The laminin receptor is a receptor for Micropterus salmoides rhabdovirus

ABSTRACT

Micropterus salmoides rhabdovirus (MSRV) is an important pathogen of largemouth bass. Despite extensive research, the functional receptors of MSRV remained unknown. This study identified the host protein, laminin receptor (LamR), as a cellular receptor facilitating MSRV entry into host cells. Our results demonstrated that LamR directly interacts with MSRV G protein, playing a pivotal role in the attachment and internalization processes of MSRV. Knockdown of LamR with siRNA, blocking cells with LamR antibody, or incubating MSRV virions with soluble LamR protein significantly reduced MSRV entry. Notably, we found that LamR mediated MSRV entry via clathrin-mediated endocytosis. Additionally, our findings revealed that MSRV G and LamR were internalized into cells and co-localized in the early and late endosomes. These findings highlight the significance of LamR as a cellular receptor facilitating MSRV binding and entry into target cells through interaction with the MSRV G protein.

IMPORTANCE

Despite the serious epidemic caused by Micropterus salmoides rhabdovirus (MSRV) in largemouth bass, the precise mechanism by which it invades host cells remains unclear. Here, we determined that laminin receptor (LamR) is a novel target of MSRV, that interacts with its G protein and is involved in viral attachment and internalization, transporting with MSRV together in early and late endosomes. This is the first report demonstrating that LamR is a cellular receptor in the MSRV life cycle, thus contributing new insights into host-pathogen interactions.

INTRODUCTION

Micropterus salmoides rhabdovirus (MSRV), a negative-stranded RNA virus belonging to the genus Siniperhavirus of the family Rhabdoviridae, is one of the primary pathogens affecting largemouth bass (1). MSRV infection is highly lethal in juvenile fish, with mortality rates of up to 100%, leading to huge economic losses in the largemouth bass industry (2, 3). MSRV particles consist of nucleic acid, nucleocapsid, and envelope, measuring approximately 110–500 nm in length and 60–200 nm in diameter. The MSRV genome, comprising around 11.5 kb nucleotides, encodes five essential proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) (4). Typical symptoms observed in infected fish include surface hemorrhaging, corkscrew swimming, and body deformity (5).

Viral infection starts with specific interactions of virion constituents with host cellular receptors. The binding between cellular receptors and viral attachment proteins determines tissue tropism and the host range of viruses (6, 7). To comprehend and manage viral diseases effectively, it is imperative to identify viral receptors and understand their interaction mechanisms (8–10). During rhabdoviruses infection, G proteins are accountable for receptor binding (11). After binding to its receptor, the virus is internalized into the host cell and transported to the target site via endosomes. The acid environment of the endosome triggers conformational changes in the G protein, leading to the fusion of the endosome membrane and the viral envelope (4, 12). Till now, receptor identification in rhabdoviruses has primarily focused on the vesicular stomatitis virus (VSV) and the rabies virus (RABV). The low-density lipoprotein receptor (13) and leucine-rich repeat-containing G protein-coupled receptor 4 (14) have been identified as receptors for VSV. The acetylcholine receptor subunit alpha (15), metabotropic glutamate receptor subtype 2 (16), low-affinity nerve growth factor receptor (17), and neural cell adhesion molecule (18) have been identified as receptors for RABV. In fish rhabdoviruses, fibronectin has been identified as a receptor for viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus (19, 20). However, few studies have been conducted on the specific receptor of MSRV.

In this study, the laminin receptor (LamR) was identified as an interacting partner of MSRV G. LamR is an extracellular matrix glycoprotein that has been identified as a receptor for various viruses (21–23). The role of LamR in MSRV infection was subsequently demonstrated, and further analyses revealed that it is involved in MSRV attachment and internalization processes, as well as in virus transport via the endosome/lysosome pathway.

