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European Journal of Medical Research logoLink to European Journal of Medical Research
. 2026 Jan 23;31:309. doi: 10.1186/s40001-025-03785-9

TMPRSS2 is dispensable for lethal SARS-CoV-2 BA.5 infection in humanized hACE2 golden hamsters

Bowen Wang 1,#, Jinwei Zhang 1,#, Yuhan Li 2,#, Haodong Wang 1, Mei Dong 1, Qiu Chen 1, Aimin Shi 1, Aihua Zhang 1,✉, Wentao Zeng 1,✉, Xuefeng Wang 1,2,✉, Jianmin Li 1,✉
PMCID: PMC12911335  PMID: 41578405

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) can specifically bind to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface via the corresponding spike (S) protein, facilitating the entry of the virus. ACE2 is highly expressed in alveolar type II cells, respiratory epithelial cells, and various other tissues, which provides favorable conditions for the transmission of SARS-CoV-2. TMPRSS2, a transmembrane serine protease widely distributed in the respiratory tract and digestive tract, cleaves the receptor-binding domain (RBD) and S1/S2 subunit of the SARS-CoV-2 spike protein. The protein cleavage activates the virus and enhances its binding to ACE2. This study reported for the first time that Tmprss2 deficiency reduces susceptibility to SARS-CoV-2 in golden hamsters. Infection experiments were conducted with three SARS-CoV-2 variants (Beta, BA.5, and XBB). The findings revealed that compared to the wild-type control, TMPRSS2 knockout in hamsters resulted in lower viral titers in the lungs and reduced pathological damage. Our results showed that Tmprss2 KO;H11-K18-hACE2 hamsters succumbed to infection with SARS-CoV-2 (BA.5 strain) by day 5 post-inoculation, similar to H11-K18-hACE2 hamsters. These findings indicated that SARS-CoV-2-induced mortality in humanized hACE2 hamsters occurs independently of TMPRSS2.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-03785-9.

Keywords: ACE2, SARS-CoV-2 infection, TMPRSS2, Inflammation

Introduction

The novel coronavirus SARS-CoV-2 triggered a global pandemic at the end of 2019 [1–3], becoming a severe public health crisis in the twenty-first century. Like other coronaviruses, SARS-CoV-2 initiates infection by interacting with the host cell ACE2 receptor via its surface spike protein [4]. However, the spike protein requires proteolytic activation to achieve efficient infectivity [5], a process largely dependent on transmembrane serine protease 2 (TMPRSS2), which is expressed on the host cell surface [6]. TMPRSS2 cleaves the S1 and S2 subunits of the spike protein to facilitate viral envelope fusion with the host cell membrane, thereby promoting viral entry through membrane fusion [7]. Therefore, TMPRSS2 plays a key role in early viral infection and represents a primary pathway for viral entry into host cells.

The Tmprss2 gene is expressed in multiple human tissues, especially in the respiratory tract, lungs, and gastrointestinal system [8–11]. Its level of expression is correlated with the severity of the initial viral infection and the extent of pathological damage. Clinical data indicate that individuals with high Tmprss2 expression have a greater risk of experiencing severe SARS-CoV-2 symptoms [12, 13], suggesting that targeted inhibition of Tmprss2 may be a promising therapeutic strategy [7, 14].

As a classic experimental animal model, the golden hamster (Mesocricetus auratus) is considered a preferred system for studying infection because of its high susceptibility to the virus and its human-like pathological features [15–17]. Unlike mice, golden hamsters can be infected with SARS-CoV-2 without genetic mutations, and they exhibit characteristic pulmonary inflammation similar to that in humans [17]. In this study, a Tmprss2-knockout golden hamster model (Tmprss2m1) was constructed using CRISPR-Cas9 gene editing technology [18], which is a valuable tool for investigating the functions of TMPRSS2 in vivo. We evaluated the effects of Tmprss2 deficiency on viral replication, pathological damage to tissues, and the immune response to three SARS-CoV-2 variants (Beta, BA.5, and XBB) [19, 20].

