziness, and headache, influenza infection can cause rarer, yet more dangerous symptoms such as pneumonia and myocarditis. A major source of the symptoms and major illnesses that come from influenza infection can be attributed to the immune response. This can be an issue when unregulated, pro-inflammatory molecules are produced out of control, leading to more severe symptoms. This process is heavily regulated by the JAK-STAT pathway, which is responsible for the activation of many pro-inflammatory molecules. Common viral signals are recognized by cells, which leads to signaling events that activate JAK proteins. Influenza antiviral drugs primarily target the viral replication process, while ignoring the burden the immune response can have on a patient. This highlights the need for antivirals that can target both the viral replication and immune response elements of IAV infection (2).

By nature of the atypical genome of IAV, it requires its own special enzyme that can produce its transcripts, called an RdRp. The IAV RdRp is also capable of stealing essential portions of cellular machinery in a mechanism called “cap snatching, ” which is essential for its replication. The PB2 subunit of RdRp contains the cap-binding region required to steal caps, and thus is a suitable target for drugs that seek to inhibit cap snatching, and by extension, viral replication (2).

The authors of this paper were able to screen for drugs that either inhibit JAK proteins or drugs that inhibit the PB2 domain of IAV RdRp. They identified two drugs with similar structures: decernotinib, which inhibits JAK, and pimodivir, which inhibits PB2. Their goal was to synthesize a drug that mimics both structures and is able to inhibit both JAK and PB2. The paper describes their process of synthesizing and testing this molecule, which they call PB05, against various influenza infection conditions (2).

Synthesis

Figure 1. Visual Representation of PB05. (b) Images of pimodivir and decernotinib in the cap binding domain of PB2. (c) Visual representation of the workflow to create a pimodivir and decernotinib-like molecule, with possible modifications to the green “R” group in the pink box

Both drugs shared a main skeleton of carbon and nitrogen-based rings, which interact with portions of PB2 (Figure 1b,c). Fluorine substituents present on pimdinovir were incorporated into the final molecule, while the modified carbon chain of decernotinib was incorporated as well. They synthesized 5 drugs with this structure, with slight structural variations named PB01, PB02, PB03, PB04, and PB05. These molecules were tested for IAV inhibition, JAK inhibition, and PB2 inhibition. From these tests, the authors were able to determine that the PB05 drug performed the best (2).

Antiviral Activity of PB05

Figure 2 PB05 and decernotinib binding visualization. (c) (Left) PB05 and decernotinib in the active region of JAK2. (Right) PB05 and decernotinib in the cap binding domain of PB2.

The effectiveness of PB05 was tested against the H1N1 and H3N2 strains of IAV, which demonstrated that PB05 was capable of reducing the levels of IAV proteins and genetic material, but about 10x less efficiently than pimodivir, the PB2 inhibitor. Their imaging technology revealed that PB05 was directly binding to PB2 (Figure 2c). As for JAK-STAT inhibition, PB05 was also able to decrease the levels of transcripts responsible for producing inflammatory molecules, and was also found to be interacting with JAK (Figure 2c). In mice, inflammation-induced damage was also severely decreased. From these experiments, they were able to prove that PB05 was an effective antiviral therapy against IAV, which worked by both inhibiting the immune response to reduce inflammation-based damage, but also by inhibiting a key IAV enzyme in viral replication (2). 

Pharmacokinetics

Aside from a drug's ability to act on its target, another key aspect of drug design is making a drug that can be taken into the body without interference from common pathways of degradation and removal. This means that digestion, excretion, and distribution of the drug can also become a problem. Thankfully, they determined that PB05 was able to be taken orally with a reasonable dosage that wasn't harmful to the mice they tested on, and stayed in the body long enough to be effective when taken twice daily (2).

Conclusion

This paper demonstrated the need for drugs that both inhibit the immune response to influenza infections to reduce the injuries caused by inflammation, and inhibit viral replication to assist the immune system in successfully clearing the infection. They were able to find two drugs that performed these actions with structural similarities, and synthesize a single drug that combined the properties of these two. They claim that PB05 is an important first step in developing better IAV treatments, but more studies are needed on its interactions with the body's metabolism and its ability to circulate. It functions by blocking the JAK pathway and the cap snatching enzyme of IAV, demonstrating the dual action ability of this drug.