RESULTS

MSRV G interacts with LamR

The viral G protein, the only envelope protein encoded by MSRV and other rhabdoviruses, is recognized as the key protein involved in binding the cell receptor (24, 25). Epithelioma papulosum cyprinid (EPC) cells are susceptible to MSRV and are widely used in MSRV research (2, 4, 26). To identify the possible MSRV G-interacting host proteins, a co-immunoprecipitation (Co-IP) assay was performed using anti-hemagglutinin (HA) magnetic beads in pcDNA3.1-HA or pcDNA3.1-HA-G transfected EPC cells. Silver staining for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the Co-IP samples showed several specific bands in the precipitated proteins of the HA-G group (Fig. 1A). Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed 24 candidate proteins potentially interacting with MSRV G (Table S1). Among them, LamR was selected for further investigation due to its important role in the early stages of viral infection (21–23, 27). The interaction of MSRV G and LamR was then confirmed using Co-IP and indirect fluorescent assay (IFA) in Hela cells (Fig. 1B through D). Importantly, MSRV G and LamR co-localized on the cell membrane in the attachment assay. After 30 min of internalization, MSRV G and LamR co-localized in the cytoplasm (Fig. 1E). These results confirmed that LamR interacts directly with MSRV G.

Fig 1.

MSRV G interacts with LamR. (A) Screening of host proteins interacting with MSRV G. EPC cells were transfected with pcDNA3.1-HA-G or pcDNA3.1-HA for 48 h. The cells were collected and subjected to Co-IP assay. The eluted samples were then analyzed by western blotting (WB) and silver staining. The HA-G was indicated by a red arrow. (B–D) The interaction of MSRV G and LamR. Hela cells were co-transfected with pEGFP-G and pcDNA3.1-Flag-LamR. The cell lysates were collected and subjected to Co-IP and IFA. (E) Co-localization of endogenous MSRV G and LamR in EPC cells. Cells were incubated with MSRV [multiplicity of infection (MOI) = 50] at 4°C for 1 h, followed by three washes with phosphate-buffered saline (PBS). Cells were incubated at 25°C for 30 min. IFA was performed using LamR or MSRV G antibodies. The upper panel shows EPC without MSRV infection, the middle panel represents the localization of the MSRV and LamR after attachment onto EPC cells, and the bottom panel represents the localization of the MSRV and LamR after 30 min of internalization. The white arrow indicates the representative co-localization of MSRV G with LamR. Scale bar = 10 µm.

LamR is required for MSRV infection

To determine the possible role of LamR as a receptor for MSRV, we knocked down its expression in EPC cells using siRNA-mediated silencing. A significant decrease of LamR mRNA and protein was observed in siLamR transfected EPC (Fig. 2A). Compared to that of control siRNA (siCtrl), MSRV-induced cytopathic effect (CPE) in siLamR group was significantly weaker (Fig. 2B). Moreover, the siCtrl-treated cells exhibited a stronger MSRV N protein signal (red) compared to the siLamR-treated cells. Subsequent analysis showed that the knockdown of LamR significantly decreased the fluorescence intensity of MSRV N protein signal in MSRV-infected cells (Fig. 2D).

Fig 2.

LamR involves in MSRV infection in EPC cells. (A) siLamR downregulated LamR expression. Transfect EPC with siLamR or control siRNA (siCtrl), real-time quantitative PCR (RT-qPCR) and WB were used to detect the knockdown efficiency 48 h post-transfection. (B–D) LamR knockdown inhibited MSRV infection. Cells were transfected with siLamR or siCtrl and infected with MSRV (MOI = 0.1). The infected cells were collected for RT-qPCR (C) and IFA (D). Data represent means ± standard deviation. *P < 0.05 and **P < 0.01.

In addition, to investigate the receptor characteristics of MSRV in different cells, we examined the role of LamR in grass carp ovary (GCO) cell line (a cell line susceptible to MSRV) (3, 5) and M. salmoides heart (MSH) cell line (28). The results showed that LamR knockdown significantly decreased MSRV-induced CPE and N protein expression both in GCO and MSH cells (Fig. S1). These findings collectively supported the involvement of LamR in MSRV infection.