Material and methods

Animal models

The golden hamsters used in this study were obtained from the Medical Experimental Animal Center of Nanjing Medical University and housed in a standard barrier environment (SPF) facility. The breeding environment and experimental conditions were as follows. The temperature ranged from 20 to 26 °C, the humidity ranged from 40 to 70%, the illumination time was 14 h (6:00–20:00), and darkness was maintained for 10 h (20:00–6:00 the following day). All animal breeding experiments were approved by the Experimental Animal Ethics Committee of Nanjing Medical University and complied with the national experimental animal standard GB/T 35892-2018, ethics code (IACUC): 2401011. The Institutional Animal Care and Use Committee of Chanchun Veterinary Research Institute (CVRI), Chinese Academy of Agricultural Sciences, approved the animal infection experimental procedures (Approval No. IACUC AMMS-11-2023-032). The experimental procedures and protocols were discussed with biosafety officers and facility managers at the CVRI. Viral infection and experiments on hamsters were conducted in an animal biosafety level 3 (ABSL-3) laboratory.

Viruses and cells

The SARS-CoV-2 variants BA.5, Beta, and XBB were passaged within Cercopithecus aethiops kidney cells (Vero E6, ATCC® CRL-1586) as described in another study [21]. All virus-related procedures were conducted in the laboratory’s certified BSL-3 isolation facilities. Routine PCR-based testing confirmed that the cultured cells were free of Mycoplasma contamination. Viral propagation and titration were performed using Vero E6 cells. Vero E6 cells were cultivated in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotic–antimycotic solution (Anti-Anti, Invitrogen, USA) at 37 °C with 5% CO2.

Golden hamster infection

Golden hamsters were anesthetized with 250 mg/kg tribromoethanol (Avertin), followed by intranasal infection with 1 × 104 TCID50 (for naive infection) SARS-CoV-2 variants (BA.5, XBB, or beta virus) contained in DMEM (50 μL each). All animals were classified into the following five groups: the mock group (n = 3), which received DMEM inoculation; the pathology progression group, which received the BA.5 virus; the XBB inoculation group (KO, each n = 3); and the control group, which received the BA.5 inoculation virus (WT, n = 3). Accidental deaths or uninfected golden hamsters were eliminated from subsequent analyses. The weights of the golden hamsters were measured, and their clinical signs were monitored every day during the experimental process. Animal euthanasia was performed 3, 5, and 10 days post-infection (DPI), and those presenting a greater than 25% reduction in body weight were humanely euthanized with isoflurane and recorded as deceased. Lung and brain tissues were harvested for further analysis.

Generation of Tmprss2 knockout golden hamsters

We generated TMPRSS2 knockout golden hamsters using the following procedure. First, sgRNAs were designed for golden hamsters with CRISPOR (http://crispor.tefor.net/) and CRISPRdirect (http://crispr.dbcls.jp/). Next, the complementary target oligonucleotides were inserted into the pUC57-T7-sgRNA-trcRNA vector via the BsaI restriction site. After PCR amplification of sequences that contained the sgRNA and T7 promoter, they were used as templates for producing sgRNAs via in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S). After treatment with lithium chloride precipitation to purify these sgRNAs, they were preserved at − 80 °C.

Donor and recipient hamsters were prepared and microinjection procedures were performed following the methods described in another study [22]. Briefly, 50 ng/µL CRISPR-Cas9 protein (IDT) and 50 ng/µL sgRNA were coinjected into two-cell embryos under a microscope equipped with a red filter in a red-light room. The injected embryos were cultured in mHECM9 medium supplemented with human serum albumin (HSA) at 37.5 °C in a humidified 10% CO2 environment for 15–30 min and subsequently transferred to the oviducts of 0.5-day pregnant recipients, with 15–30 embryos per recipient. Founder pups were mated with WT males or females to produce the F1 generation. Founder and F1 pups were genotyped using a one-step mouse genotyping kit following specific protocols (Vazyme, PD101-01).

