Abstract
Baculovirus is an obligate parasitic virus of the phylum Arthropoda. Baculovirus including Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been widely used in the laboratory and industrial preparation of proteins or protein complexes. Due to its large packaging capacity and non-replicative and non-integrative natures in mammals, baculovirus has been proposed as a gene therapy vector for transgene delivery. However, the mechanism of baculovirus transduction in mammalian cells has not been fully illustrated. Here, we employed a cell surface protein-focused CRISPR screen to identify host dependency factors for baculovirus transduction in mammalian cells. The screening experiment uncovered a series of baculovirus host factors in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and NPC intracellular cholesterol transporter 1 (NPC1). Further investigation illustrated that EXTL3 affected baculovirus attachment and entry by participating in heparan sulfate biosynthesis. In addition, NPC1 promoted baculovirus transduction by mediating membrane fusion and endosomal escape. Moreover, in vivo, baculovirus transduction in Npc1−/+ mice showed that disruption of Npc1 gene significantly reduced baculovirus transduction in mouse liver. In summary, our study revealed the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape in mammalian cells, which is useful for understanding baculovirus transduction in human cells.
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Introduction
Baculoviruses are enveloped, double-strand DNA viruses comprising large genomes ranging from 80 to 180 kilobase pairs1. As the first baculovirus with full sequence information, AcMNPV has a genome of 133,894 nucleotides in size that contains 154 predicted non-overlapping open reading frames2. In nature, AcMNPV produces two structurally and functionally distinct virion forms, budded virions (BVs) and occlusion-derived virions (ODVs)3. BVs occur primarily in a loose viral envelope containing a single nucleocapsid, while ODVs have more than one nucleocapsid in a single envelope4. Although the two virion forms may share some common nucleocapsid proteins such as P39, the envelope components are largely different. BVs have envelope glycoproteins GP64, whereas ODVs contain polyhedral envelope protein P32-34, polyhedrin P294,5, and per os infectivity factors (PIFs) that are required for the infection in insect gut6,7. BVs capture cell membranes as their envelopes and transmit within the hosts. By contrast, ODVs acquire envelopes from cell nuclear membrane and transmit between hosts8,9. BVs enter cells through receptor-mediated endocytosis10,11, while ODVs appear to fuse directly with the plasma membrane12,13.
Like other enveloped viruses, the interactions between envelope glycoproteins and host cell receptors are important for baculovirus entry. GP64 and F protein are the major envelope glycoproteins of lepidopteran nucleopolyhedrovirus (NPVs)14. In group I NPVs such as AcMNPV, GP64 functions as a fusion protein mediating viral entry into host cells10, while the F protein Ac23 seemed dispensable for infection in insect cells14,15. Different from group I NPVs, group II NPVs such as Spodoptera exigua MNPV and Lymantria dispar MNPV do not have GP64 protein and utilize F protein as the fusion protein for virus entry16,17,18.
As a class III fusion protein, AcMNPV GP64 protein has 512 amino acids19,20 constituting five domains or structurally distinct regions, among which ___domain I contains fusion peptide and receptor binding peptide10,19. Different from class I and class II fusion proteins, GP64 does not undergo proteolytic cleavage20,21. The membrane fusion process of AcMNPV is triggered by a reversible, pH-dependent conformational change of GP6422, which remains trimeric in pre-fusion and post-fusion states19. Despite the existing studies in insect cells, the cellular receptor for GP64 binding in mammalian cells has not been reported.
AcMNPV is one of the most widely used baculoviruses in laboratory research and industry23. Baculovirus expression vector system (BEVS) was developed in the 1980s by using BVs as a transgene delivery vector to insect cells24. BEVS has now become one of the most widely used expression systems in the laboratory and industry for production of recombinant proteins25, virus-like particles26,27, and baculovirus-based vaccines28,29,30,31,32,33.
In addition to gene transfer into insect cells, baculovirus has been employed as gene delivery vectors in mammalian cells34,35,36. Baculovirus is capable of delivering a variety of genome editing tools into human cells, including zinc finger nucleases (ZFNs)37,38,39, TALENs40, CRISPR-Cas41, and CRISPR-based prime editors42. Genome editing in primary neurons and induced pluripotent stem cells (iPSCs) has also been achieved by baculovirus delivery of CRISPR-Cas9. Compared to other viral vectors43,44, the non-replicative and non-integrative natures render baculovirus a potentially safe gene therapy vector. Another notable advantage of baculovirus as a gene transfer vector lies in its large packaging capacity, which enables the delivery of large genome-editing tools such as prime editors in a single virion42.
More importantly, baculovirus has been exploited for in vivo gene delivery in mammals. Direct injection of baculovirus in mouse and rat brains could efficiently transduce neural cells45. Intravitreal baculovirus injection mediated gene transfer in mouse and rabbit eyes46,47. In cancer mouse models, intratumoral baculovirus injection was shown to be capable of delivering antiangiogenic proteins for cancer therapy48,49. Engineering efforts have shown that the transduction efficiency of baculovirus in mammalian cells in vitro and in vivo could be enhanced50,51,52,53.
Despite the widespread applications, the mechanism of baculovirus transduction in mammalian cells is yet poorly understood. Previous studies have shown that polybrene and heparin can inhibit baculovirus transduction in mammalian cells, suggesting an important role of electrostatic interactions54,55. Syndecan-1 (SDC-1) was also found to be important for baculovirus binding on mammalian cells56. Other studies suggested that baculovirus attachment on mammalian cells depended on cell surface phospholipids57 and cholesterol58,59. Meanwhile, restriction factors that limit baculovirus transduction in mammalian cells have also been identified60. Nevertheless, there is yet no systematic investigation on baculovirus host factors in mammalian cells.
Genetic screen using clustered regularly interspaced short palindromic repeats (CRISPR) has been widely used in the dissection of host-pathogen interactions61. In a previous study, we established a cell surface protein-focused CRISPR library, so-called surfaceome CRISPR (SfCRISPR), and showed that this library could efficiently identify rhinovirus host factors62. In the present study, we employed this sfCRISPR library to perform forward genetic screen on a recombinant baculovirus carrying EGFP transgene (Bac-EGFP). Two rounds of negative selection identified several candidate host dependency factors for baculovirus transduction in human cells, including exostosin-like glycosyltransferase 3 (EXTL3) and Niemann-Pick C1 (NPC1). In-depth analyses illustrated the role of EXTL3 and NPC1 for the in vitro and in vivo baculovirus transduction in mammalian cells.
Results
Identification of baculovirus host factors in HEK-293T cells using SfCRISPR screen
To identify cell surface host factors of baculovirus in mammalian cells, we performed genetic screen using a previously described cell surfaceome CRISPR library (SfCRISPR) that contained sgRNAs targeting to 1344 cell surface protein-coding genes in human genome62. We examined several cell lines for baculovirus transduction and found that HEK-293T cells exhibited highest transduction rate (Supplementary Fig. 1a), which was important for reducing the background in the negative EGFP selection (Fig. 1a). HEK-293T cells were transduced with lentivirus (LV) carrying the SfCRISPR library. Next-generation sequencing (NGS) analysis showed that this cell library had full coverage of sgRNAs (Supplementary Fig. 1b) with a uniform distribution as predicted (Supplementary Fig. 1c). In addition, we analyzed the shift of sgRNAs targeting essential and nonessential genes by comparing the sgRNAs at 5 days and 12 days after LV transduction and found that sgRNAs targeting essential genes were significantly depleted (Supplementary Fig. 1d) as described63. These results suggested that the library was constructed successfully and was qualified for downstream phenotypic screen.