LamR antibody and soluble rLamR inhibit MSRV entry

To evaluate the function of LamR during MSRV entry, a blocking assay was performed using anti-LamR antibody (LamR-Ab) and rabbit IgG (negative control). The viabilities of EPC, GCO, and MSH cells were unaffected by the concentrations employed in the blocking assay (data not shown). To facilitate tracking of virus particles, MSRV was labeled with lipophilic dye 1,1′-diocta­decyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD). Real-time quantitative PCR (RT-qPCR) analysis confirmed a dose-dependent decrease in MSRV RNA post-LamR-Ab treatment. The EPC cells exhibited a blocking percentage of 31.84% in 2.5 µg/mL, 53.49% in 5 µg/mL, and 56.98% in 10 µg/mL (Fig. 3A); the GCO cells showed a percentage of 30.93% in 2.5 µg/mL, 36.05% in 5 µg/mL, and 64.14% in 10 µg/mL (Fig. S2A); and the MSH cells showed a percentage of 25.04% in 2.5 µg/mL, 57.66% in 5 µg/mL, and 64.56% in 10 µg/mL (Fig. S2C). Furthermore, the DiD-MSRV fluorescence intensity in the LamR-Ab group was significantly lower than that in the rabbit IgG group (Fig. 3C; Fig. S2B and D).

Fig 3.

LamR antibody and soluble rLamR inhibits MSRV entry into EPC cells. (A and C) LamR antibody blocks MSRV entry. Cells were incubated with various doses of diluted LamR-Ab (2.5, 5, and 10 µg/mL) at 25°C for 4 h. Subsequently, the cells were exposed to DiD-MSRV (MOI = 50) at 4°C for 1 h and incubated at 25°C for 1 h. The cells were then collected for RT-qPCR and fluorescence detection. (B and D) Soluble LamR inhibits MSRV entry. DiD-MSRV was incubated with rLamR (50, 100, and 200 µg/mL) at 4°C for 1 h and then exposed to EPC cells at 4°C for 1 h. After incubating at 25°C for 1 h, the non-internalized virus was removed using trypsin. The cells were collected for RT-qPCR and fluorescence detection. The fluorescence signal of DiD-MSRV particles was quantified by ImageJ. Forty cells were quantified in each group. Scale bar = 10 µm. *P < 0.05 and **P < 0.01.

Next, soluble rLamR was utilized to investigate the effects of LamR on MSRV entry. Prokaryotic expression and purification of soluble rLamR were shown in Fig. S3. RT-qPCR results revealed dose-dependent inhibition of MSRV entry mediated by rLamR. The inhibition percentages in EPC cells were 30.17% in 50 µg/mL, 52.47% in 100 µg/mL, and 58.36% in 200 µg/mL (Fig. 3B); in GCO cells, they were 29.14% in 50 µg/mL, 42.52% in 100 µg/mL, and 66.06% in 200 µg/mL (Fig. S4A); and in MSH cells, they were 30.52% in 50 µg/mL, 51.01% in 100 µg/mL, and 62.90% in 200 µg/mL (Fig. S4C). This inhibition was also demonstrated by measuring the fluorescence intensity of DiD-MSRV entering the cells (Fig. 3D; Fig. S4B and D). These results showed that LamR effectively inhibited the entry of MSRV to EPC, GCO, and MSH cells.

LamR is essential for MSRV attachment and internalization

Viral entry is a multistep process, including attachment, internalization, and membrane fusion (29). LamR has been identified as a receptor for various viruses (21), indicating its potential role in the initial stages of MSRV infection. To investigate the role of LamR in MSRV attachment, MSH, EPC, and GCO cells were transfected with siLamR or siCtrl for 24 h, followed by incubation with DiD-MSRV at 4°C for 1 h. Compared to the siCtrl-transfected group, siLamR-transfected cells showed a significant decrease in viral RNA attachment and internalization in EPC cells (Fig. 4A and C). Furthermore, LamR knockdown led to a noticeable reduction in fluorescence intensity of DiD-MSRV in EPC cells (Fig. 4B and D). In addition, similar results were seen in GCO and MSH cell lines (Fig. S5). These findings suggested that LamR is essential for MSRV attachment and internalization.

Fig 4.

Role of LamR in MSRV attachment and internalization. EPC cells with LamR knockdown were incubated with DiD-MSRV (MOI = 50) at 4°C for 1 h (for attachment) or at 25°C for 1 h (for internalization), followed by detection using RT-qPCR (A and C) and confocal microscopy (B and D). Scale bar = 10 µm. *P < 0.05 and **P < 0.01.

