Blood Pressure Drug Diltiazem Blocks Rabies Virus Entry and Saves Infected Mice

Rabies remains one of the deadliest infectious diseases known to medicine. Once the virus reaches the central nervous system and clinical symptoms appear, mortality approaches 100 percent, and the World Health Organization estimates that roughly 59,000 people die of the disease each year, with nearly half of the victims children under the age of fifteen. Although effective vaccines and rabies immunoglobulins exist, their cost and chronic global supply shortages leave many people in rural Asia and Africa dangerously unprotected after exposure to a rabid animal. A new study published in PLOS Pathogens now offers a strikingly practical way forward: researchers report that diltiazem, a cheap and widely used FDA-approved blood pressure medication, blocks the entry of rabies virus into cells and rescued approximately 70 percent of mice from a lethal infection.

The research, led by Ziruo Sun, Jinliang Wang, Zhigao Bu and colleagues at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, began with a systematic search for the host machinery that rabies virus depends on to invade cells. In a previous genome-wide screen, the team had used 64,755 small interfering RNAs targeting 21,585 human genes in HEK293 cells to identify host factors required for rabies virus replication. Among the hits were sixteen genes related to potassium channels, and the strongest effect came from silencing KCNMA1, the gene that encodes the pore-forming subunit of the large conductance calcium- and voltage-activated potassium channel known as KCa1.1, a seven-transmembrane protein sitting in the plasma membrane.

To confirm that this channel genuinely matters for infection, the researchers knocked down KCa1.1 expression in both mouse neuroblastoma N2a cells and human HEK293 cells using specific siRNAs. Quantitative PCR verified that the channel’s mRNA was substantially reduced, and when the silenced cells were infected with a recombinant rabies virus strain expressing enhanced green fluorescent protein, the amount of infectious virus released into the culture supernatant dropped significantly at both 24 and 48 hours after infection. The team then turned to paxilline, a selective chemical inhibitor of KCa1.1 that binds the inner pore of the channel and blocks potassium conductance. In N2a, HEK293, and human SK-N-SH neuroblastoma cells, paxilline had no effect on cell viability but suppressed rabies virus replication in a clear dose-dependent manner, and time-course experiments confirmed the inhibition persisted across the tested period.

Because KCa1.1 is expressed primarily at the plasma membrane, the investigators hypothesized that it might act during viral entry rather than during later stages of the viral life cycle. Entry involves two separable steps: attachment of virus particles to the cell surface and their subsequent internalization. By incubating cells with virus at 4 degrees Celsius, a temperature that permits binding but prevents internalization, and then quantifying attached viral RNA, the team showed that neither KCa1.1 silencing nor paxilline treatment changed how much virus bound to cells. When the temperature was shifted to 37 degrees to allow entry, however, and surface-bound virus was stripped away with trypsin before analysis, the amount of viral RNA inside treated or silenced cells fell dramatically. A complementary microscopy assay, which labeled viral glycoprotein under conditions that distinguish particles remaining on the surface from those that had entered, showed that paxilline-treated cells accumulated more virus on their surface while their total particle count stayed the same. Together, these experiments demonstrated that activated KCa1.1 is specifically required for the internalization step of rabies virus entry.

A co-immunoprecipitation assay revealed that KCa1.1 does not physically interact with the rabies virus glycoprotein, the viral surface protein that drives attachment and clathrin-mediated endocytosis. That result pointed to an intermediary. Prior work had shown that KCa1.1 assembles into macromolecular complexes with voltage-gated calcium channels, including Cav1.2, and that calcium influx through these channels can activate KCa1.1. Notably, Cav1.2 was the only calcium channel that emerged from the team’s earlier genome-wide screen. Co-immunoprecipitation confirmed a specific interaction between Cav1.2 and KCa1.1, and silencing Cav1.2 in N2a and HEK293 cells significantly reduced rabies virus titers, establishing that the L-type calcium channel is also required for productive infection.

The mechanistic picture became sharper when the researchers examined the role of the viral glycoprotein itself. Co-immunoprecipitation showed that Cav1.2 specifically interacts with the rabies virus glycoprotein, and a pull-down assay using purified virus particles demonstrated that Cav1.2 captures rabies virions in a dose-dependent manner. Binding assays revealed that Cav1.2 silencing or blockade did not reduce how much virus attached to cells, but internalization assays showed far less viral RNA inside treated cells. In other words, the glycoprotein engages Cav1.2, and this engagement is required to activate the channel and trigger the internalization machinery, even though the virus still docks normally on the cell surface when the channel is disabled.