a Flowchart showing the negative selection approach for baculovirus host factor identification using surfaceome CRISPR screen. Candidate gene hits identified from the first (b) and second (c) rounds of selection. d The effects of knockout of the seven candidate genes on Bac-EGFP transduction, as determined by flow cytometry analyses. The p-value between Nontarget and EXTL3-KO-1, EXTL3-KO-2, NPC1-KO-1, NPC1-KO-2, MBTPS2-KO-1 and MBTPS2-KO-2 groups are 1e-5, 1e-5, 6e-6, 1e-5, 4e-5 and 4e-5, respectively. e Fluorescence imaging of Bac-EGFP infected cells. The experiment is repeated three times independently with similar results. f RT-qPCR quantification of EGFP mRNA in the lysate of EXTL3 and NPC1 knockout cells at 48 h post-Bac-EGFP transduction. β-actin is used as an internal control. g Evaluation of the effects of EXTL3 and NPC1 knockdown on Bac-EGFP transduction. The p-value between SiRNA-nontarget and SiRNA-NPC1-2 groups is 1e-5. h Evaluation of the effects of NPC1 inhibitor U18666A on Bac-EGFP transduction. For (d, f, and g), Data are presented as mean ± SD (n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t-test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
The validated cell library containing cell surface protein knockouts was then infected with baculovirus harboring enhanced green fluorescent protein transgene (Bac-EGFP). The Bac-EGFP-transduced cells were sorted by flow cytometry for enriching EGFP negative cells (Fig. 1a). This negative selection approach allowed enrichment of cells deficient in baculovirus host factors. Two rounds of negative selection were performed and sgRNA enrichment was observed (Supplementary Fig. 1e). With the process of negative selection, we observed an increased frequency of EGFP negative cells in round 2 selection, suggesting of successfully imposed selection pressure (Supplementary Fig. 2a, b).
The cells after round 1 and round 2 selection were collected and the genomic DNA was extracted and analyzed by NGS. The genes with enriched sgRNAs were determined using modified robust rank aggregation (a-RRA) analyses in the MAGeCK pipeline (MAGeCK score) and candidate gene hits with an FDR cutoff of 0.05 were displayed (Fig. 1b, c and Supplementary Data File 1). Seven candidate hits were consistently enriched in both rounds of selection, including exostosin-like glycosyltransferase 3 (EXTL3), Niemann-Pick C1 (NPC1), SREBP cleavage activating protein (SCAP), FAM20B glycosaminoglycan xylosylkinase (FAM20B), membrane-bound transcription factor site-2 protease (MBTPS2), glucuronic acid epimerase (GLCE) and transmembrane 9 superfamily member 3 (TM9SF3). For each candidate gene, two sgRNAs were designed to construct knockout cells in HEK-293T cells (Supplementary Fig. 3a–g).
It was found that the knockout of EXTL3, NPC1, SCAP, MBTPS2 and GCLE with both sgRNAs significantly reduced Bac-EGFP transduction (Fig. 1d). Importantly, EXTL3 and NPC1 knockout reduced EGFP-positive cells to less than 50% of that in the non-targeting sgRNA group (Fig. 1d). Thus, subsequent experiments were focused on studying the function of EXTL3 and NPC1. EXTL3 contains a glycosyltransferase ___domain and plays a critical role in the biosynthesis of heparan sulfate, which can facilitate viral attachment and entry64,65,66. NPC1 is a known host factor for various clinically important viruses67,68. Therefore, in the subsequent experiments, we focused our investigation on the functions and mechanisms of EXTL3 and NPC1 in baculovirus transduction.
Validation of EXTL3 and NPC1 as host dependency factors of baculovirus
In consistency with the flow cytometry experiments, fluorescent microscopy imaging showed that EXTL3 or NPC1 knockout reduced Bac-EGFP transduction (Fig. 1e). Moreover, RT-qPCR analysis revealed decreased mRNA expression of EGFP transgene in EXTL3 and NPC1 knockout cells (Fig. 1f). Similar to Bac-EGFP transduction, baculovirus carrying tdTomato (Bac-tdTomato) also exhibited reduced transduction efficiency in EXTL3 and NPC1 knockout HEK-293T cells (Supplementary Fig. 4a, b). In addition, we designed EXTL3- and NPC1-targeting siRNAs (Supplementary Fig. 4c, d) and found that knockdown of EXTL3 and NPC1 could also suppress baculovirus transduction albeit with lesser impact than EXTL3 and NPC1 knockout (Fig. 1g). The function of NPC1 in baculovirus transduction was further validated by U18666A, a small molecule inhibitor of NPC1. It was found that U18666A inhibited Bac-EGFP transduction in a dose-dependent manner in HEK-293T cells (Fig. 1h). Collectively, these results demonstrated that EXTL3 and NPC1 were host dependency factors for baculovirus transduction in human cells.
Evaluation of the functions of EXTL3 and NPC1 in baculovirus attachment, entry, and endosomal escape
Similar to the results in HEK-293T cells, EXTL3 and NPC1 knockout in HeLa cells (Supplementary Fig. 5a) inhibited Bac-EGFP transduction (Fig. 2a and Supplementary Fig. 5b). The NPC1 inhibitor U18666A reduced EGFP fluorescence in Bac-EGFP-transduced HeLa cells (Fig. 2b and Supplementary Fig. 5c). Next we constructed single clones of HeLa cells for EXTL3 and NPC1 knockouts respectively. These single clones were confirmed to contain genomic modifications at both alleles of EXTL3 and NPC1 (Supplementary Fig. 5d). Consistent with the above results, EXTL3−/− and NPC1−/− clones exhibited reduced Bac-EGFP transduction, as determined by EGFP expression at both protein (Fig. 2c and Supplementary Fig. 5e) and mRNA (Fig. 2d) levels. Moreover, rescue experiments by overexpressing EXTL3 and NPC1 in corresponding knockout single clones (Supplementary Fig. 5f, g) restored baculovirus transduction (Fig. 2c, d and Supplementary Fig. 5h).
a Evaluation of the effects of EXTL3 and NPC1 knockouts on Bac-EGFP transduction, as determined by flow cytometry analyses. The p-value between Nontarget and NPC1-KO-2 groups is 2e-5. b Evaluation of the effects of NPC1 inhibitor U18666A on Bac-EGFP transduction. Evaluation of the effects of EXTL3 and NPC1 knockouts and overexpression rescue on Bac-EGFP transduction, as determined by flow cytometry (EGFP protein) (c) and RT-qPCR (EGFP mRNA) (d). β-actin is used as an internal control for RT-qPCR. For (c), the p-values between Nontarget and EXTL3−/− and NPC1−/− groups are 5e-8 and 5e-6, respectively. The p-value between EXTL3−/− and EXTL3 rescued groups is 2e-5. The p-value between NPC1−/− and NPC1 rescued groups is 1e-5. For (d), the p-value between Nontarget and EXTL3−/− and NPC1−/− groups are 4e-7 and 7e-8, respectively. Evaluation of the effects of EXTL3 or NPC1 knockout and overexpression rescue on Bac-EGFP attachment (e) and entry (f). g qPCR quantification of the intracellular baculovirus genome GP64 gene after Bac-EGFP transduction in non-targeting sgRNA, NPC1−/− and NPC1 rescued HeLa cells. Bac-EGFP is incubated with cells at an MOI of 2 for 1 h and then removed from the medium. The total GP64 in each well is quantified. The p-values between NPC1−/− and Nontarget and NPC1 rescued group at 47 h are 5e-5 and 3e-5, respectively. For (a), (c–g), Data are presented as mean ± SD (n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t-test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
To determine the roles of EXTL3 and NPC1 during baculovirus transduction, we investigated their functions in viral attachment and entry. For viral attachment assay, Bac-EGFP was incubated with cells at 4 °C for 1 h. The cells with attached baculovirus were harvested and lysed for qPCR quantification of viral loads. For virus entry assay, Bac-EGFP was incubated with cells first at 4 °C for 1 h and then at 37 °C for 45 min to initiate the internalization of Bac-EGFP. Surface-bound Bac-EGFP was removed by trypsin treatment and the internalized virus was quantified by qPCR. It was found that EXTL3−/− cells significantly reduced baculovirus attachment and entry, which could be restored by EXTL3 overexpression (Fig. 2e, f). By contrast, NPC1 knockout or overexpression rescue did not significantly affect baculovirus attachment or entry (Fig. 2e, f). Taken together, these results suggested that EXTL3, but not NPC1, was involved in baculovirus attachment and entry.
