James Weger-Lucarelli, an associate professor in the Department of Biomedical Sciences and Pathobiology at the Virginia-Maryland College of Veterinary Medicine, received a five-year, $2.01 million grant from the National Institute of General Medical Sciences in July. The funding will support his investigation into how virus-infected cells live and die, and what chemical messages they send to their neighbors in the process. The research could eventually inform the development of better antiviral drugs and more effective virus-based cancer therapies.

The award is an R35 Maximizing Investigators' Research Award, or MIRA, a funding mechanism that differs fundamentally from the standard research grant. Rather than supporting a single, narrowly defined hypothesis, a MIRA funds an investigator's entire research program over a long horizon. The National Institute of General Medical Sciences, the institute within the National Institutes of Health charged with supporting basic research that underlies all of medicine, reserves these awards for scientists it considers worth backing long-term, giving them latitude to pursue unexpected findings wherever they lead.

"An R01 is more like you're saying exactly what you're going to do, almost experiment by experiment," Weger-Lucarelli said. "This is more conceptual. You have a little more freedom to be exploratory, more innovative."

The distinction is more than administrative. Under a conventional R01 award, researchers commit in advance to a specific set of aims and experiments, and significant deviations typically require written approval from the funding agency. That structure works well for projects where the science is predictable, but it can penalize investigators whose most interesting results are the ones they did not anticipate. By contrast, the MIRA mechanism was designed around the recognition that scientific progress often comes from following surprise rather than plan. For an early-stage program like Weger-Lucarelli's, the difference can determine whether an unexpected observation becomes a dead end noted in a report or the seed of an entirely new research direction.

That flexibility matters for the kind of questions his lab is asking. When a virus infects a cell, the cell can trigger its own shutdown, a form of programmed self-destruction that halts the virus before it can replicate and escape to infect other cells. This is one of the most ancient defenses in biology, a form of cellular altruism in which the individual sacrifices itself to protect the collective. Viruses, in turn, have evolved countermeasures to interfere with that decision, deploying viral proteins that block the cell's death machinery or disable the alarm systems that would otherwise alert the immune system, keeping their host cells alive long enough to produce new viral particles.

And when an infected cell does die, it can release chemical signals into the surrounding tissue — a kind of biological warning broadcast that either rallies the immune response to contain the infection or, if a virus has found a way to redirect those signals, accelerates the spread instead. Dying cells can release inflammatory molecules that recruit immune cells to the site of infection, or they can release signals that dampen inflammation, depending on how they die and what the virus permits them to release. The distinction can mean the difference between a localized, quickly controlled infection and a systemic one.

"The cell is trying to stop the virus, and the virus is trying to stop the host," Weger-Lucarelli said. "It's kind of like a tennis match between the two."

The metaphor captures an arms race that has been running for hundreds of millions of years. Every virus that successfully infects humans today has already survived countless rounds of selection in which cells that died promptly eliminated viral lineages, and viruses that kept cells alive spread more widely. What remains in circulation is a snapshot of that ongoing negotiation, and the molecular details of it remain surprisingly poorly mapped, particularly in tissue environments that resemble the body rather than the laboratory.

Understanding that back-and-forth requires studying it in conditions that approximate a real body, and a central thrust of the funded work involves moving experiments out of flat laboratory dishes and into three-dimensional tissue models. In these 3D systems, multiple cell types can interact with one another the way they do in living tissue, rather than in the artificial uniformity of a standard cell culture. In a conventional dish, a single cell type grows in a thin layer, bathed in nutrient-rich medium, with unlimited access to oxygen and no structural architecture. In three-dimensional models, cells pack together, exchange nutrients through diffusion gradients, and communicate across cell-type boundaries much as they do in an organ.

The difference, it turns out, is not subtle. In Weger-Lucarelli's laboratory, viruses kill cells in conventional two-dimensional culture within two days. In three-dimensional models, the very same cells survive a week or more under otherwise identical conditions.

"If we were doing it in 2D, we would have missed that entirely," he said.