To obtain hACE2-Tmprss2 knockout golden hamsters (K18-hACE2;Tmprss2m1/m1), we first crossed Tmprss2m1/m1 hamsters with H11-K18-hACE2 hamsters generated by our laboratory (ref) and then mated K18-hACE2;Tmprss2m1/+ hamsters with Tmprss2m1/m1 hamsters to generate K18-hACE2;Tmprss2m1/m1 golden hamsters.

Viral titer measurement in tissues

One day before utilization, Vero-E6 cells were inoculated in 24-well plates, followed by homogenization of the infected tissues with DMEM. After 1 h of cell inoculation using tissue dilutions, the inoculum was discarded, and the cells were subsequently inoculated with 0.9% methylcellulose for five days. Finally, after crystal violet staining, the plaque-forming units (PFUs) were calculated to determine the viral titers.

Histological analysis

Tissues were immersed in 4% paraformaldehyde, followed by paraffin embedding and sectioning into 3.5-μm sections to conduct hematoxylin–eosin (H&E) staining to analyze tissue histology.

Real-time RT-qPCR

The Premix Pro Taq HS qPCR Kit (Accurate Biology) was used to detect SARS-CoV-2 RNA through real-time RT-qPCR using the following primers and probes:

RT-IFNr-F (5ʹ-GGCCATCCAGAGGAGCATAG-3ʹ),RT-IFNr-R(5ʹ-TTTCTCCATGCTGCTGTTGAA-3ʹ),RT-IL1β-F(5ʹ-GGCTCCAAAAGATGAAGAGCTG-3ʹ),RT-IL1β-R (5ʹ-AGCTTCTCCACAGCCACAAT-3ʹ), RT-IL6-F (5ʹ-TGTCTTCTTGGGACTGCTGC-3ʹ), RT-IL6-R (5ʹ-CCAAACCTCCGACTTGTTGA-3ʹ), RT-IL10-F (5ʹ-GGTTGCCAAACCTTATCAGAAATG-3ʹ), RT-IL10-R (5ʹ-TTCACCTGTTCCACAGCCTTG-3ʹ), RT-TNFα-F (5ʹ-AGAATCCGGGCAGGTCTACT-3ʹ), and RT-TNFα-R (5ʹ-TATCCCGGCAGCTTGTGTTT-3ʹ). The optimal PCR amplification procedure included 5 min of predenaturation at 95 °C, 5 s of denaturation at 95 °C, and 30 s of annealing and extension at 60 °C for 40 cycles. A LightCycler 480 (Roche, Basel, Switzerland) was used for this assay. LightCycler 480 software (version 1.5.1) (Roche) was used to analyze the data.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism 9. Two-tailed Student’s t-tests and two-way ANOVA were performed to determine differences. The data are presented as the mean ± SEM. All differences among and between groups were considered to be statistically significant at p < 0.05 (*p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Results

Establishment of TMPRSS2 knockout golden hamsters

To determine the function of TMPRSS2 in golden hamsters, we used previously reported methods in the laboratory to generate Tmprss2-knockout (KO) golden hamsters. First, wild-type (WT) female golden hamsters were injected with pregnant mare serum gonadotropin (PMSG) to induce superovulation, which increased the number of embryos available for microinjection and improved the success rate of obtaining KO golden hamsters (Fig. 1A). Based on the structure of the Tmprss2 gene, exon 3 was selected as the target site, and a sgRNA was designed in which Tmprss2m1/m1 (Founder) with a two-base pair deletion was obtained by microinjecting the CRISPR component into two-cell embryos (Fig. 1B). This mutation caused frame shifts that resulted in premature stop codons in the Tmprss2 mRNA. After hybridization between heterozygotes, homozygotes were obtained, and homozygotes were mated to produce homozygotes with normal fertility. The genotype ratio of the offspring conformed to Mendelian inheritance laws.