To explore whether NPC1 participated in any transduction process following attachment and entry, we sought to analyze the function of NPC1 on endosomal escape. It is known that baculovirus does not replicate in mammalian cells69, thus we monitored the decay rate of intracellular baculovirus genome in an approach similar to a previous study62. Non-targeting sgRNA-treated, NPC1−/− and NPC1 rescued HeLa cells were transduced with Bac-EGFP for 1 h and then the virus-containing medium was replaced with fresh medium. The total GP64 quantity in each well was monitored over a course of 47 h after the removal of virus. It was found that baculovirus DNA decreased in a time-dependent manner (Fig. 2g), suggesting that the cellular DNA degradation machinery was active toward baculovirus genome. Importantly, NPC1−/− cells retained significantly higher level of baculovirus genome than non-targeting sgRNA-treated cells starting from 8 h after removal of virus (Fig. 2g). Consistently, NPC1 overexpression rescue significantly reduced baculoviral genome quantity at 35 h and 47 h in comparison to NPC1−/− cells. To exclude the possibility that the difference in intracellular viral genome was due to differential cell proliferation, we examined the proliferation of non-targeting sgRNA, NPC1−/− and NPC1 rescued cells and observed minor difference between each group (Supplementary Fig. 5i). These data showed that knockout of NPC1 could extend the persistence of intracellular baculovirus genome and thus indicated a potential role of NPC1 in baculovirus endosomal escape.
The above results prompted us to expand the investigation on the mechanism of retention of Bac genome. Bafilomycin A1 (BafA1) is a specific inhibitor of V-type ATPases70 and has been reported to be capable of inhibiting endosomal escape of viruses by suppressing endosomal acidification71,72. In the present study, it was found that pre-treatment of cells with BafA1 could prolong the retention of intracellular Bac genome (Supplementary Fig. 6a), similar to the effects of NPC1 knockout. In addition, BafA1 treatment had minor impact on cell proliferation (Supplementary Fig. 6b), suggesting that the above-observed effects of BafA1 on intracellular Bac genome were unlikely due to its side effects on cells. Collectively, these data established a link between NPC1-mediated endosomal escape and the exposure and decay of Bac genome in cytoplasm.
EXTL3 participates in baculovirus entry through heparan sulfate biosynthesis pathway
EXTL3 (Exostosin-like protein 3) belongs to EXT protein family, which includes EXT1, EXT2, EXTL1, EXTL2, and EXTL373. EXTL3 encodes a glycosyltransferase responsible for the biosynthesis of heparan sulfate (HS). Two glycosyltransferase domains, GT47 and GT64, of EXTL3 in the Golgi luminal region are the functional domains for HS biosynthesis74 (Fig. 3a). We constructed EXTL3−/− HEK-293T cells (Supplementary Fig. 7a) to explore the function of EXTL3-mediated HS biosynthesis in baculovirus transduction. It was found that baculovirus attachment and entry in EXTL3−/− cells were markedly reduced as compared to non-targeting sgRNA cells (Fig. 3b). These results were consistent with those in HeLa cells and illustrated an important role of EXTL3 in baculovirus attachment and entry.
a Structural organization of EXTL3 protein. b Evaluation of baculovirus attachment and entry in EXTL3−/− cells. GP64 gene was quantified by qPCR. The p-value between Nontarget and EXTL3−/− in entry groups is 4e-5. c Western blotting detection of heparan sulfate proteoglycan 2 (HSPG2) in wild-type and EXTL3−/− cells. The experiment is repeated three times independently with similar results. d Analysis of the effects of heparin sodium on Bac-EGFP transduction, as determined by flow cytometry quantification of EGFP-positive cells. e Analysis of the effects of heparin sodium on Bac-EGFP attachment and entry, as determined by qPCR quantification of attached or internalized baculovirus GP64 gene. f Evaluation of the effects of HPSE on baculovirus transduction. The p-value between Nontarget and Nontarget+HPSE groups is 2e-5. g Schematic presentation of HSPG biosynthesis. h Evaluation of the effects of EXT2 knockout on baculovirus transduction. The p-value between Nontarget and EXT2-KO groups is 7e-6. i Investigation of the relationship between EXTL3 and NPC1 functions in baculovirus transduction. The p-value between No heparin sodium and With heparin sodium in NPC1−/− groups is 1e-6. The p-value between No heparin sodium and With heparin sodium in NPC1 rescued groups is 2e-6. For (b, e, f, h, and i), Data are presented as mean ± SD (n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t-test. Mock group is the PBS treatment without baculovirus. Source data are provided as a Source Data file.
Given that HS is a known baculovirus attachment and entry factor in mammalian cells54,55,56, we sought to explore whether EXTL3 affected baculovirus attachment and entry by affecting HS biosynthesis. We thus examined the expression of heparan sulfate proteoglycan 2 (HSPG2) protein in EXTL3−/− HEK-293T cells and found that EXTL3 knockout abolished the production of HSPG2 (Fig. 3c). In addition, we found that Bac-EGFP transduction could be inhibited by heparin sodium in a dose-dependent manner (Fig. 3d and Supplementary Fig. 7b), which is a competitive inhibitor for cellular HS65,75. Similarly, 2 μg/μL heparin sodium could significantly inhibit baculovirus attachment and entry (Fig. 3e). Moreover, 24 h pretreatment with heparanase (HPSE) could significantly reduce baculovirus transduction in non-targeting sgRNA control cells or in overexpression-rescued EXTL3−/− cells (Fig. 3f). These results collectively suggested that HS was important for baculovirus attachment and entry and that EXTL3 promoted baculovirus transduction by involving in HS biosynthesis.
Next, we sought to investigate whether other members in the EXT protein family have similar roles in baculovirus transduction. HS biosynthesis is initiated by the attachment of xylose to specific serine residues in HSPG core proteins, followed by the formation of a linkage tetrasaccharide, glucuronic acid-galactose-galactose-xylose (GlcA-Gal-Gal-Xyl). EXTL3 links the first N-acetyl-D-glucosamine (GlcNAc) residue to GlcA and an enzyme complex composed of EXT1 and EXT2 adds GlcA-GlcNAc disaccharide repeats to the nascent chain, followed by a series of processing reactions76 on the chain (Fig. 3g). We constructed EXT2 knockout HEK-293T cells (Supplementary Fig. 7c) and found that EXT2 knockout could also affect baculovirus transduction (Fig. 3h) though the degree of inhibition was not as prominent as those with EXTL3 knockout. These results suggested that different moieties or forms of HSPG may have differential effects on baculovirus transduction.
Next, we sought to explore the relationship between EXTL3 and NPC1 functions in baculovirus transduction. We found that in the presence of heparin sodium, the efficiency of baculovirus transduction in NPC1−/− HeLa cells was further reduced (Fig. 3i and Supplementary Fig. 7d). In addition, NPC1 overexpression rescue did not eliminate the inhibitory activity of heparin sodium on baculovirus transduction (Fig. 3i and Supplementary Fig. 7d). These results suggested that NPC1 could affect baculovirus transduction through a EXTL3-independent pathway and that the functions of NPC1 and EXTL3 in baculovirus transduction were additive to each other.
NPC1 participates in baculovirus endosomal escape mainly through I and C ___domain-mediated membrane fusion
To elucidate the mechanism of action of NPC1 in baculovirus endosomal escape, we first used DiOC18 dye to label baculovirus particles (DiOC18-Bac-EGFP). The fluorescence of DiOC18 is self-quenched in labeled viruses and dequenched when membrane fusion occurs77,78. DiOC18-staining experiments showed that the membrane fusion of baculovirus was significantly suppressed in NPC1−/− HeLa cells which could be restored by NPC1 overexpression, as analyzed by confocal microscopy and flow cytometry experiments (Fig. 4a, b). These results are consistent with the above findings and strongly suggested that NPC1 promoted baculovirus endosomal escape by involving in membrane fusion.