That observation carries implications well beyond his own lab. Cell culture in flat dishes has been a cornerstone of virology for decades, and countless antiviral strategies have been evaluated on the assumption that what happens in a dish reflects what happens in tissue. If infected cells behave so differently when embedded in a three-dimensional architecture — surviving several times longer, signaling to a more diverse cast of neighboring cells — then some conclusions drawn from traditional cultures may need re-examination. The extended survival of infected cells in 3D models suggests that the live-or-die decision Weger-Lucarelli studies may unfold on a different timeline, and through different molecular conversations, than the field has generally appreciated.

It also suggests a possible explanation for clinical observations that have long puzzled virologists, such as why some infections linger in tissues for extended periods or why antiviral drugs that perform well in cell culture sometimes underperform in patients. A drug tested against cells that die within days in a dish may look highly effective against a process that, in a real tissue, plays out over weeks. Better models of the infected tissue environment could help close that translational gap, reducing the attrition that has historically plagued antiviral development.

The grant also strengthens an existing collaboration with Samy Lamouille at the Fralin Biomedical Research Institute at VTC focused on oncolytic viruses — viruses engineered to seek out and kill tumor cells — as a potential treatment for glioblastoma, the aggressive brain cancer that remains one of the most difficult malignancies to treat. Glioblastoma is the most common malignant brain tumor in adults, and despite decades of research, median survival after diagnosis remains measured in months rather than years. The tumor's characteristic diffuse infiltration into surrounding brain tissue makes complete surgical removal nearly impossible, and the blood-brain barrier complicates drug delivery, leaving a pressing need for fundamentally different therapeutic approaches.

The logic of oncolytic therapy inverts the usual virological arms race: instead of trying to help cells survive infection, researchers deploy a virus designed to push cancer cells decisively toward death. Many cancer cells, including glioblastoma cells, have acquired defects in their antiviral defenses precisely because those defenses can otherwise trigger cell death — a vulnerability that oncolytic viruses are engineered to exploit. But the strategy is only as good as the cell-death and signaling programs the virus activates. A tumor cell killed quietly may release signals that suppress the immune response, while one killed in a way that broadcasts alarm may provoke a broader immune attack on the tumor. Understanding how infected cells decide their own fate, and what they signal when they die, is directly relevant to making such therapies work more reliably.

Positioned within a veterinary college gives the glioblastoma work an unusual advantage. Dogs are among the few animals that develop glioblastoma naturally, and their tumors develop in ways that closely resemble the human disease. That creates a clinical testing pathway that would be far harder to access from a conventional research setting. A laboratory mouse implanted with a human tumor offers a controlled but artificial system; a pet dog presenting at a veterinary hospital with a spontaneously arising brain tumor brings the full complexity of the disease — its genetic heterogeneity, its interaction with a functioning immune system, its growth in a real brain of real size — into the research program.

"There's a huge advantage of being at a vet school," Weger-Lucarelli said, "especially with brain cancer. Dogs have naturally occurring brain cancer that develops very similar to humans. Much easier than going into human clinical trials."

Comparative oncology of this kind has gained traction in recent years precisely because spontaneous tumors in pets recapitulate the biological complexity of human cancers — the heterogeneous cell populations, the three-dimensional tissue architecture, the immune environment — that laboratory models often strip away. Veterinary clinical trials can enroll more patients than early-phase human trials, proceed more quickly through regulatory pathways, and generate data on dosing, toxicity, and response in an outbred population that is far more representative of human patients than inbred laboratory mice. Findings from canine patients can inform the design of eventual human trials, while the dogs themselves may benefit from experimental treatments that would otherwise be unavailable.

The fit between the two halves of the program is deliberate. The same fundamental question — how a cell responds to viral infection, and what message that response sends — governs whether an antiviral defense succeeds in normal tissue and whether an oncolytic virus succeeds in a tumor. Insights from the 3D tissue models can feed directly into the glioblastoma collaboration, and observations from canine patients can point the fundamental research toward the biology that matters most in disease.