Fig. 1.

Fig. 1

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Establishment and expression validation of Tmprss2 knockout golden hamsters. A The microinjection protocol for Tmprss2 KO golden hamsters is illustrated. B A schematic diagram of the target and protein domain changes in the Tmprss2-KO gene editing golden hamster. C. Validation of Tmprss2 gene knockout at the transcriptional level via qPCR detection. D. Electrophoretic validation results of Tmprss2 gene knockout

Quantitative PCR (qPCR) analysis revealed the relative mRNA levels of Tmprss2 in WT and Tmprss2m1/m1 golden hamsters. The mRNA level in the WT group was normalized to about 1.0 and was used as a reference, whereas the Tmprss2m1/m1 group presented near-zero expression (Fig. 1C), indicating successful knockout of the Tmprss2 gene. In the electrophoresis image, the WT sample showed distinct bands, confirming the presence of Tmprss2 transcription products, whereas no bands were found in the Tmprss2m1/m1 sample, which confirmed that the gene knockout of Tmprss2 was successful (Fig. 1D).

Experimental design of Tmprss2 SARS-CoV-2 infection

TMPRSS2 is the core molecular target of SARS-CoV-2 involved in SARS-CoV-2 infection. TMPRSS2 can cleave the receptor-binding domain (RBD) in the viral spike protein (S protein), thereby activating it and facilitating viral entry by promoting the interaction between the ACE2 receptor and host respiratory epithelial cells. To assess how TMPRSS2 gene knockout affects SARS-CoV-2 infection, we conducted intranasal infection experiments on WT and Tmprss2m1/m1 golden hamsters (Fig. 2A). This experiment included three SARS-CoV-2 variants: Beta, BA.5, and XBB. On day 0, both groups were inoculated with SARS-CoV-2. Hamster lung, brain, small intestine, and other tissues were obtained at 3 dpi and 5 dpi for histopathological examination and quantitative PCR detection of inflammatory factor (qPCR) expression. The experiment ended at 10 dpi.

Fig. 2.

Fig. 2

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Experimental scheme and pathogenicity of Tmprss2 KO golden hamsters infected with SARS-CoV-2. A. Tmprss2 KO golden hamsters were infected intranasally with different strains of the virus and killed at 3, 5, and 10 DPI, and lung tissue samples were collected. B. The percentage change from the initial weight was monitored daily for 10 days of infection with different strains in WT and Tmprss2 KO golden hamsters. C. Viral replication in infected WT and Tmprss2 KO golden hamsters at 3 dpi is presented

During the early phase of WT BA.5 infection, the body weight initially slightly decreased around day 5 and then recovered gradually. By 10 dpi, body weight increased by about 7% compared to the initial value. In contrast, BA.5-infected KO animals presented minimal fluctuations in body weight, which remained close to baseline throughout the observation period, with no notable increase or decrease by day 10. The KO group infected with the Beta variant showed transient weight gain followed by a continuous decline, resulting in a slight weight reduction by day 10 compared to the initial value. Following XBB infection, the weight of the KO animals showed a continuous downward trend, which was significantly lower than the initial value at 10 dpi, and the decrease was more significant than that in the other KO groups (Fig. 2B).

Additionally, the viral titers in the WT group were consistently greater than those in the KO groups, indicating that deletion of the Tmprss2 gene decreases the degree of viral replication in golden hamsters (Fig. 2C).

Tmprss2 KO golden hamsters infected for three days show inflammatory infiltration in the lungs

The Mock group, comprising TMPRSS2 KO hamsters without viral infection, exhibited normal morphology with intact alveolar structures and no evident inflammatory cell infiltration or tissue damage (Fig. 3A), serving as a baseline for healthy lung tissue. At 3 dpi, all golden hamsters infected with different variants presented different degrees of inflammatory infiltration (Fig. 3B–E).