DiOC18-staining experiment for illustration of NPC1 function in baculovirus membrane fusion, as analyzed by confocal microscopy (a) or flow cytometry analyses (b). The p-value between DiOC18 only and Nontarget groups is 1e-5. The p-value between NPC1−/− and NPC1 rescued groups is 4e-6. c The structural organization of NPC1. d Design of NPC1 constructs. Analyses of the interactions between HA-tagged GP64 and Myc-tagged full-length NPC1, NPC1-A, NPC1-C, NPC1-I (e), NPC1-ΔA, NPC1-ΔC or NPC1-ΔI (f). g Investigation of the effects of overexpression rescue with full-length or truncation constructs of NPC1 on baculovirus transduction, as determined by the EGFP-positive cells via flow cytometry. Mock, PBS treatment without baculovirus. The p-values between NPC1−/− and Nontarget and NPC1 rescued groups are 8e-8 and 1e-7, respectively. h Neutralization of Bac-EGFP transduction in HEK-293T cells by recombinant protein of NPC1 C ___domain, as determined by flow cytometry analyses. i ESI-MS confirmation of purified GST-I protein (n = 1). j Far-western analysis of the interaction between GP64 and GST-I. For (b, g, and h), Data are presented as mean ± SD (n = 3) from three biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t-test. For (e, f, and j), The experiment is repeated three times independently with similar results. Source data are provided as a Source Data file.
NPC1 contains 13 transmembrane helices (TM) and 3 distinct lumenal domains A, C, and I79 (Fig. 4c). It has been reported that NPC1 affects the endosomal escape of Ebola virus through its C ___domain67,80. To assess which luminal ___domain of NPC1 interacts with GP64, we performed co-immunoprecipitation experiments. We designed constructs encoding individual domains of A, C, and I (NPC1-A, NPC1-C, and NPC1-I), and constructs with deletions of individual ___domain (NPC1-ΔA, NPC1-ΔC, and NPC1-ΔI) (Fig. 4d). Co-IP analyses of the interactions between GP64 and NPC1-A, NPC1-C or NPC1-I showed that both NPC1-C and NPC1-I could interact with GP64 (Fig. 4e). Additional Co-IP analyses showed that GP64 could interact with full-length NPC1 and truncation constructs NPC1-ΔA, NPC1-ΔC and NPC1-ΔI (Fig. 4f), suggesting that the A, C or I domains might have redundant functions for GP64 binding. Collectively, these Co-IP experiments suggested that the C and I domains of NPC1 could both involve in the interactions with GP64.
To understand the roles of individual NPC1 domains in supporting baculovirus transduction, we performed a rescue experiment by generating stable cell lines harboring full-length NPC1 or truncation constructs on the basis of NPC1−/− HeLa cells (Supplementary Fig. 8a). It was found that the truncation construct NPC1-ΔA could partly restore the susceptibility of cells to baculovirus transduction whereas NPC1-ΔC or NPC1-ΔI did not significantly restore baculovirus transduction (Fig. 4g and Supplementary Fig. 8b). These results were consistent with the Co-IP results in that C and I domains play more important roles in baculovirus transduction than A ___domain. In addition, we noted that while full-length NPC1 could restore baculovirus transduction to a level similar to that in non-targeting sgRNA group, none of the truncation constructs could achieve a comparable rescuing efficiency (Fig. 4g). This suggested that the intact structural organization of NPC1 could be important for GP64 binding and baculovirus transduction. Collectively, the above results suggested that C and I domains played predominant roles in NPC1 binding with GP64 while the intact structure of NPC1 might be also important.
Next, we sought to investigate whether purified proteins of NPC1 C and I domains could interact with baculovirus or baculovirus proteins. We first performed a neutralization experiment with a commercial recombinant protein of NPC1 C ___domain (residues R372 to F622). It was found that NPC1 C ___domain could inhibit Bac-EGFP transduction in a dose-dependent manner in HEK-293T cells (Fig. 4h). Because the function of NPC1-I ___domain was rarely reported in viral infection and no commercial source could be found, we expressed and purified GST tagged NPC1-I ___domain protein from BL21 (DE3) Escherichia coli cells. Despite of an unknown impurity band (Supplementary Fig. 8c), the identity of the target protein band was confirmed by mass spectrometry (Fig. 4i). We set a far WB assay to investigate the interaction between NPC-I and GP64, where GST-tagged NPC1-I ___domain was resolved by SDS-PAGE, transferred to a PVDF membrane and then probed with purified GP64 protein. It was found that NPC1-I ___domain could directly interact with GP64 in vitro (Fig. 4j). Interestingly, we found that NPC1-C and NPC1-I shared 30% protein sequence similarity as analyzed by ClustalW (Supplementary Fig. 8d). It was thus likely that NPC1-I resembled NPC1-C for virus interaction and endosomal escape, as reported in Ebola virus67,80,81.
Baculovirus transduction in mouse liver is dependent on NPC1
To understand baculovirus transduction in mice, we first examined the toxicity of intravenously administrated baculovirus. We administrated Bac-EGFP to C57BL/6J mice via tail vein and found that Bac-EGFP resulted in a transient loss of body weight in the first four days post injection (Supplementary Fig. 9a), which was eventually recovered. Similarly, a transient change of blood routine was observed in Bac-EGFP-treated mice (Supplementary Fig. 9b).
We then analyzed the vulnerability of different tissues to baculovirus transduction in wild-type mice and found that liver displayed highest degree of Bac-EGFP transduction (Fig. 5a, d). It was noted that wild-type mice liver exhibited highest expression of Npc1 (Fig. 5b). To investigate whether baculovirus transduction in mouse liver was related with Npc1 expression, we sought to construct Npc1 knockout mice. Unfortunately, Npc1−/− C57BL/6J mice showed notably reduced birth rate and body weight (Supplementary Fig. 9c). A previous study also showed that homozygous Npc1 knockout in mice resulted in reduced general health and pathologic changes in multiple organs82. By contrast, Npc1−/+ mice did not show defects in birth rate or body weight (Supplementary Fig. 9c), nor did liver show pathologic changes as compared to wild-type mice (Supplementary Fig. 9d). Therefore, we used Npc1−/+ heterozygous C57BL/6J mice for subsequent analyses. The gene disruption of Npc1 was validated in the mRNA levels (Fig. 5b). Importantly, Bac-EGFP transduction in liver tissues of Npc1−/+ mice was significantly reduced (Fig. 5c) as compared to that in wild-type mice, suggesting that baculovirus transduction in mouse liver was dependent on Npc1 expression. It was also noted that in Npc1−/+ mice, EGFP mRNA could not be detected in organs other than liver (Fig. 5d).
a Investigation of the tissue tropism of Bac-EGFP in wild-type mice at 72 h post intravenous injection, as determined by EGFP expression at protein (n = 3). The fluorescence images are acquired using confocal microscopy on flash-frozen tissues that are fixed with optimal cutting temperature compound (OCT). b RT-qPCR quantification of NPC1 mRNA in the lysate of liver, kidney, lung, brain, and spleen in wild-type and Npc1−/+ mice. The p-value between WT and Npc1−/+ in spleen groups is 1e-8. RT-qPCR quantification of EGFP mRNA in the lysate of liver (c) and other tissues including kidney, lung, brain, and spleen (d) in wild-type and heterozygous Npc1−/+ knockout mice at 72 h post-Bac-EGFP injection. The EGFP mRNA in the lysates of kidney, lung, brain, and spleen (d) in heterozygous Npc1−/+ knockout mice is below the limit of detection. For (b–d), β-actin is used as an internal control. The data in this figure are from independent biological replicates. The significant difference is analyzed by two-tailed unpaired Student’s t-test. For (b and d), Data are presented as mean ± SD of biological replicates (n = 5 for (b) and = 3 for (d)). For c, Data are collected from nine biological replicates (n = 9). The thick horizontal dashed line represents the median and thin horizontal dashed line indicates interquartile range between 25th and 75th percentile. Source data are provided as a Source Data file.