LamR facilitates MSRV internalization via clathrin-mediated endocytosis

Previous studies have reported that MSRV enters EPC cells through clathrin-mediated endocytosis (4). Furthermore, LamR regulates the process of cell endocytosis (30, 31). Therefore, we hypothesized that LamR is involved in MSRV endocytosis. To test this hypothesis, we transfected EPC cells with siCtrl or siLamR and analyzed the uptake of Alexa fluor 555 conjugated transferrin (AF-Tf), a marker for clathrin-mediated endocytosis (32), using confocal microscopy. Our results showed that LamR knockdown significantly inhibited AF-Tf uptake (Fig. 5A). Notably, treatment with chlorpromazine (CPZ), a specific clathrin-mediated endocytosis inhibitor (33), exhibited effects similar to those of LamR knockdown on AF-Tf uptake, and their combination exhibited synergistic inhibitory effects (Fig. 5B). These findings suggested that LamR is involved in clathrin-mediated endocytosis.

Fig 5.

LamR facilitates MSRV internalization via clathrin-mediated endocytosis. (A) Knockdown of LamR impairs the uptake of transferrin. EPC cells were transfected with siLamR or siCtrl for 24 h, followed by treatment with Alexa fluor 555 conjugated transferrin (AF-Tf) (50 µg/mL) at 25°C for 45 min. The cells were detected by confocal microscopy. (B) LamR knockdown impairs transferrin uptake through clathrin-mediated endocytosis. EPC cells were treated with siRNA and CPZ, followed by AF-Tf incubation. (C and D) Effect of LamR knockdown on MSRV internalization. EPC cells were treated with siRNA and CPZ. Subsequently, the cells were exposed to DiD-MSRV (MOI = 50) at 4°C for 1 h. After incubation at 25°C for 1 h, the cells were detected using RT-qPCR and confocal microscopy. Scale bar = 10 µm. *P < 0.05 and **P < 0.01.

To test whether LamR affects the endocytosis of MSRV, LamR knockdown EPC cells were treated with CPZ and subsequently infected with MSRV. The results showed that LamR knockdown and CPZ treatment had similar effects on MSRV internalization and exhibited a synergistic inhibitory effect on MSRV internalization (Fig. 5C and D). In summary, these results indicated that LamR is involved in MSRV internalization through clathrin-mediated endocytosis.

MSRV and LamR are transported together in early and late endosomes

MSRV enters EPC cells through clathrin-mediated endocytosis and is transferred through the endosome/lysosome pathway (4). Here, we investigated the co-transportation of MSRV G and LamR within early and late endosomes in EPC cells. Transfection with pEGFP-Rab5 and pEGFP-Rab7 enabled the labeling of early and late endosomes, respectively, whereas MSRV G and LamR were labeled with specific antibodies. The results displayed co-localization of MSRV G, LamR, and Rab5 (Fig. 6A and E) or Rab7 (Fig. 6C and F) within the EPC cells (white spots in the merged image). Furthermore, 3D rendered images generated using Imaris software confirmed the co-localization of the MSRV G-LamR complex with Rab5 in early endosomes (Fig. 6B) and with Rab7 in late endosomes (Fig. 6D).

Fig 6.

MSRV and LamR transport together in early and late endosomes. (A and C) Co-localization of MSRV G and LamR within early and late endosomes. EPC cells were transfected with pEGFP-Rab5 (A) or pEGFP-Rab7 (C) and infected with MSRV (MOI = 50). MSRV G (red) and LamR (light blue) were detected using specific antibodies. The co-localization of the MSRV G-LamR complex with Rab5 or Rab7 was indicated by white arrows. (B and D) Imaris software was used to generate 3D rendered images. The Venn diagram represents statistics on the co-localization among MSRV G, LamR, Rab5 (E), and Rab7 (F). (G) MSRV G interacted with LamR under acidic conditions. pEGFP-G and pcDNA3.1-Flag-LamR were co-transfected in Hela cells for 48 h, and GFP-G interacted with Flag-LamR in Co-IP assay at pH 5.5 and pH 7.4.