The five-year funding window also shapes the human side of the laboratory. Weger-Lucarelli is currently recruiting a postdoctoral researcher and plans to work at the bench alongside whoever joins the lab, rather than managing from a distance. The long grant cycle allows him to recruit and retain graduate students and postdocs without the uncertainty that accompanies shorter, annually renewed funding, giving early-career scientists time to pursue questions that may take years to answer fully. In a research environment where many trainees live grant cycle to grant cycle, that stability is itself a scientific resource.

Traditionally, the CRISPR-Cas mechanism functions much like a GPS navigation system: an RNA guide serves as the address directing the Cas enzyme “vehicle” to a specified DNA target. This principle underlies several well-known diagnostic platforms, such as SHERLOCK and DETECTR, which rely on RNA guides to achieve high specificity in DNA targeting. However, the HKUST team inverted this paradigm by engineering a DNA-guided Cas12a system, conferring the unprecedented ability to target and cleave RNA with programmable precision.

The cornerstone of this innovation is the design of a synthetic CRISPR DNA guide (crDNA) that effectively reprograms the Cas12a protein. Unlike the natural system where PAM sequences simultaneously serve as the molecular “activation” signal and informational address, the researchers cleverly decoupled these functions. By constructing a short DNA strand mimicking the PAM-containing duplex, they created a functional deoxyribonucleoprotein complex capable of recognizing any selected RNA molecule for targeted cleavage. This strategic decoupling allowed for a novel mode of RNA recognition previously unseen in CRISPR biology.

To validate this revolutionary concept, the team utilized an integrative approach combining AlphaFold-guided modeling, molecular dynamics simulations, and high-resolution cryo-electron microscopy (cryo-EM). The cryo-EM structural data, collected under the supervision of Prof. Zhai Yuanliang and Dr. Lam Wai-Hei, revealed atomic-level confirmation of the synthetic DNA guide interacting within the Cas12a complex, closely matching computational predictions. This synergy of AI-driven structural prediction and empirical validation underscores the importance of cutting-edge computational tools in accelerating discoveries in gene editing.

One of the defining advantages of substituting RNA guides with DNA lies in the dramatic increase in molecular stability. DNA is inherently chemically more stable than RNA, mitigating the fragile nature of RNA molecules which often necessitate stringent cold-chain requirements for storage and handling. This robustness facilitates more cost-effective, portable, and accessible diagnostic platforms that function reliably at ambient temperatures, making them particularly suitable for deployment in resource-limited settings such as remote clinics and border entry points.

Cost considerations further accentuate the impact of this development. Synthetic DNA guides are significantly less expensive to manufacture due to simpler chemical synthesis protocols and the elimination of cold-chain logistics. While a formal cost-comparison study remains pending, it is widely recognized within biotechnology that RNA molecules require additional chemical protection steps during synthesis, contributing to higher manufacturing costs. The DNA-guided system stands poised to democratize molecular diagnostics by making these tools more affordable and scalable worldwide.

Precision and safety are also markedly enhanced with the DNA-guided CRISPR-Cas12a system. Beyond the enhanced stability, the platform can detect and discriminate single-nucleotide polymorphisms in RNA targets, achieving a level of specificity that RNA interference (RNAi) technologies often fail to match. Moreover, preliminary cellular studies indicate significantly reduced off-target RNA cleavage compared to RNA-targeting CRISPR tools such as Cas13, suggesting a safer profile for future therapeutic applications where minimizing collateral effects is critical.

Importantly, this novel CRISPR configuration transcends the limitations of conventional RNA interference, which primarily targets protein-coding mRNAs. The DNA-guided Cas12a system can be programmed to target a broad spectrum of RNA molecules, including non-coding RNAs such as microRNAs and long non-coding RNAs. These molecules play vital roles in gene regulation and disease pathology, expanding potential applications of this platform to areas previously considered challenging for RNA-targeting technologies.

The team demonstrated the exceptional sensitivity of their system by applying the SLEUTH platform—their name for “Specific Locus Evaluation Utilizing Targeted Hydrolysis”—to clinical samples of SARS-CoV-2. They achieved attomolar-range detection sensitivity for both RNA and DNA targets under diverse conditions, validating the platform’s robustness. This positions SLEUTH as a promising point-of-care diagnostic tool capable of facilitating rapid viral detection without relying on expensive infrastructure or cold-chain preservation.