Fig. 3.

Fig. 3

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Histopathological phenotypes of WT and Tmprss2 KO golden hamster lungs three days after inoculation with different SARS-CoV-2 variants. A Normal pathology of the lungs of Tmprss2 KO golden hamsters three days after intranasal inoculation with PBS (mock). B Pathology of the lungs of WT hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for three days. C Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for three days. D Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 beta variant for three days. E Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 XBB variant for three days. Scale bar: up 200 μm; down: 30 μm

In WT golden hamster viral infection with the BA.5 strain, a pronounced immune response characterized by significant infiltration of inflammatory cells and interstitial edema was observed, indicating severe lung tissue damage. This response highlighted the role of TMPRSS2 in facilitating viral entry and subsequent pathogenesis (Fig. 3B).

In contrast, TMPRSS2 KO hamsters infected with the same BA.5 strain presented considerably lower inflammatory cell infiltration (Fig. 3C), with relatively preserved alveolar architecture and minimal tissue damage. These findings suggested that the absence of TMPRSS2 decreases viral infectivity and mitigates lung inflammation.

Similarly, TMPRSS2 KO hamsters infected with the beta and XBB variants presented milder pulmonary lesions than their WT counterparts (Fig. 3D–E), with partial inflammatory cell infiltration but largely intact alveolar structures and low degrees of tissue damage.

These findings showed that TMPRSS2 expression is a critical determinant of SARS-CoV-2 infectivity and lung pathology severity in golden hamsters. TMPRSS2 deficiency is correlated with reduced viral replication and attenuated pulmonary inflammation, highlighting that TMPRSS2 may be used to treat SARS-CoV-2 infections.

To characterize the role of TMPRSS2 in the host immune response, we assessed inflammatory cytokine levels inside lung tissues in two groups of infected golden hamsters at different time points via RT-qPCR. At 3 dpi, golden hamsters lacking TMPRSS2 presented reduced levels of several inflammatory factors (IFN γ, IL-1β, IL-10, and TNFα) following infection with the BA.5 variant (Fig. 4A–E), indicating that the absence of TMPRSS2 impairs the immunological response to SARS-CoV-2. TMPRSS2-deficient hamsters presented only a slight increase in IFN-γ expression after BA.5 infection and an increase in IL-1β expression after XBB infection at 3 dpi. The level of expression of other inflammatory markers remained largely unchanged in the absence of TMPRSS2.

Fig. 4.

Fig. 4

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Expression of inflammatory factors in the lungs of WT and Tmprss2 KO golden hamsters three days after inoculation with different SARS-CoV-2 variants. A–E IFNγ, IL-1β, IL-6, IL-10, and TNFα levels in the lungs of WT and Tmprss2 KO golden hamster lungs three days after inoculation with diverse SARS-CoV-2 variants

After five days of Tmprss2 KO golden hamster infection, lung inflammation improved

At 5 dpi, all infected golden hamsters exhibited resolution of major lung lesions. In WT golden hamsters infected with the BA.5 variant, histological analysis revealed reduced inflammatory cell infiltration and preserved alveolar architecture, although residual tissue damage persisted in peribronchial regions. Similarly, Tmprss2-deficient golden hamsters infected with Beta, BA.5, or XBB variants showed decreased inflammatory infiltration in their lung tissues, despite the presence of low-grade inflammation (Fig. 5A–E). At the immunological level, minimal changes in the expression of pro-inflammatory cytokines and chemokines were found between WT and Tmprss2-deficient golden hamsters infected with the BA.5 strain at 5 dpi (Fig. 6A–E). When responses to different variants were compared, Tmprss2-deficient animals showed stable expression of most inflammatory markers across all infections, except for a marked upregulation of IL-10 following infection with the XBB variant (Fig. 6D). These immunological findings matched the histopathological observations, indicating concordance between molecular and morphological features during the resolution of infection.