The team also identified the downstream cellular process that the channel axis manipulates. Rabies virus enters cells through clathrin-mediated endocytosis with the help of actin, and previous work had shown that KCa1.1 promotes influenza virus entry by regulating F-actin polymerization. Using phalloidin staining to visualize filamentous actin, the researchers found that rabies virus entry induces F-actin polymerization in HEK293 cells, and that this polymerization was markedly reduced when Cav1.2 or KCa1.1 were knocked down or when cells were treated with diltiazem or paxilline. The authors therefore propose a sequential model: rabies virus glycoprotein binds Cav1.2 and triggers its activation; activated Cav1.2 then interacts with and activates KCa1.1; and activated KCa1.1 drives F-actin polymerization that powers the internalization of the virus particle.

The therapeutic implications rest on diltiazem, a benzothiazepine-class L-type calcium channel blocker approved in the United States in 1982 and long prescribed for hypertension, angina, and atrial arrhythmias. In cell culture, diltiazem blocked rabies virus infection in a dose-dependent manner without affecting viability, and four other FDA-approved Cav1.2 antagonists, the phenylalkylamine verapamil and the dihydropyridines nifedipine, nicardipine, and nimodipine, also inhibited the virus in vitro. The decisive test came in mice. Six-week-old C57BL/6J mice were inoculated intramuscularly with ten 50 percent mouse lethal doses of the rabies street virus GX/09, and six hours later they began receiving daily intramuscular injections of diltiazem at 25 or 50 milligrams per kilogram, or a phosphate buffer control. All 19 control mice died within 9 to 16 days with typical rabies signs such as body tremors and hind limb paralysis. At the lower dose, 5 of 19 mice survived, roughly 26 percent, while at 50 milligrams per kilogram, 13 of 19 mice survived, approximately 70 percent, and the survivors remained completely asymptomatic throughout a 28-day observation period.

Measurements of viral burden supported the idea that diltiazem works by blocking entry before the virus reaches the brain. No viral RNA was detectable in the brains of treated or control mice at day 4, but by day 6, viral RNA was present in all six control animals examined and in only two of six diltiazem-treated animals, and immunohistochemistry at day 8 detected rabies antigen in all control brains but only one of three treated brains. When the researchers delayed diltiazem administration until days 6 and 7, after viral replication was already underway in the brain, they observed no significant reduction in brain viral RNA, indicating that the drug’s protection is unlikely to stem from direct suppression of established central nervous system infection. Notably, the 50 milligram per kilogram mouse dose corresponds, after body surface area normalization under FDA guidelines, to a human equivalent dose of roughly 4 milligrams per kilogram, or about 240 milligrams per day for a 60-kilogram adult, which falls squarely within the range already used clinically for cardiovascular indications.

The authors argue that diltiazem could serve as an affordable, mass-produced complement to existing post-exposure prophylaxis, offering a stopgap where rabies immunoglobulin is unavailable and a potential enhancement where it is not, since it is estimated that only 1 to 10 percent of category III exposure patients currently receive the recommended treatment. The findings also extend a growing body of evidence that ion channels are conserved entry factors for diverse viruses: Cav1.2 has been implicated in SARS-CoV-2 and influenza virus entry, KCa1.1 in influenza virus endocytosis, and diltiazem has shown activity against rotavirus, porcine reproductive and respiratory syndrome virus, and porcine deltacoronavirus. With no approved drugs currently targeting KCa1.1, the potassium channel remains a target for future drug development, while Cav1.2, already served by three structural classes of clinically validated antagonists, stands out as a promising foundation for broad-spectrum antiviral strategies. Further studies will be needed to evaluate the in vivo efficacy of the other calcium channel blockers and to determine whether diltiazem concentrations in the central nervous system reach antiviral levels, but the prospect of repurposing a decades-old cardiovascular pill against one of humanity’s most lethal pathogens gives the global effort to eliminate rabies deaths a genuinely new weapon to test.

Subject of Research: Repurposing the calcium channel blocker diltiazem to block rabies virus entry via the Cav1.2-KCa1.1 ion channel axis

Article Title: Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection

Article References: Sun, Z., Wang, J., Wen, Z., Shuai, L., Sun, W., Yang, M., Wang, J., Chen, J., Mi, T., Zhou, X., Zhou, P., Lv, N., Zhao, Z., Ge, J., Chen, W., Wang, X., Wang, C., Bu, Z., & Wang, J. (2026). Targeting the Cav1.2-KCa1.1 axis prevents rabies virus internalization and protects against lethal infection. PLOS Pathogens, 22(10), e1014675. https://doi.org/10.1371/journal.ppat.1014675

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014675

Keywords: rabies virus, diltiazem, Cav1.2, KCa1.1, ion channels, viral entry, endocytosis, drug repurposing, F-actin polymerization, post-exposure prophylaxis, PLOS Pathogens, antiviral therapy