Beyond diagnostics, the technology heralds significant potential for next-generation antiviral therapies. Given that many pathogenic viruses, including influenza, SARS, and COVID-19, utilize RNA genomes or RNA intermediates during replication, the ability to selectively target and cleave such RNA species offers a platform for innovative antiviral interventions. Such therapies could revolutionize responses to future pandemics, providing precise molecular weapons against viral pathogens.

Looking forward, the research team, including PhD candidate Wu Xiaolong, envisions extending this DNA-guided CRISPR platform’s utility to a broader suite of RNA-based diagnostics and therapeutics. The lab is actively pursuing expansion of the SLEUTH platform to detect additional respiratory viruses and exploring applications in liquid biopsy to identify circulating RNA biomarkers associated with cancers. Additionally, HKUST has filed provisional patents in the United States to secure intellectual property rights for this transformative technology.

This groundbreaking work aligns strategically with HKUST’s recent establishment of a School of Medicine and the institution’s accelerated commitment to translational medicine and RNA-based therapeutic development. By harnessing structural biology, AI-driven design, and bioengineering, the team showcases a cutting-edge approach to gene editing that stands to reshape the biomedical landscape profoundly.

In summary, the introduction of a DNA-guided RNA-targeting CRISPR-Cas12a system represents a paradigm shift in the CRISPR field. It offers a new molecular toolkit blending enhanced stability, precision, safety, and cost-effectiveness with broad applicability across diagnostics, therapeutics, and research. As this technology advances toward clinical translation, it promises to open transformative avenues for precise RNA manipulation in both health and disease.

Subject of Research:
Not applicable

Article Title:
DNA-guided CRISPR–Cas12a effectors for programmable RNA recognition and cleavage

News Publication Date:
1-May-2026

Web References:
https://www.nature.com/articles/s41587-026-03120-5

References:
10.1038/s41587-026-03120-5

Image Credits:
HKUST

Keywords

CRISPR, Cas12a, DNA-guided CRISPR, RNA targeting, gene editing, molecular diagnostics, antiviral therapy, synthetic guide RNA, SLEUTH platform, cryo-EM, AlphaFold, RNA cleavage

Viruses are renowned for their ability to exert profound impacts on their hosts with minimalist genomes. The rabies virus, for instance, encodes only five proteins, a stark contrast to the roughly 20,000 proteins produced by human cells. This disparity has long puzzled virologists: how can so few proteins orchestrate the takeover of complex cellular processes? The new research identifies dynamic conformational changes and RNA-binding capabilities as key strategies that enable viral proteins to function with extraordinary versatility.

Central to this discovery is the observation that the P protein can adopt multiple distinct shapes, or conformations, enabling it to interact with various cellular components in different contexts. This structural plasticity defies the traditional modular view of proteins as linear assemblies of domains, each with fixed functions. Instead, the rabies P protein’s domains exhibit context-dependent folding and interaction patterns, leading to emergent properties such as RNA binding, which had not been fully appreciated before.

RNA molecules within cells are not mere passive carriers of genetic information; they engage in intricate networks that regulate gene expression, immune responses, and the assembly of cellular machinery. The study reveals that the P protein’s ability to bind RNA is a critical factor underpinning its multifunctionality. By attaching to RNA, the protein can infiltrate and exploit membrane-less organelles—liquid-like compartments formed through phase separation—that coordinate essential cellular activities.

Phase separation, a physical phenomenon where biomolecules demix to form concentrated droplets within the cytoplasm or nucleus, is emerging as a fundamental organizing principle in cell biology. The rabies P protein’s capacity to toggle between different physical phases allows it to enter these specialized compartments, such as nucleoli, and manipulate cellular processes including protein synthesis, intracellular signaling, and immune evasion. This ability essentially transforms the infected cell into a highly efficient virus-producing factory.