Discussion
The large packaging capacity allows baculovirus to carry multiple transgene cassettes for the expression of large protein complexes in insect cells83,84,85. For the same reason, baculovirus has been an attractive gene therapy vector for delivering large or multiple gene cassettes in single viral particles, which can overcome the packaging limitation associated with conventional viral vectors. However, despite of the existing studies on baculovirus entry mechanism in mammalian cells54,55,57,58,59, there has been no systematic investigation on baculovirus host factors in mammalian cells. In the present study, we performed a forward genetic screen using the SfCRISPR library that carries approximately 1400 cell surface proteins. Two proteins, EXTL3 and NPC1, were identified as host dependency factors and were shown to involve in baculovirus attachment and entry, and endosomal escape processes respectively.
Investigation of EXTL3 suggested that it promoted baculovirus attachment and entry by mediating heparan sulfate (HS) biosynthesis. EXT2, another enzyme involved in HSPG biosynthesis, was also shown to affect baculovirus transduction. These results were consistent with the previous findings54,55 and confirmed the importance of HS for baculovirus transduction. As HS is widely distributed across different cell types and tissues, we did not further explore the dependency of baculovirus transduction on HS. However, it is likely that different cells or tissues display different expression profiles of EXT family proteins or proteins related to HS biosynthesis, which can be important determinants for baculovirus transduction.
Moreover, it was found in the present study that NPC1 participated in baculovirus membrane fusion and endosomal escape, but not entry or attachment. These findings were consistent with the function of NPC1 in infection of filoviruses86 and African swine fever virus (ASFV)87. Unlike the case in EBOV where the C ___domain of NPC1 interacted with EBOV glycoprotein86, it was found in the present study that both the C and I domains of NPC1 interacted with baculovirus GP64 protein. Additionally, an NPC1 inhibitor U18666A that can block EBOV infection was found to be efficient in inhibiting baculovirus transduction in mammalian cells. The structural basis of baculovirus GP64 interaction with NPC1-C and -I domains and the mechanism of U18666A-mediated inhibition of baculovirus transduction require further investigation. In consistency with these observations in mammalian cells, previous studies suggested that Bombyx mori NPC1 and NPC2 proteins promoted Bombyx mori nucleopolyhedrovirus (BmNPV) infection in insect cells by facilitating membrane fusion and endosomal escape88,89. More interestingly, BafA1 treatment experiments suggested the NPC1-mediated endosomal escape could release Bac genome into cytoplasm for transgene expression, the process of which will also accelerate Bac genome degradation.
One major discovery in the present study is that partial knockout of Npc1 reduced baculovirus transduction in mouse liver. To the best of our knowledge, this was the first study revealing host factors for in vivo baculovirus transduction in mammals. This result implied that the expression of NPC1 and its function in endosomal escape could be a critical determinant for baculovirus transduction in mammals. This knowledge can be exploited to design novel strategies of targeted baculovirus delivery for tissues or cells with high NPC1 expression. In addition, our study shed light on engineering baculovirus GP64 protein for enhanced interactions with mammalian NPC1. Nevertheless, although the present and previous studies revealed general safety of baculovirus administration in mice, the safety of baculovirus in humans remains unknown and should be carefully addressed in future studies.
In addition to EXTL3 and NPC1, our studies revealed several other genes that might involve in baculovirus transduction in HEK-293T cells. Among these genes, MBTPS2 seemed to have a major impact on the transduction efficiency. MBTPS2 encodes site-2 protease (S2P), which is a hydrophobic zinc metalloprotease. S2P can cleave transmembrane proteins to release their nucleus-localizing components that regulate the transcription of genes involved in lipid biosynthesis90 and ER stress response91. It has been reported that S2P can mediate the activation of antiviral proteins through regulated intramembrane proteolysis (RIP) process during HCV infection92. Thus, it could be interesting to investigate in future studies whether MBTPS2 plays similar functions during baculovirus transduction in mammalian cells.
One limitation in our study was that the HEK-293T cells used in the screening process were deficient of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. While the high baculovirus transduction efficiency in HEK-293T could reduce the background signal in the negative EGFP selection, the lack of cGAS-STING pathway eliminated the possibility of uncovering host factors involved in cGAS-STING signaling Recent studies showed that cGAS-STING signaling could impede baculovirus transduction in mammalian cells by affecting interferon (IFN) production60. Interestingly, cGAS-STING-mediated IFN production in mammalian cells could be inhibited by AcMNPV P26 protein93. In future studies, it would be interesting to perform CRISPR screen on cGAS-STING-competent cells to uncover baculovirus host factors that act in the context of IFN signaling.
Methods
Cell culture
HeLa cells were obtained from the American Type Culture Collection (ATCC). HEK-293T, U87-MG, Club, HK-2, U251, and A549 cells were obtained from the Cell Bank of Shanghai Institutes for Biological Science (SIBS). All the cells used in this study were validated by VivaCell Biosciences (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermo, Pittsburgh, USA) supplemented with 10% fetal bovine serum (FBS, Thermo) and 1% penicillin-streptomycin (Thermo) and maintained at 37 °C in a fully humidified incubator containing 5% CO2. All cells were confirmed by PCR to be free of mycoplasma contamination.
Lentivirus production and transduction
To produce lentivirus (LVs), HEK-293T cells at a confluence of 70–90% were transfected with LV packaging plasmid pMD2.G, envelope plasmid psPAX, and transfer plasmid pLentiCRISPR-v2 that carried Cas9 gene and a single sgRNA or pooled sgRNA plasmid library with a mass ratio of 1: 1.5: 2 using Lipofectamine 3000 (Thermo). In the case of overexpression, the transfer plasmids were pLenti-EF1α-IRES-NPC1-Bsd, plenti-EF1α-IRES-EXTL3-Bsd, pLenti-EF1α-IRES-NPC1ΔA-Bsd, pLenti-EF1α-IRES-NPC1ΔC-Bsd and pLenti-EF1α-IRES-NPC1ΔI-Bsd). At 6 h after transfection, the medium was replaced with fresh medium. The medium supernatant containing LVs was harvested at 48–60 h post-transfection by centrifugation at 2000 rpm (420 × g) for 10 min, filtrated through a 0.45 μm filter (Merck, Darmstadt, Germany) and stored at −80 °C.
HeLa cells and HEK-293T cells were transduced with LVs in the presence of polybrene (10 μg/mL, Merck) using spinfection through centrifugation at 2000 rpm (420 × g) for 2 h. At 24 h post-transduction, LV-containing medium was removed and cells were cultured in fresh medium in the presence of 1–2 μg/mL puromycin for 3 to 5 days or 2–5 μg/mL blasticidin (Thermo) for 7–10 days to remove empty cells containing no LVs. Finally, survived cells were collected, aliquoted, and stored in liquid nitrogen.
Baculovirus production and transduction
Bac-EGFP recombinant viruses were generated using Bac-to-Bac baculoviral expression system (Thermo, Cat. No. A11101). Transgene plasmid pFastBac carrying EGFP gene was transformed into DH10Bac E. coli competent cells that harbored the parental bacmid to form a recombinant bacmid encoding the EGFP transgene. The recombinant bacmid was then transfected into insect cells for production of recombinant baculovirus particles. At 72 h post-transfection, the culture supernatant containing baculovirus particles was harvested and concentrated by Optima XPN-100 Ultracentrifuge (Beckman Coulter, California, USA). Virus titer was determined using the 50% tissue culture infectious dose (TCID50) assay94.
For baculovirus transduction, HEK-293T or HeLa cells were seeded at a cell density of 40%. At 24 h after seeding, the cells were transduced with baculovirus at an MOI of 1 or 2 for HEK-293T or HeLa respectively. MOIs other than 1 and 2 were indicated in figure legends. At 24 h after baculovirus transduction, baculovirus-containing medium was removed and the cells were incubated in DMEM (Thermo) supplemented with 10% FBS (Thermo) for another 24 h. Thereafter, the cells were collected and the efficiency of baculovirus transduction was quantified by flow cytometry (CytoFLEX, Beckman Coulter, California, USA) and visualized by fluorescence microscope (EVOS M5000, Thermo). The expression of EGFP mRNA was quantified by RT-qPCR using specific primers (Supplementary Table 2).