Additionally, the interaction between MSRV G and LamR was confirmed by Co-IP assay under acidic conditions (pH 5.5) (Fig. 6G), consistent with our observation that the MSRV G and LamR complex was present within the late endosome. Collectively, these findings demonstrated the binding of MSRV G to LamR and its concurrent transport within intracellular compartments.

Soluble rLamR neutralizes MSRV infection in largemouth bass

We next sought to assess the effect of LamR on MSRV infection in largemouth bass. Different concentrations of rLamR (50 and 200 µg/mL) were mixed with MSRV and intracerebrally injected into largemouth bass. rLamR at 200 µg/mL exhibited a survival rate of 31.5%, while the survival rate of 50 µg/mL rLamR was 7.86%. In comparison, all bovine serum albumin (BSA)-treated largemouth bass were dead (Fig. 7A). These findings provided evidence that rLamR promotes the survival rate of largemouth bass infected with MSRV in a dose-dependent manner.

Fig 7.

Soluble rLamR protects largemouth bass from MSRV challenge. (A) Survival rates of largemouth bass after intracerebrally injected with the mixture of MSRV and rLamR (50 and 200 µg/mL). (B) The histopathological analysis in spleen, liver, and kidney at 2 days post-infection (dpi). (C–E) Viral titers in the spleen, liver, and kidney measured by RT-qPCR at 1 and 2 dpi. Scale bar = 50 µm. *P < 0.05 and **P < 0.01.

To evaluate the effects of LamR on viral replication and histopathology, three fish per group were euthanized at 1 and 2 days post-infection. rLamR treatment resulted in inhibited viral titers in the spleen, liver, and kidney tissues (Fig. 7C through E). Furthermore, compared to the control group, LamR treatment alleviated histopathological abnormalities in the spleen, liver, and kidney, including coagulation necrosis and fragmentation in the spleen, degeneration and necrosis in the liver, monocyte infiltration, vacuolization and disintegration in the kidney (Fig. 7B). These in vivo findings indicated that soluble rLamR effectively neutralized MSRV infection in largemouth bass.

DISCUSSION

MSRV infection causes a highly contagious viral disease, resulting in huge economic losses to the largemouth bass industry worldwide (1). The host tropism of rhabdoviruses is primarily determined by the interaction between G protein and its cell receptor (29, 34). Despite previous investigations on host factors, the specific interaction between MSRV G and host membrane protein has not been studied, resulting in limited knowledge about the early stages of MSRV invasion. In this study, Co-IP and LC-MS/MS assays were performed to identify host proteins potentially interacting with MSRV G. The in vitro results from protein interaction, siRNA silencing, soluble protein neutralization, antibody blocking, immunofluorescence, as well as in vivo results from soluble protein neutralization assays, strongly suggest that LamR is a cellular receptor for MSRV infection. More importantly, we found that MSRV and LamR internalized into cells and transported to early and late endosomes together.

LamR is an extracellular matrix glycoprotein that provides cell adhesion to the basement membrane (21). LamR can be translated into two distinct forms: one located in the cytoplasm at approximately 37 kDa, and the other located in the membrane at approximately 67 kDa. The 37 kDa LamR is considered to be the precursor for generating the 67 kDa LamR, with its maturation potentially involving the dimerization and acylation of the precursor. However, verification of this process remains pending. LamR functions exhibit remarkable diversity, including laminin binding, participation in ribosomal biogenesis and translation, modulation of cellular migration, invasion, viability, and growth, regulation of cytoskeleton organization, as well as binding to chromatin and histones. Moreover, LamR is employed as a receptor for several pathogenic agents, such as Treponema pallidum (35), white spot syndrome virus (36), classical swine fever virus (CSFV) (22), grass carp reovirus I (37), Venezuelan equine encephalitis virus (38), adeno-associated virus (AAV) serotypes 8, 2, 3, and 9 (23), tick-borne encephalitis virus (39), dengue virus (40), sindbis virus (41), and prion protein (27).