Microscopic imaging using confocal microscopy has vividly demonstrated these interactions in human cells. The P protein forms liquid-like droplets inside the nucleus, localizing to nucleoli—key hubs of ribosome biogenesis—and associates with microtubules, the structural scaffold of the cell. Such spatial and functional targeting exemplifies the virus’s strategy to exploit existing cellular infrastructure for viral replication and assembly.

Beyond rabies, the findings have significant implications for other high-priority pathogens like Nipah and Ebola viruses. These pathogens also encode relatively few proteins but exhibit broad cellular control and immune modulation. It is plausible that they too leverage conformational adaptability and RNA-binding to hijack host cellular systems. Understanding these shared viral strategies could unlock broad-spectrum antiviral approaches that disrupt this functional versatility.

The study also challenges prevailing conceptual frameworks in virology, which often liken multifunctional viral proteins to train carriages—distinct modules each responsible for a single task. This research posits a more dynamic model in which protein shape-shifting and intra-domain interactions generate a repertoire of functions from a single polypeptide chain, highlighting a sophisticated biophysical and biochemical strategy.

This deeper understanding of how viral proteins manipulate the physical chemistry of the host cell environment opens new avenues for drug development. Targeting the conformational dynamics or RNA-binding interfaces of viral proteins may yield therapies that incapacitate their multifunctionality, thereby hampering viral replication and pathogenesis. This approach could complement existing antiviral strategies, which mostly focus on viral enzymes or entry mechanisms.

The multidisciplinary study brought together expertise from molecular virology, structural biology, and biophysics, leveraging cutting-edge techniques such as live-cell imaging, biophysical assays, and advanced microscopy. Collaborators included the Australian Synchrotron and several research institutions across Australia, underscoring the collaborative nature of this discovery.

Ultimately, this research elevates our comprehension of viral protein multifunctionality and illustrates a paradigm shift in how we conceptualize viral infection mechanisms. By revealing the interplay between protein conformation, RNA binding, and phase separation, it not only expands the fundamental biological understanding but also sets the stage for translational research aimed at combating some of the world’s deadliest viruses.

The insights gleaned from this work underscore the sophistication of viral evolution and the elegant simplicity with which viruses exploit cellular systems. Future studies will likely explore whether the principles uncovered here apply more broadly across viral families and how they might be targeted therapeutically to prevent or mitigate viral diseases.

Subject of Research: Cells

Article Title: Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein

News Publication Date: 5-Nov-2025

Web References:
https://www.nature.com/articles/s41467-025-65223-y
http://doi.org/10.1038/s41467-025-65223-y

References:
Rawlinson, S., Moseley, G., Gooley, P., et al. (2025). Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein. Nature Communications.

Image Credits:
Stephen Rawlinson, Monash University

Keywords: Human health, Diseases and disorders

Viruses, fundamentally simple yet cunning pathogens, rely heavily on their hosts’ cellular machinery to propagate. Because viral genomes are minimalistic, encoding only essential components, viruses must hijack host cellular processes to replicate and spread. One key evolutionary adaptation in herpesviruses involves the molecular mimicry of host kinases—enzymes that regulate the cell cycle and numerous signaling pathways through phosphorylation, a reversible chemical modification that switches protein activities on or off.

Cyclin-dependent kinases (CDKs) are central regulators of the eukaryotic cell cycle. They consist of two lobes (N- and C-lobes) with distinct structural and functional features. Phosphorylation at conserved serine, threonine, or tyrosine residues within the N-lobe modulates kinase activity, thus dictating cellular progression through specific cell cycle phases. Interestingly, certain herpesvirus protein kinases, termed conserved herpesvirus protein kinases (CHPKs), structurally mimic cellular CDKs, suggesting an evolutionary strategy to subvert host cell cycle controls.

Herpes simplex virus type 2 (HSV-2) is notorious for causing genital infections, meningitis, and severe neonatal diseases. It establishes lifelong latency in sensory neurons with intermittent reactivation episodes. Understanding the molecular underpinnings of HSV-2’s latency and reactivation cycles is pivotal for devising enduring antiviral interventions. The Japanese team, led by Professor Yasushi Kawaguchi and Assistant Professor Naoto Koyanagi, embarked on elucidating how HSV-2’s UL13 kinase—a CHPK—emulates CDK functionality and regulation via phosphorylation.