Construction of surfaceome CRISPR library
The human surfaceome CRISPR library was described in our previous work62, which contained 16,975 sgRNAs targeting 1314 surface protein genes and 1000 non-targeting sgRNAs. To construct surfaceome CRISPR library in HEK-293T, the cells were transduced with LV library at an MOI of 0.3 using spinfection as described above. Cells of more than 500-fold coverage of the library size were collected, aliquoted, and stored in liquid nitrogen.
CRISPR screening for baculovirus host factors and next-generation sequencing (NGS) analyses of sgRNA enrichment
The HEK-293T cell library of 1 × 107 cells was seeded onto 15 cm petri dishes. At 24 h after seeding, the cells were incubated with recombinant baculovirus carrying an EGFP transgene (Bac-EGFP) of 3 × 107 infective units (IU) for 24 h. The medium containing virus was then removed and cells washed with phosphate-buffered saline (PBS) for three times and then cultured in fresh medium for another 24 h. Then the treated cells were harvested and sorted using flow cytometry (Moflo, Beckman Coulter, California, USA) for EGFP-negative cells. Genomic DNA of the sorted cells was extracted using phenol: chloroform: isoamyl alcohol (v/v/v, 25:24:1) and then purified using ethanol precipitation. Genome-integrated sgRNAs were amplified from the collected genomic DNA by PCR using primers containing Illumina adapters (Supplementary Table 1). PCR amplicons were analyzed by Genewiz (Suzhou, Jiangsu, China) using next-generation sequencing (NGS) on Illumina HiSeq 3000 platform. After removing the adapters, the 20 bp sgRNA was mapped to the reference sgRNA with 1 bp mismatch allowed. The raw read counts were subjected to MAGeCK analyses to determine the enriched sgRNA and gene knockouts. A false discovery rate (FDR) of less than 0.05 was applied to identify significantly enriched sgRNAs and candidate gene knockouts.
Generation of gene knockout cells
The LV transfer plasmids pLentiCRISPR-v2-sgRNA carrying single sgRNAs (Supplementary Table 3) for knockout cell line construction were generated as previously described above62. The LVs were packaged and transduced onto cells as described above. To evaluate the knockout efficiency, the genomic DNA of knockout cells was extracted using Quick Extraction kit (Lucigen, Wisconsin, USA) and the modified genomic sites were PCR amplified using corresponding primers (Supplementary Table 4). The PCR amplicons were sequenced by Sanger sequencing (Genewiz) and gene disruption efficiency was analyzed by TIDE website (https://tide.nki.nl/)95. Single clones were obtained by cell sorting using flow cytometry (Moflo, Beckman) and genotyped by Sanger sequencing to determine the mutations at each allele.
RT-qPCR
Total RNA from cultured cells was extracted using TRIzol (Thermo), chloroform (Sinopharm, Ningbo, China) and purified using isopropanol precipitation. RNA was then reverse transcribed into cDNA by PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). The mRNA levels were determined by reverse transcription quantitative PCR (RT-qPCR) using SYBR green dye on Applied Biosystems Q6 Real-Time PCR cycler (Thermo) and specific primers (Supplementary Table 2). All SYBR Green primers were validated with dissociation curves. The expression of genes was normalized to β-actin or RPLP0.
For RT-qPCR on mouse tissues, the tissue samples were collected, frozen in liquid nitrogen, and homogenized using grinding beads through tissue grinding device (Jingxin, Shanghai, China) in TRIzol reagent (Thermo). The extraction and quantification processes in tissue RNA were the same to that in cell culture samples as described above.
qPCR quantification of baculovirus genome
Genomic DNA of baculovirus or baculovirus-containing cells was extracted using phenol: chloroform: isoamyl alcohol (v/v/v, 25: 24: 1) and then purified using ethanol precipitation. The total DNA concentration was determined for each sample. For quantitative PCR (qPCR) reaction, 2 ng total DNA was added into each reaction as the template. GP64 DNA, which was used as indicator of viral genome content, was quantified by qPCR using SYBR green dye on Applied Biosystems Q6 Real-Time PCR cycler (Thermo) and specific primers (Supplementary Table 2). All SYBR Green primers were validated with dissociation curves. The genomic DNA level was normalized to β-actin.
Gene knockdown using siRNA
HEK-293T cells were seeded onto 6-well plates with a density of 5 × 105 cells per well. At 24 h after seeding, cells were transfected with 100 pmol siRNA (Genepharma, Shanghai, China) (Supplementary Table 5) using 7.5 μL Lipofectamine 2000 (Thermo) for 6 h, then washed with PBS and cultured in fresh DMEM (Thermo) supplemented with 10% fetal bovine serum (FBS, Thermo). At 48 h post-transfection, cells were infected with baculovirus at an MOI of 1 for 24 h, then washed with PBS for three times and cultured in fresh medium for 24 h. The cell samples were harvested and lysed for total RNA extraction, and the mRNA levels of EGFP, EXTL3, and NPC1 in cell lysate were determined using RT-qPCR as described above.
Virus attachment and entry assays
Virus attachment and entry assays were performed as described96 with minor modifications. HEK-293T cells or HeLa cells were seeded onto 12-well plates at a density of 200,000 cells per well and incubated overnight. For virus attachment assay, cells were incubated with baculovirus at an MOI of 20 in cold medium without FBS on ice for 60 min, then washed with cold Dulbecco’s phosphate-buffered saline (DPBS) for three times and harvested. The baculovirus genomic DNA was extracted from the cells containing attached virus and quantified by qPCR as described above. For virus entry assay, cells were incubated with baculovirus at an MOI of 20 in cold medium on ice for 60 min, washed by cold DPBS for three times, treated with pre-warmed medium containing FBS, and then incubated at 37 °C for 40 min. The treated cells were washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. The genomic DNA of internalized baculovirus was extracted and quantified by qPCR as described above.
Construction of stable cell lines harboring overexpressed genes
EXTL3 and NPC1 genes were codon-optimized for expression in human cells and synthesized by Genewiz (Supplementary Tables 6, 7). The 20 bp sgRNA-targeting sites and PAM sequences were mutated with silent mutations. Myc and FLAG tags were added to the C-terminus of these genes for WB detection. These genes were cloned into the XbaI and BamH1 sites of pLV-EF1α-IRES-Bsd plasmid. The lentiviral overexpression plasmids of NPC1 truncation mutants (NPC1-ΔA, NPC1-ΔC, and NPC1-ΔI) were constructed from the full-length gene in pLV-EF1ɑ-IRES-NPC1-Bsd. For LV packaging, the transgene plasmid containing EXTL3, NPC1, NPC1ΔA, NPC1ΔC or NPC1ΔI was co-transfected into HEK-293T cells with helper plasmids pMD2.G and psPAX as described above. The transgene-containing LVs were transduced into EXTL3−/− or NPC1−/− cells, and the cells were subjected to selection with 2 μg/mL blasticidin (Thermo) for 7 to 10 days to purge empty cells containing no LVs. Finally, survived cells were collected, aliquoted, and stored in liquid nitrogen.
Analysis of the intracellular baculovirus genomic DNA
The experiment was performed in an approach similar to a previous study62. Non-targeting sgRNA-treated, NPC1−/− and NPC1 overexpression rescued HeLa cells were seeded onto 24-well plates at a density of 80,000 cells per well and incubated overnight. The next day the cells were incubated with Bac-EGFP at an MOI of 2 for 1 h. The virus-containing medium was removed and the cells were washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. These treated cells were supplemented with pre-warmed DMEM medium containing FBS and then incubated at 37 °C for indicated time. Intracellular baculovirus genomic DNA was extracted from each well. The total baculovirus GP64 amount in each well was determined by qPCR and then normalized to the zero time point.