Fig. 5.

Fig. 5

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Histopathological phenotypes of WT and Tmprss2 KO golden hamster lungs five days after inoculation with different SARS-CoV-2 variants. A Normal lung pathology in Tmprss2 KO golden hamsters five days after intranasal inoculation with PBS (mock). B. Pathology of the lungs of WT hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for five days. C Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for five days. D Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 beta variant for five days. E. Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 XBB variant for five days. Scale bar: up 200 μm; down: 30 μm

Fig. 6.

Fig. 6

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Expression of inflammatory factors in the lungs of WT and Tmprss2 KO golden hamsters five days after inoculation with different SARS-CoV-2 variants. A–E IFNγ, IL-1β, IL-6, IL-10, and TNFα levels in the lungs of WT and Tmprss2 KO golden hamster lungs five days after inoculation with different SARS-CoV-2 variants

After 10 days of Tmprss2 KO golden hamster infection, lung inflammation was alleviated

At 10 dpi, WT golden hamsters retained partial lung damage and exhibited reduced inflammatory infiltration. In contrast, the lung inflammatory lesions of TMPRSS2 KO golden hamsters were mostly resolved, and lung tissue damage was significantly alleviated or completely absent (Fig. 7A–E). This pathological difference suggests that BA.5 viruses continue to infect and induce lung damage via TMPRSS2, while deletion of the Tmprss2 gene impedes viral infection. These findings support the conclusion that inhibiting Tmprss2 can alleviate lung damage in golden hamsters, providing theoretical support for clinical interventions such as the development of TMPRSS2-targeted small-molecule inhibitors.

Fig. 7.

Fig. 7

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Histopathological phenotypes of WT and Tmprss2 KO golden hamster lungs 10 days after inoculation with different variants of SARS-CoV-2. A Normal lung pathology of Tmprss2 KO golden hamsters 10 days after intranasal inoculation with PBS (mock). B Pathology of the lungs of WT hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for 10 days. C Pathology of the lungs of Tmprss2 KO golden hamsters after intranasal inoculation with the SARS-CoV-2 BA.5 variant for 10 days. D Pathology of the lungs of Tmprss2 KO golden hamsters after the SARS-CoV-2 beta variant was inoculated intranasally for 10 days. E Pathology of the lungs of Tmprss2 KO golden hamsters after the SARS-CoV-2 XBB variant was inoculated intranasally for 10 days. Scale bar: up 200 μm; down: 30 μm

Using different SARS-CoV-2 variants, we subsequently detected the levels of multiple immune factors at 10 dpi (Fig. 8A–E). Statistical analysis revealed that Tmprssem1/m1 golden hamsters were infected with the BA.5 variant, which resulted in lower overall expression of inflammatory factors than the WT control. Additionally, in Tmprssem1/m1 golden hamsters infected with different variants, the expression of inflammatory factors remained largely unchanged during the late stage of infection, which indicated that all tested SARS-CoV-2 viral strains had been effectively cleared from the lungs.

Fig. 8.

Fig. 8

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Expression of inflammatory factors in the lungs of WT and Tmprss2 KO golden hamsters 10 days after inoculation with different variants of SARS-CoV-2. A–E The expression of IFNγ, IL-1β, IL-6, IL-10, and TNFα in the lungs of WT and Tmprss2 KO golden hamsters 10 days after inoculation with different variants of SARS-CoV-2