Employing cutting-edge molecular virology techniques, the researchers demonstrated that UL13 kinase undergoes phosphorylation at a conserved tyrosine residue (Tyr-162) within its N-lobe motif. This post-translational modification negatively regulates UL13’s catalytic activity. Experimental infections with wild-type HSV-2, UL13-deleted mutants, and a phosphorylation-deficient UL13-Y162F mutant revealed that phosphorylated UL13 is prevalent during later stages of viral replication, emphasizing a temporal regulatory role.

Notably, phosphomimetic mutations of this tyrosine residue attenuated kinase activity by diminishing phosphorylation of UL13 substrates. This fine-tuning effect was conserved across other herpesvirus subfamilies, underscoring a shared evolutionary mechanism of CDK mimicry and regulation. Functional assays showed that regulated phosphorylation modulates viral replication and pathogenicity during the lytic phase, particularly influencing viral virulence in murine brain infection models.

Intriguingly, while phosphorylation of UL13’s tyrosine residue suppressed acute viral replication, it was indispensable for viral reactivation from latency in guinea pigs. This duality suggests that UL13-mediated CDK mimicry orchestrates the delicate balance herpesviruses maintain between active lytic infection and latent persistence, optimizing viral survival and transmission over the host’s lifetime.

Phosphorylation-mediated regulation of viral kinases reflects a sophisticated layer of viral control, separate from but analogous to host cellular systems. This viral CDK mimicry does not merely copy enzymatic function but also incorporates intricate feedback loops via post-translational modifications, allowing herpesviruses to adapt dynamically to the intracellular environment and immune pressures.

The conservation of this motif and regulatory mechanism among diverse herpesviruses points to an ancient and successful evolutionary strategy. Moreover, the detection of similar conserved tyrosine phosphorylation motifs in viral kinases encoded by poxviruses suggests that this form of regulatory mimicry may extend beyond herpesviruses, revealing a broader paradigm in viral-host molecular interactions.

Professor Kawaguchi highlights the significance of these findings, emphasizing that uncovering the regulatory complexity of CHPK kinases not only advances the fundamental understanding of herpesvirus biology but also opens avenues for targeted antiviral drug design aimed at disrupting kinase regulation. Such approaches could interfere with viral replication dynamics without harming host kinase functions, offering precision therapeutic options.

Beyond immediate translational impacts, this work underscores the utility of viruses as biological probes. By decoding viral strategies such as CDK mimicry, researchers glean unique insights into cellular regulatory networks that are otherwise challenging to study. This reciprocal illumination enriches both virology and cell biology, fostering novel research trajectories and integrative biomedical innovations.

As herpesviruses continue to pose global health challenges through recurrent infections and associated diseases, deepening our comprehension of their molecular arsenal remains paramount. The delicate interplay of phosphorylation and kinase mimicry delineated in this study exemplifies the evolutionary ingenuity of viruses and represents a critical step toward disrupting their lifecycle through next-generation therapeutics.

Subject of Research: Cells

Article Title: Regulatory Mimicry of Cyclin-Dependent Kinases by a Conserved Herpesvirus Protein Kinase

News Publication Date: 16-Apr-2025

Web References:
https://www.pnas.org/doi/10.1073/pnas.2500264122

References:
Koyanagi, N., Hengphasatporn, K., Kato, A., Nobe, M., Takeshima, K., Maruzuru, Y., Maenaka, K., Shigeta, Y., & Kawaguchi, Y. (2025). Regulatory Mimicry of Cyclin-Dependent Kinases by a Conserved Herpesvirus Protein Kinase. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.2500264122

Image Credits:
Prof. Yasushi Kawaguchi from The University of Tokyo, Japan

Keywords:
Viruses, Herpesviruses, Enzymes, Kinases, Molecular Biology, Immunology, Cellular Processes, Phosphorylation, Viral Infections