During the initial stage of envelope virus infection, the interaction between the virus envelope protein and host cell membrane protein mediates viral entry into the cell (29). The G protein, being the exclusive envelope protein in MSRV particles, potentially assists viral entry by binding to host cell membrane protein (42). Here, the interaction between the host membrane protein LamR and MSRV G protein was identified through IP-MS screening. LamR acts as a cell surface receptor for multiple viruses and plays a vital role in viral binding and entry by interacting with viral proteins (22, 23, 27, 36, 40, 41). Importantly, suppressing LamR expression impeded MSRV infection in vitro, and pre-incubation of MSRV particles with soluble LamR protein also inhibited MSRV infection both in vivo and in vitro. These findings indicate that LamR plays an important role in the infection process of MSRV.

The attachment and internalization of viruses mainly rely on host factors on the target cell membrane. As a host cell receptor for multiple viruses, the function of LamR undergoes differentiation. In the case of CSFV infection in porcine kidney cell lines, LamR exclusively influences the process of viral attachment, while having no impact on viral internalization (22). However, during infection of AAV (23) and prion protein (43), LamR has been shown to modulate both viral attachment and internalization. As LamR mediates MSRV proliferation, we further investigated whether it is involved in the attachment and internalization of MSRV. Silencing LamR expression impeded MSRV aggregation on the EPC, GCO, and MSH cell surfaces, indicating that LamR could affect the attachment of MSRV. Similar experiments confirmed the influence of LamR on MSRV internalization. Further studies revealed that the administration of LamR antibody and soluble rLamR effectively impeded MSRV entry in a dose-dependent manner. Interestingly, blocking LamR partially inhibited viral entry, suggesting the potential existence of co-receptors similar to those found in other viruses (22, 40). Overall, these results highlight the crucial role of LamR in both MSRV attachment and internalization processes.

LamR predominantly localizes in lipid raft and is associated with endocytosis of the virus (44), epigallocatechin gallate (30), and amyloid beta peptides 42 (45). However, the specific process by which LamR regulates endocytosis is currently unclear. There are multiple pathways for viruses to enter cells through endocytosis, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, and micropinocytosis (29). Notably, a previous study from our group revealed that MSRV enters EPC cells via clathrin-mediated endocytosis (4), prompting us to investigate whether LamR could modulate MSRV entry by influencing this specific pathway. Our findings indicate that suppressing LamR expression reduces clathrin-mediated endocytosis, highlighting the capacity of LamR to impact clathrin-mediated endocytosis. Further experimentation utilizing a CPZ inhibitor and LamR-specific siRNA resulted in the synergistic inhibition of clathrin-mediated endocytosis and MSRV entry into cells (Fig. 5). Thus, our research revealed the influence of LamR on the endocytosis of MSRV through clathrin-mediated endocytosis in EPC cells.

During the endocytosis process of enveloped viruses, the virus is first endocytosed into the intracellular compartments and subsequently transported through specific pathways before membrane fusion and entry into the cytoplasm (29). Previous research has indicated that endocytosed MSRV particles are transported through early endosomes, late endosomes, and lysosomes (4). We observed co-localization of LamR with MSRV in early and late endosomes. Notably, we demonstrated an interaction between MSRV G and LamR under acidic conditions (pH 5.5), suggesting the involvement of LamR in MSRV intracellular transport. However, the underlying mechanism requires further investigation. Notably, rhabdoviruses endocytosis involves conformational changes in the G protein due to the acidic environment of the endosome, leading to the fusion of the endosome membrane and viral envelope protein (4, 12). Furthermore, our study demonstrated the interaction between MSRV G and LamR under acidic conditions (pH 5.5) (Fig. 6G), indicating that MSRV G and LamR complexes were co-transported to early and late endosomes. This suggests that LamR is involved in the intracellular transport of MSRV. However, its fundamental mechanism needs further investigation.

In conclusion, our study demonstrates that LamR regulates MSRV proliferation by interacting with MSRV G as its receptor. In addition, LamR participates in MSRV attachment and influences MSRV internalization through clathrin-mediated endocytosis. Further analysis revealed the involvement of LamR in the trafficking of MSRV through endosome/lysosome pathway. These findings provide novel insights into MSRV pathogenesis and offer new perspectives for the management of MSRV infections.

MATERIALS AND METHODS

Cells and viruses

EPC, GCO, and MSH cells were grown in M199 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 25°C. MSH cells were generously gifted by Prof. Weiwei Zeng of Foshan University. Hela cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) (Gibco) at 37°C and 5% CO₂.