SARS-CoV-2 has other routes of infection

To assess whether SARS-CoV-2 uses alternative TMPRSS2-independent pathways in host cells, we generated hACE2;Tmprss2m1/m1 transgenic golden hamsters by crossing Ace2-overexpressing (H11 K18 hACE2) hamsters with Tmprss2m1/m1 hamsters (Fig. 9A). We hypothesized that loss of TMPRSSE might attenuate the lethal phenotype associated with the overexpression of hACE2 following viral infection. However, hACE2;Tmprss2m1/m1 plants intranasally infected with SARS-CoV-2 (BA.5 strain) all died by day 5 post-inoculation (Fig. 9B). Histopathology revealed different degrees of inflammatory cell infiltration in both the Tmprss2m1/m1 and hACE2;Tmprss2m1/m1 hamster groups (Fig. 9C). The TMPRSS2-deficient hamsters presented significantly greater viral loads, stronger innate immune activation, more extensive inflammatory infiltration, and more pronounced tissue injury, including interstitial edema, than their TMPRSS2-proficient counterparts. These findings indicated that deletion of TMPRSS2 does not abrogate the lethal outcome driven by the overexpression of ACE2 and that SARS-CoV-2 can exploit TMPRSS2-independent mechanisms of cell entry.

Fig. 9.

Fig. 9

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Infection experiments with Tmprss2 KO/H11 K18 hACE2 hybrid golden hamsters. A. Tmprss2 KO and Tmprss2 KO/H11 K18 hACE2 BA.5 infection experiments were conducted for five days. B. Survival curve at 5 dpi. C. Five days after lung infection with BA.5, Tmprss2 KO golden hamsters, and Tmprss2 KO/H11 K18 hACE2 hybrid golden hamsters developed pulmonary HE. Scale bar: up 200 μm; down: 20 μm

Discussion

In this study, we found that TMPRSS2 plays a key role in SARS-CoV-2 infection using a novel Tmprss2 knockout (KO) golden hamster model. Our findings provided valuable insights into viral pathogenesis and therapeutic targets. By demonstrating that Tmprss2 deficiency reduces viral titers, mitigates lung pathology, and weakens inflammatory responses across three major variants (Beta, BA.5, and XBB), our findings aligned with the established mechanism in which TMPRSS2 activates viral spike proteins to facilitate host cell entry [7, 23]. Notably, although we performed pathological staining analysis on extrapulmonary tissue (brain), we did not observe brain tissue lesions in hamsters infected with different subvariants (Additional file 1: Figure S1). This finding further supports the conclusion that the lung is the primary target organ of the virus. The golden hamster serves as a robust translational model and recapitulates key features of human SARS-CoV-2 pathology, including pulmonary inflammation and viral tropism, thereby enhancing the clinical relevance of our results [24–26]. The consistent reduction in viral replication and tissue damage across different variants highlights TMPRSS2 as a conserved host factor, irrespective of specific spike mutations. These findings support previous reports that TMPRSS2-mediated spike cleavage at the S1/S2 and RBD regions is a key step in viral invasion, highlighting that TMPRSS2 may be a broad-spectrum target for disease treatment [27–30].