Cell Counting Kit-8 assay (CCK-8)
Non-targeting sgRNA, NPC1−/−, and NPC1 rescued HeLa cells were seeded onto 96-well plates at a density of 10,000 cells per well and incubated at 37 °C overnight. The cells were incubated with Bac-EGFP at an MOI of 2 for 1 h, washed with PBS for three times and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. At indicated time points, the supernatant was removed and cells incubated with complete medium containing 10% CCK-8 reagent (MeilunBio, Liaoning, China) at 37 °C for 1 h. Cell proliferation was quantified by measuring absorbance at 450 nm using microplate reader (SpectraMax iD3, Molecular Devices, Shanghai, China). The background signal in empty wells without cells was subtracted from each sample.
Bafilomycin A1 (BafA1) treatment
Wild-type HeLa cells were seeded on to 24-well plates at a density of 80,000 cells per well and incubated overnight. The next day the cells were pre-incubated with 100 nM BafA1 (MedChemExpress, New Jersey, USA, Cat. No. HY-100558) for 1 h, followed by transduction with Bac-EGFP at an MOI of 2 for 1 h in the presence of 100 nM BafA1. The virus-containing medium was then removed, the cells washed three times with PBS and then treated with 0.25% trypsin (Thermo) for 30 s to remove surface-bound baculovirus particles. These cells were incubated in DMEM medium containing FBS and 10 nM BafA1 for indicated time. The genomic DNA of intracellular baculovirus was extracted from cells and quantified by qPCR as described above.
For CCK-8 assay, cells were seeded on to 96-well plates at a density of 10,000 cells per well. The next day, the cells were pre-incubated with 100 nM BafA1 and transduced with Bac-EGFP to mimic the treatment as described above. At 48 h after removal of virus, cell proliferation was quantified by CCK-8 as described above at indicated time points.
WB analyses
For WB analyses, cells were lysed with RIPA buffer (Beyotime Biotechnology, Beijing, China) on ice for 10 min and centrifuged at 12,000 rpm (13,523 × g) at 4 °C for 10 min to remove cell debris. The total protein concentration in cell lysate was determined using the BCA Protein Assay Kit (Thermo). Cell lysate was mixed with SDS-PAGE loading buffer (Takara) containing 200 mM dithiothreitol (DTT), incubated at 95 °C for 10 min, and resolved on 4–12% PAGE gels (GenScript, Nanjing, China). Protein samples were transferred onto nitrocellulose membranes (Merck) using an iBlot gel transfer system (Thermo). The following primary and secondary antibodies were used in WB including anti-Myc rabbit antibody (CST, Cat. No. 2272S), anti-HA rabbit antibody (CST, Cat. No. 3724S), anti-Heparan Sulfate Proteoglycan 2/Perlecan antibody (Abcam, Cambridge, UK, Cat. No. ab255829) and HRP-conjugated anti-rabbit IgG (CST, Cat. No. 5127S). Anti-β actin antibody conjugated with HRP (CST, Cat. No. 5125S) was used as an internal control.
Co-immunoprecipitation (Co-IP)
HEK-293T cells were seeded onto 6-well plates at a density of 1 × 106 cells per well. After the confluency reached 70% to 80%, plasmid was transfected into cells. Plasmids containing Myc-labeled NPC1 or NPC1 mutant and plasmids containing HA-labeled GP64 (Supplementary Table 8) were co-transfected into HEK-293T cells by Lipofectamine 3000 (Thermo). At 24 h after transfection, cells were resuspended with co-IP lysis buffer. One-half of the lysate was used to incubate with magnetic beads (Thermo) containing anti-HA or anti-Myc antibodies at room temperature for 1 h. Magnetic beads were collected with a magnetic rack (Thermo), then proteins bound to the magnetic beads were eluted by 1x SDS-PAGE loading buffer containing DTT. Finally, total proteins and co-immunoprecipitated proteins were detected by WB as described above.
Recombinant NPC1 protein neutralization assay
HEK-293T cells were seeded on to 48-well plates at a density of 1 × 105 cells per well. After Bac-EGFP incubation with recombinant protein of NPC1 C ___domain (residues R372-F622) (SinoBiological, Beijing, China, Cat. No. 16499-H32H) at indicated concentrations at 4 °C for 30 min. Thereafter, HEK-293T cells were transduced with Bac-EGFP at an MOI of 1 for 24 h in the presence of NPC1-C ___domain protein. Then baculovirus-containing medium was removed and the cells were incubated in DMEM (Thermo) supplemented with 10% FBS (Thermo) for 24 h and then harvested. The efficiency of baculovirus transduction was quantified by flow cytometry (CytoFLEX, Beckman Coulter, California, USA).
Expression and purification of the human NPC1-I ___domain
NPC1-I ___domain was codon-optimized for expression in Escherichia coli, synthesized by Genewiz, and cloned into pGEX4T-1-GST plasmid. The recombinant pGEX4T-1-GST-NPC1-I plasmid was transformed into chemically competent BL21 (DE3) E. coli cells. After overnight culturing, single colonies were selected and amplified in Luria–Bertani (LB) medium containing (100 mg/mL) ampicillin overnight. The next day, the culture was inoculated into fresh LB medium at 1: 100 dilution and cultured at 37 °C for about 3 h until OD600 reached 0.6–0.8. Protein expression was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22 °C overnight. The cells were then collected by centrifugation at 4,000 rpm (1681 × g) for 15 min at 4 °C.
For protein purification, the cell pellet was resuspended with PBS and then lysed by sonication. The cell lysate was centrifuged at 12,000 rpm (13,523 × g) for 30 min at 4 °C and the supernatant was isolated. The supernatant was run through a glutathione resin affinity chromatography column (Merck) for protein capture. The resin was sufficiently washed with PBS and the protein was eluted with wash buffer supplemented with reduced glutathione. The eluted proteins were concentrated and further purified by size exclusion chromatography (Superdex 200 Increase 10/300GL) with a buffer containing 20 mM Tris pH 8.0, 250 mM NaCl. The fractions containing target proteins were pooled and concentrated for mass spectrometry analysis.
Mass spectrometry validation of purified NPC1-I protein
The purified NPC1-I protein was validated by Orbitrap Fusion MS (Thermo Fisher, San Jose, CA) using Proteome Discoverer 2.2 and Xcalibur analysis software at the Analytical Chemistry Platform at SIAIS, ShanghaiTech University. The entire gels were rinsed with water for 3–4 h, then the bands of GST (control, n = 1) and NPC-I (sample, n = 1) were excised with a clean scalpel. The two excised bands were cut into cubes (1 mm × 1 mm) separately. The gel slices were transferred into two microcentrifuge tubes and spun down by a microcentrifuge. For in-gel reduction, alkylation, and de-staining, the gels were incubated with 100 μL de-staining solution (100 mM ammonium bicarbonate buffer containing 50% acetonitrile, vol/vol) for 30 min at 37 °C, and then incubated with 500 μL neat acetonitrile for 10 min until gel slices shrank and became opaque and stiff. These gel slices were span to remove all liquid and 40 μL of 10 mM DTT solution was added to cover gel slices for 30 min at 56 °C in an air thermostat. The tubes were chilled down to room temperature, and incubated with 500 μL acetonitrile for 10 min and then all liquid was removed. The gels were incubated with 40 μL of 55 mM iodoacetamide solution for 20 min at room temperature in the dark, treated with acetonitrile, and then all liquid removed. The dry gels were incubated with 70 μL trypsin buffer containing 13 μg/mL trypsin for 100 min at 4 °C and then incubated with 20 μL 100 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile at 37 °C overnight. The next day the samples were incubated with 200 μL extraction buffer (1 vol: 2 vol of 5% formic acid: acetonitrile) for 15 min at 37 °C in a shaker. Then the supernatant was collected into a PCR tube, dried in a vacuum centrifuge, and stored at 4 °C for further analysis.