The MSRV strain MSRV-YH01 was generously gifted by Prof. Jiayun Yao of the Zhejiang Institute of Freshwater Fisheries. MSRV was grown in EPC cells and purified using ultracentrifugation as previously described (4).

Regents and antibodies

CPZ was purchased from Sigma (St. Louis, USA). LamR recombinant rabbit monoclonal antibody, anti-Flag magnetic beads, Lipofectamine 3000, Opti-MEM, and AF-Tf were purchased from Thermo (Carlsbad, USA). Mouse anti-N and anti-G polyclonal antibodies were prepared and stored in our laboratory. GFP monoclonal antibody was purchased from Santa Cruz (Texas, USA). Mouse anti-Flag, anti-HA, anti-ACTB antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG, and HRP-conjugated goat anti-mouse IgG were supplied by ABclonal (Wuhan, China). Alexa fluor 555-labeled donkey anti-rabbit IgG (H + L), Alexa fluor 555-labeled donkey anti-mouse IgG (H + L), anti-GFP magnetic beads, anti-HA magnetic beads, and enhanced chemiluminescence (ECL) reagent were purchased from Beyotime (Hangzhou, China).

Plasmid construction and transfection

The open reading frame (ORF) of G protein (GenBank accession no: MK397811.2) was amplified by the cDNAs reverse-transcribed from MSRV-infected EPC cells and cloned into pCDNA3.1-HA and pEGFP-N1. The ORF of LamR (GenBank accession no: XM_042725451.1https://www.ncbi.nlm.nih.gov/nuccore/XM_042725451.1/) was cloned into pCDNA3.1-Flag and pET-28a. The primers are listed in Table S2.

Cells that reached 80%–90% confluence in dishes were transfected using Lipofectamine 3000. The siRNA or plasmid and P3000 were diluted in Opti-MEM and combined with Lipofectamine 3000, followed by incubation at room temperature for 15 min. The mixed suspension was then transferred to the cell dishes. At 6 h post-transfection, the culture medium was replaced by M199 or DMEM containing 10% FBS.

LC-MS/MS analysis

After transfection of EPC cells with pcDNA3.1-HA-G or pcDNA3.1-HA, the cells were collected and lysed using lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM EDTA, pH 7.4). Subsequently, 25 µL of anti-HA magnetic beads was added to the lysate and incubated the mixture at 4°C for 8 h. Elution was achieved using HA peptide eluent (400 µg/mL HA peptide in Tris buffered saline). The eluent was then subjected to SDS-PAGE separation and visualized through silver staining. Staining bands were excised and digested with trypsin, followed by analysis using LC-MS/MS.

Co-IP assays

Co-IP assays were performed as described previously (46). Hela cells were cultured in 25 cm² dishes and co-transfected with plasmid as indicated. Cells were lysed by lysis buffer. The lysates were centrifuged at 13,000 × g for 20 min, and the supernatants were mixed with the anti-Flag or anti-GFP magnetic beads in a rotation wheel for 6 h. The beads were washed three times with wash buffer using a magnetic separation device to remove unbound proteins and contaminants. The final immunoprecipitates were analyzed using the indicated antibodies.

Western blotting

The samples were lysed with lysis buffer and separated using 10% SDS-PAGE. Following the transfer of the protein to the polyvinylidene fluoride (PVDF) membrane, it was blocked with 5% skimmed milk and then exposed to the primary antibody. Subsequently, the PVDF underwent three washes with PBST (phosphate buffer saline with 0.1% Tween-20) and incubated with secondary antibody. The protein bands were detected using the ECL reagent as described previously (47).

RT-qPCR

Total mRNA was extracted from collected tissues or cell lines using TRIzol (TaKaRa, Dalian, China). RNA concentration was measured using NanoDrop 2000 (Thermo). The reverse transcription was conducted with the PrimeScript RT Master Mix (TaKaRa). Relative cDNA expression was quantified using TB Green Real-time PCR Master Mix (Takara). The β-actin gene served as an internal control. The qPCR condition was as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 30 s, followed by 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 50°C for 30 s. Relative fold change was calculated by applying the 2^−ΔΔCt method compared to the corresponding control.