Notably, Tmprss2 knockout hamsters presented attenuated inflammatory cytokine expression (e.g., IFNγ, IL-1β, and TNFα) during the acute phase of infection, suggesting that TMPRSS2 not only facilitates viral entry but also promotes the amplification of pro-inflammatory pathways. A reduction in interstitial edema and preserved alveolar structure in Tmprss2-deficient lungs further indicated that TMPRSS2 inhibition may attenuate direct viral cytopathology and immune-mediated tissue injury. These findings complement clinical observations linking elevated TMPRSS2 expression to severe SARS-CoV-2 outcomes, reinforcing the rationale for developing TMPRSS2 inhibitors (e.g., nafamostat) to block early infection and mitigate disease severity [28, 31, 32]. The hybrid experiment using K18-hACE2; Tmprss2m1/m1 hamsters revealed that knocking out Tmprss2 did not abrogate the lethal phenotype associated with increased ACE2 availability. SARS-CoV-2 invades host cells through the binding of its spike (S) protein to host cell receptors followed by protease-mediated activation. Among these processes, TMPRSS2-mediated cleavage of the S protein at the S2' site represents the canonical activation pathway. However, distinct Omicron subvariants, such as BA.5, have evolved a reduced dependency on this pathway during their evolutionary trajectory. The unique furin cleavage site at the S1/S2 boundary of the SARS-CoV-2 S protein provides a direct recognition target [33]. Studies have demonstrated that in Omicron subvariants including BA.5, furin-mediated cleavage of the S1/S2 site can occur either during the viral particle assembly and release phase or at the early stage of viral binding to host cells. This allows the virus, following binding to the ACE2 receptor, to rapidly expose the fusion peptide and trigger membrane fusion, or enter the endosomal pathway to complete subsequent infection, without the need for further activation by TMPRSS2 [34, 35]. Meanwhile, basic amino acid residues such as Arg403 in the receptor-binding domain (RBD) of the BA.5 S protein form stable interactions with groups on heparan sulfate chains through electrostatic forces [36, 37], inducing conformational changes in the S protein and promoting its high-affinity binding to the ACE2 receptor. The existence of these alternative pathways not only explains why Omicron subvariants can efficiently spread across diverse tissue cells but also provides novel insights for the development of antiviral strategies, which may serve as potential approaches to block Omicron subvariants.This implied that alternative pathways of entry (e.g., cathepsin-mediated endocytosis) may partially compensate for TMPRSS2 deficiency. This highlights the complexity of viral entry mechanisms and the need for combinatorial therapeutic approaches targeting TMPRSS2 and other proteases to achieve optimal efficacy.

While our study provided robust in vivo evidence for the role of TMPRSS2, its limitations include a focus on pulmonary infection (excluding extrapulmonary tissues) and a short observation period (up to 10 days). Future studies should investigate the role of TMPRSS2 in long-term sequelae, intertissue viral spread, and interactions with host immune cells. Additionally, assessing whether TMPRSS2 deficiency influences viral evolution or immune evasion could improve our understanding of host-virus coadaptation. To summarize, this study established TMPRSS2 as an important factor mediating the SARS-CoV-2 pathogenic mechanism in a clinically relevant model, offering preclinical validation for TMPRSS2-targeted interventions. By disrupting viral spike activation and reducing downstream inflammation, such strategies may represent a promising avenue for combating existing and emerging coronavirus variants.

Supplementary Information

Additional file 1. (3.1MB, docx)

Acknowledgements

This study was supported by grants from the National Key R&D Program of China (2021YFF0702500), the Key Program of State Key Laboratory of Reproductive Medicine and Offspring Health (SKLRM-2024A2).

Author contributions

JM Li, BW Wang, XF Wang conceived and designed the study. JM Li and WT Zeng conceived and development the method for genome editing of golden hamster. WT Zeng, JW Zhang, and BW Wang generated the TMPRSS2 KO golden hamsters. BW Wang, HD Wang, M Dong analyzed the phonotype of golden hamsters. With the help of YH Li, XF Wang, Q Chen, AH Zhang and AM Shi, virus infection was carried out.BW Wang conducted subsequent experiments. JM Li, BW Wang, AH Zhang, and WT Zeng interpreted the data of experiments and wrote the manuscript.

Funding

This work was supported by grants from the National Key R&D Program of China (2021YFF0702500), the Key Program of State Key Laboratory of Reproductive Medicine and Offspring Health (SKLRM-2024A2).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Aihua Zhang, Wentao Zeng, Xuefeng Wang and Jianmin Li: Lead contact.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Bowen Wang, Jinwei Zhang and Yuhan Li have contributed equally.

Contributor Information

Aihua Zhang, Email: aihuazhang@njmu.edu.cn.

Wentao Zeng, Email: wentaozeng@njmu.edu.cn.

Xuefeng Wang, Email: xuefeng_wangNUDT@outlook.com.

Jianmin Li, Email: jianminli@njmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1. (3.1MB, docx)

Data Availability Statement

No datasets were generated or analysed during the current study.

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