Works Cited

1. Kyokha Ameen Y. Seasonal Influenza: A Narrative Review of Epidemiology, Clinical Features, and Preventive Strategies. Cureus. 2025 Oct 24;17(10):e95336. doi: 10.7759/cureus.95336. PMID: 41287674; PMCID: PMC12640676.

2. Rong B, Yang Y, Lu K, Zhou X, Zheng P, Lin X, Wen Y, Lin S, Deng X, Zhou Q, Liu S. Dual Inhibition of PB2 and JAK2 for Influenza: A Strategy Combining Antiviral and Host-Directed Immune Modulation. Molecules. 2026 Feb 17;31(4):696. doi: 10.3390/molecules31040696. PMID: 41752472; PMCID: PMC12943364.

The Molecular Kidnapping: How Reoviruses are Taking Advantage of Our ATXN2L protein to Produce Their Babies

 Mammalian reovirus is a diverse family of virus that has segmented double-stranded RNA genome contained in two protein shells, which distinguishes them from many other viral families. Despite causing disease in wide range of mammalian hosts, reovirus is not typically considered as a human pathogen due to the absence of obvious symptoms or known clinical impact. Because of this, it becomes a perfect 'model organism' for studying cellular processes that can be safely handled in the lab, including translation, which is the main subject of this paper. It is the last step in converting DNA into proteins that are the actual workers of our cells.

In our cells, translation is made possible by the presence of a hat (5'cap) and a tail (poly-A tail) appended to mRNAs (the temporary carrier of genetic information), which helps them to exit the nucleus, protect them from enzymes that can degrade them, and help initiate translation (circularize the mRNA and reload translation machinery). Then mRNA will be converted into protein in the cytoplasm. In comparison, reovirus RNA genome gets translated within viral factories, which are little houses with no walls (often in membrane-less compartments called liquid-liquid phase separation) containing necessary tools for baby viruses to assemble and mature. Reovirus µNS protein forms the scaffold of these houses and interact with the core, where viral mRNAs are transcribed with the hat but without the tail. Surprisingly, these viral mRNAs can be readily translated in infected cells without the assistance and protection of the poly-A tail! This paper seek to find out the host cell factor that reoviruses has 'kidnapped' to produce viral proteins for themselves.

Since µNS protein is the important building block of viral factories where viral translation takes place, the 'manipulated' cellular protein probably interact with µNS protein. Based on this logic, the researchers used a technique called proximity-dependent Biotin identification, which is basically a spray-paint process. µNS protein is tagged with the spray---biotin protein ligase, which will enable the biotinylation of nearby proteins---paint. The 50 painted proteins labeled with biotin are identified and underwent a protein-protein interaction cluster analysis. There's one cluster that matches well with the expectations: proteins associated with SG (stress granule) and translation initiation. Followed by a long process of screening that identified proteins that would lead to failure in viral replication if deleted and if temporarily suppressed through CRISPR and siRNA whole genome screening respectively, a single common candidate that pop out in all filtered results is identified: ATXN2L.

Then the hypothesis that ATXN2L is the essential mediator of viral translation without poly A tail is tested stepwise. If ATXN2L indeed facilitates the translation of viral protein, it should be indispensable for viral replication. The researchers hence used CRISPR/Cas9 gene editing technique, which basically messes up the nucleotides at the specific desired site (gene ATXN2L) and render it nonfunctional, and it resulted in greatly impaired viral replication compared to the control group with undisrupted ATXN2L gene (WT) and with ATXN2L gene re-introduced (KO+). The green fluorescence is the virus-specific the antibody staining, indicating the infectivity. 

Additionally, if ATXN2L plays an essential role in viral translation, it probably interact with viral mRNA in some ways. Therefore, the researchers then tried to confirm this hypothesis and identify the specific interacting region. By creating ATXN2L with different combinations of deletions of regions that potentially participates in RNA binding (LSm and LSmAD) or contacts with poly-A-tail-binding protein (PAM2 motif) (shown in the image below, Δ meaning deletion), they found out that removal of either LSm and LSmAD alone or together significantly decreases ATXN2L's interaction with µNS and viral yield, with LSm being the primary one. This result suggests that RNA binding domains of ATXN2L are required for viral replication and it's highly possible that ATXN2L physically contact with mRNA at these specific sites. 