For liquid chromatography (LC) analysis, the above samples were supplemented with 20 μL of 0.1% (vol/vol) formic acid, vortexed, and centrifuged for 5 min at 12,000 × g. These treated peptides were loaded onto an analytical column (Ionopticks, AUR2-25075C18A, 25 cm × 75 μm, C18, 1.6 μm) connected to an Easy-nLC1200 UHPLC-Orbitrap Fusion (Thermo Fisher Scientific). The elution gradient and mobile phase constitution used for peptide separation were set as follows: 0–60 min, 5–30% buffer B; 60–70 min, 30–45% buffer B; 70–75 min, 45–100% buffer B; 75–80 min, 100% buffer B at a flow rate of 300 nL/min. Mobile phase in buffer A was 0.1% formic acid in water and mobile phase in buffer B was 0.1% formic acid in 80% acetonitrile.
Peptides eluted from the LC column were directly electro-sprayed into the mass spectrometer with the application of a distal 2.1 kV spray voltage. Survey full-scan mass spectra (from m/z 350 to 1800) were acquired in the Orbitrap analyzer (Orbitrap Fusion, Thermo Fisher Scientific) with resolution r = 60,000 at m/z 400. The cycle time of the MS-MS2 events was 3 s, sequentially generated and selected from the full mass spectrum at a 32% normalized collision energy. The dynamic exclusion time was set to 10 s. The acquired MS/MS data were analyzed using the AA sequence of protein GST, using Protein Discoverer 2.2 with the parameter settings as follows: precursor and fragment mass tolerance of 10 ppm and 0.02 Da and dynamic modifications of + 15.995 Da for Oxidation (Met) and + 42.011 Da for Acetyl (N-terminus), and + 57.021 Da for Carbamidomethyl (C terminus) as static modifications. To accurately estimate peptide probabilities and filter the false discovery, the fixed value PSM validator node was used and set to have a maximum delta Cn of 0.05. Trypsin was defined as cleavage enzyme and the maximal number of missed cleavage sites was set to two.
Far western blotting
Far western blotting was performed as previously described97. Briefly, 0.2–1 μg purified NPC1-I protein (with an unknown impurity band) or a control GST protein was mixed with SDS-PAGE loading buffer (Takara) containing 200 mM DTT, incubated at 95 °C for 10 min and resolved on 4–12% PAGE gels (GenScript, Nanjing, China). Protein samples were transferred onto nitrocellulose (PVDF) membranes (Merck) using an iBlot gel transfer system (Thermo). PVDF membrane was treated with PBS solution containing gradient concentrations of guanidine hydrochloride (GuHCl) solution (Solarbio, China) for protein unfolding and refolding. The GuHCl gradient was decreased from 6 M to 3 M and then 0.1 M with the membrane being treated with each gradient for 30 min. Finally, the membrane was treated with PBS without GuHCl at 4 °C overnight. The membrane was then blocked with PBS containing 5% milk (w/v) and incubated with 1–10 μg GP64 protein (SinoBiological, Beijing, China) overnight at 4 °C. The membrane was then treated with an anti-GP64 mouse antibody (Abcam, Cat. No. ab91214) as the primary antibody and an HRP-conjugated anti-mouse antibody (R&D, Cat. No. HAF007) as the secondary antibody. The bait protein could be detected at the ___location of the prey protein on the membrane if they interact to form a complex.
Transduction with DiOC18-labeled baculovirus
Baculovirus was labeled with DiOC18 (Thermo) at a final concentration of 2 μM in PBS. The solution was rotated at room temperature in the dark for 1 h and then filtered through a 0.22 μm pore size syringe filter (Merck) to remove unbound dyes and aggregates. Cells were infected with DiOC18-labeled baculovirus at 37 °C for 3 h, then washed with PBS three times and treated with trypsin briefly to remove free and surface-bound viruses. Thereafter, the cells were fixed with PFA and analyzed by flow cytometry (CytoFLEX, Beckman Coulter, California, USA) and confocal microscopy (LSM 710, Zeiss, Oberkochen, Germany). A control group with DiOC18 dye alone, without the addition of baculovirus, was prepared following the same filtration process as described above to assess the background fluorescence labeling of cells by free dyes in the DiOC18-Bac-EGFP-treated groups.
Baculovirus transduction in mice
Npc1−/+ and wild-type C57BL/6 mice were purchased from, bred, and raised in GemPharmatech (Nanjing, China). All mice were housed in animal facility with ambient room temperature of 20–26 °C, humidity of 50–70%, and dark/light cycle of 12 h. Baculovirus-related animal experiments were performed in OBiO-tech corporation (Shanghai, China). For baculovirus, transduction, 6–8-week-old male Npc1−/+ and wild-type C57BL/6 mice were used. The mice were maintained within a Specific Pathogen-Free (SPF) facility with free access to water and food. The housing facility for mice was under a 12:12 h light: dark cycle at temperatures 20–26 °C, humidity 40–70%. For transduction experiments, mice were randomly grouped (n = 3 to 9 per group as indicated) and injected with baculoviruses through tail vein with a dose of 4.4 × 104 IU per gram body weight. Animals were sacrificed under an anesthetic condition at the indicated time post-baculovirus transduction. The tissues including liver, brain, spleen, lung, and kidney were collected and flash-frozen in liquid nitrogen or fixed in PFA buffer for further analyses.
For tissue tropism of Bac-EGFP transduction, Bac-EGFP-transduced tissues were flash-frozen and fixed with optimal cutting temperature compound (OCT). The fixed tissues were sectioned at 5 μm, and the sections were fixed with PFA and stained with Hoechst (Thermo). The fluorescence images were acquired using confocal microscopy (LSM 710, Zeiss).
Statistics and reproducibility
All data were the results from at least three biological replicates and were shown as mean ± standard deviation unless noted otherwise. All experiments were repeated three times independently (biological replicates) unless noted otherwise. No data were excluded for analyses. Statistical analyses and graphing were performed with GraphPad Prism 7.0. The P-values were determined using two-tailed unpaired Student’s t-test unless otherwise noted.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The authors declare that all data supporting the findings of this study are available in the article, its Supplementary Data, its Source Data, or from the corresponding authors upon request. Source data are provided with this paper. The NGS data generated in this study have been deposited in the SRA database under accession code PRJNA1131913. Source data are provided with this paper.
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Acknowledgements
We thank Lishuang Zhang and Pengwei Zhang from the Discovery Technology Platform of SIAIS for the support of flow cytometry experiments, Wei Zhu from the Analytical Chemistry Platform for assistance with mass spectrometry analysis, and the Biomedical Big Data Platform for the design of sgRNA libraries and MAGeCK analyses. This research was supported by the Science and Technology Commission of Shanghai Municipality (23ZR1442100 to J.L.), the Shanghai Frontiers Science Center for Biomacromolecules and Precision Medicine at ShanghaiTech University (2022A0301-417-01 to J.L.), and the Postgraduate Scientific Research Innovation Project of Central South University (2023ZZTS0542 to Y.H.).
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J.L., J.D.L., and H.M. conceived this study, J.L., H.M., Y.H., and Y.N. designed the experiments, and Y.H., H.M., C.D., and C.Y. carried out the experiments and analyzed the data. W.W., H.M., and L.J. designed the sgRNA libraries, and Y.H., H.M., J.L, and J.D.L. wrote the manuscript. All authors contributed to the manuscript and approved the submission.
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J. Liu is the founder and shareholder of Shanghai AsiFlyer Biotechnology. The remaining authors declare no competing interests.
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All study protocols involving mice were approved by the ethical committee of ShanghaiTech University (approval number: 20230821001) and conducted in accordance with regulatory policies in China for the care and use of animals.
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Huang, Y., Mei, H., Deng, C. et al. EXTL3 and NPC1 are mammalian host factors for Autographa californica multiple nucleopolyhedrovirus infection. Nat Commun 15, 7711 (2024). https://doi.org/10.1038/s41467-024-52193-w
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DOI: https://doi.org/10.1038/s41467-024-52193-w