DiD-MSRV

DiD labeling was performed on purified MSRV particles according to our previously established protocol (4). Briefly, 10 mM DiD was incubated with MSRV particles with gentle shaking for 2 h. Unbound DiD was removed using a NAP-10 gel filtration column (GE Healthcare, USA).

Immunofluorescence

Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 10 min. Subsequently, the cells were incubated in 5% BSA for 2 h, followed by incubation with the primary antibody overnight at 4°C. After thorough washing, the cells were further incubated with the secondary antibody for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 8 min. Fluorescence images were acquired using a confocal microscope LSM880 (Carl Zeiss, Jena, Germany).

Antibody blocking assay

The LamR antibody or rabbit IgG was diluted in the M199 medium. Cells were treated with antibody suspension (2.5, 5, and 10 µg/mL) and incubated at 25°C for 4 h. Subsequently, DiD-MSRV (multiplicity of infection = 50) was added for adsorption at 4°C for 1 h. After 1 h of infection at 25°C, the non-internalized virus was eliminated by trypsin treatment. The cells were collected for RT-qPCR and IFA.

Soluble rLamR neutralization assay

Soluble rLamR was pre-incubated with the virus at 4°C for 1 h at varying concentrations (50, 100, and 200 µg/mL). The protein-virus suspensions were added to cells at 4°C for 1 h. Following three washes with pre-cooled PBS, the cells were maintained at 25°C for 1 h. The non-internalized virus was removed using trypsin, and the treated cells were collected for detection.

Transferrin uptake assay

Following transfection with siRNA or plasmid, EPC cells were treated with AF-Tf (50 µg/mL) for 20 min at 4°C. The un-adsorbed AF-Tf was then removed using pre-cooled PBS, and the cells were subsequently maintained at 25°C for 45 min. After removing the extracellular AF-Tf, the cells were fixed for 10 min with 4% paraformaldehyde, followed by permeabilization using 0.1% Triton X-100. Finally, the cell nuclei were stained with DAPI, and the internalized transferrin was visualized using confocal microscopy.

Survival assay

Largemouth bass (length 2–3 cm) were purchased from a largemouth bass farm in Huzhou City. These fish were then kept in a recirculating aquaculture system for 14 days, under controlled freshwater conditions of 22–24°C and a 12-h light/12-h dark cycle. They were fed commercial fodder daily.

Three hundred largemouth bass were randomly divided into three groups. The MSRV virus was mixed with rLamR (50 and 200 µg/mL) or an equal volume of BSA and incubated at 4°C for 1 h. Subsequently, the mixture was intraperitoneally injected into the largemouth bass. Tissue samples from the kidney, spleen, and liver were collected to measure virus titers, perform histopathological analysis (hematoxylin and eosin staining), and immunohistochemistry. Before sampling, the fish were euthanized using 0.03% (vol/vol) ethylene glycol monophenyl ether as an anesthetic. Daily observation of the mortality rate was conducted, and the survival rates among the different groups were compared.

Histopathology and immunohistochemistry

The liver, spleen, and kidney tissues were fixed in freshly prepared 4% paraformaldehyde for 72 h, paraffin-embedded, and sliced into 4 µm thickness. The staining protocol involved routine hematoxylin and eosin staining and immunohistochemical staining. After antigen retrieval, the tissue slices were blocked with 5% BSA, followed by incubation with N protein polyclonal antibody (1:200). Subsequently, the slices were incubated with HRP-labeled anti-mouse IgG. Finally, the slices were imaged using a light microscope.

Statistical analysis

GraphPad Prism 8.0 was used for statistical calculations and data plotting. Unless otherwise stated, results are shown as means ± standard deviation from three independent experiments. Statistical significance was determined using T-test. Significance values were denoted by asterisks: *P < 0.05 and **P < 0.01.

DATA AVAILABILITY

All data generated or analyzed during this study are included in the article.

ETHICS APPROVAL

All experimental procedures were conducted in compliance with the Experimental Animal Management Law of China and were approved by the Animal Ethics Committee of Ningbo University.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.00697-24.

Supplemental material. jvi.00697-24-s0001.docx.

Figures S1 to S5; Tables S1 and S2.

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