Then the researchers used Biotin-Streptavidin pull-down to find out if they two indeed interact with each other and where the region of contact is. Biotin is the small molecule that is attached to the viral mRNA and Streptavidin is a protein that acts like a high-powered magnet that can pull out Biotin and the bound mRNA along with proteins associates with it. Researchers tested three versions of viral s4 mRNA (as surrogate or the "representative" one among the ten segments): the normal version (s4 3'UTR), a version with the last ten nucleotides truncated (s4 3'UTR Δ10) and one with the last ten nucleotides scrambled. They chose the last ten nucleotides because they contain a five-nucleotide highly conserved sequence (meaning that it's found among almost all viral mRNAs), which can have evolutionary significance. They found that for both the truncated and scrambled condition, Streptavidin was not able to pull biotinylated mRNA bound by ATXN2L out from the cell lysates whereas with the control condition, they detected ATXN2L binding to the biotinylated mRNA. This suggests that mRNA and ATXN2La indeed interact with each other, and the conserved UCAUC sequence is the critical "landing pad" for ATXN2L, likely mediated by the its LSm and LSmAD domains.

Then researchers also discovered other evidences that indicated ATXN2L's participation in viral protein production, including: the absence of ATXN2L blocked the enlargement of viral factories ( appears to be punctate), which occurs when more viral proteins are contained. In the image below, KO refers to the condition where ATXN2L is knocked out. ATXN2L is also found to associate with the translation complex (polysome), including 80S ribosome as well as translation initiation factors elF4G1 and elf4G3.

Finally and most importantly, the researchers asked if ATXN2L indeed facilitates translation of mRNA WITHOUT poly-A tail. They created two mRNA that can glow when translated (NanoLuciferase translation reporter construct), one with the poly-A tail and the other without, so that they can compare the translation level through detecting luminescence exhibited. They found that the translation level of mRNA with poly-A tail is maintained regardless of the presence of ATXN2L but the translation of mRNA without the tail is impaired significantly by the absence of ATXN2L. Eventually, the researchers arrived at the conclusion that ATXN2L is the protein that's being 'kidnapped' by reovirus to complete their translation as it's doing its normal work in managing cell stress as a SG-associated gene. 

"What is the point of walking me through this extremely long research journey?" you might think. First of all, how RNA viruses translate their mRNA without a poly-A tail has long been an unresolved puzzle. This research helps us to find the molecular bridge that connects the viral factories and mRNAs as well as the probable contact sites between mRNA and ATXN2L. The researchers presented to us a very rigorous and comprehensive way of identifying the protein that's playing the major role in viral translation, from initial screening based on certain criteria and confirming its interacting partner proteins as well as its "jobs" in viral replication from different angles. Additionally, now that the important role of ATXN2L in viral translation is established, it can be used as the potential target of antivirals. If we can develop a drug that blocks either the interaction of ATXN2L with µNS or viral mRNA, the reoviruses will be deprived of their ability to produce viral proteins in our cells. Taking even another step further, since reoviruses are known for their oncolytic potential (preferentially replicate in malignant tumor cells), engineering the interactions among ATXN2L, viral factories and mRNA can also increase the efficiency of delivering therapeutic agents into the cancer cells.  Conceivable next steps of this study can be testing the role of ATXN2L in viral yield in different cell types and families of viruses as well as investigating the specific mechanisms of how ATXN2L facilitate translation, such as circularization of mRNA and reloading the ribosome.

Bibliography:

Primary research article:

Somoulay, Xayathed, et al. “Ataxin-2-like Promotes Translation of Nonpolyadenylated Reovirus MRNA.” Nature Communications, vol. 17, no. 1, 17 Dec. 2025, www.nature.com/articles/s41467-025-67547-1, https://doi.org/10.1038/s41467-025-67547-1. Accessed 21 Apr. 2026.

Other Citations:

DouvilleRenée N., et al. “Reovirus Serotypes Elicit Distinctive Patterns of Recall

Immunity in Humans.” Journal of Virology, vol. 82, no. 15, Aug. 2008, pp. 7515–7523,

https://doi.org/10.1128/jvi.00464-08. Accessed 4 May 2022.

Lemay, Guy, and Simon Boudreault. “The Reovirus μ2 Protein, an Enigmatic

Multifunctional Protein with Numerous Secrets yet to Be Uncovered.” Virology, vol.

601, Jan. 2025, p. 110275, https://doi.org/10.1016/j.virol.2024.110275. Accessed 10

June 2025.

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