Baculovirus utilizes cholesterol transporter Niemann–Pick C1 for host cell entry

The dual roles of baculovirus for the control of natural insect populations as an insecticide, and for foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for baculovirus infection in insect cells. Both pretreatment of Bombyx mori embryonic cells (BmE) with NPC1 antagonists (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV (Bombyx mori nuclear polyhedrosis virus) infectivity. Furthermore, we show that the major glycoprotein gp64 of BmNPV, responsible for both receptor binding and fusion, is able to interact predominantly with the BmNPC1 C domain, with an enhanced binding capacity at low pH conditions, indicating that NPC1 most likely plays a role during viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of hNPC1 in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of exploiting host NPC1 proteins during virus intracellular entry events. IMPORTANCE BmNPV is one of the most important members of the Baculoviridae; many viruses in this family have been frequently employed as viral vectors for foreign gene delivery or expression and as biopesticides, but their host receptors still remain unclear. Here, we describe that the intracellular cholesterol transporter BmNPC1 is indispensable for BmNPV infection in insect cells, and it interacts with the major viral glycoprotein gp64. Our study on the role of BmNPC1 in baculovirus infection has further expanded the list of the enveloped viruses that require host NPC1 proteins for entry, and will ultimately help us to uncover the molecular mechanism of the involvement of NPC1 proteins in the entry process of many enveloped viruses.

(represented by relative copy number of gp41) decreased significantly at concentrations of 50-100 µM for imipramine 109 and at concentrations of 1-10 µM for U18666A. For BmE cells pretreated with 100 µM of imipramine or 10 µM of 110 U18666A, the viral load was reduced to 27% and 10%, respectively, compared to the vehicle along treatment control 111 (Fig 2D, 2F). In summary, we conclude that blockage of BmNPC1 function by small molecules can efficiently reduce 112 BmNPV infection in BmE cells, indicating that functional BmNPC1 is required for BmNPV infection in insect cells. Furthermore, viral loads represented by relative copy number of gp41 in BmNPC1 RNAi treated cells was less than 123 10% of that in control RNAi-treated cells at 72 h p.i. (Fig 3E). The viral titer (5.6 log 10 TCID 50 /ml) at 96 h p.i. as 124 quantified by TCID 50 in BmNPC1 RNAi treated cells was significantly lower compared to that in control cells (10.8 125 log 10 TCID 50 /ml, Fig 3F). Collectively, we conclude that knocking down BmNPC1 by RNAi expression in BmE cells 126 can substantially reduce BmNPV infectivity. 127

BmNPC1 null stable cell line was resistant to BmNPV infection. To further confirm that BmNPC1 is essential 128
for BmNPV infection, we generated a BmNPC1 null stable cell line by using CRISPR-Cas9 technology. Partial 129 BmNPC1 gene was replaced by ie1-DsRed and A3-neo expression cassettes (Fig S3B), and the mutated BmNPC1 gene 130 was confirmed by PCR amplification and sequencing ( Fig S3D). We next tested the susceptibility of the NPC1-mutant 131 cells to BmNPV-GFP virus infection, and found that the percentage of GFP positive cells among BmNPC1-null cells as 132 indicated by the expression of red fluorescence was less than 2% of that of the control cells at 96 h p.i. (Fig 4A and  133 4B). Concurrently, the virus load as evaluated by relative copy number of gp41 was reduced to 20% of that in control 134 cells ( Fig 4C). Altogether, these data confirmed that BmNPC1 is required for BmNPV infection, and the cells lacking 135 functional BmNPC1 exhibit substantially reduced susceptibility for BmNPV infection. hNPC1 is able to interact with a primed form of Ebola virus glycoprotein GP, the primary glycoprotein responsible for 138 receptor binding and fusion (18, 25). To investigate whether BmNPC1 contributes to BmNPV infection by interacting 139 with glycoprotein gp64 during viral entry, we performed Co-immunoprecipitation with in-vitro expressed proteins 140 (BmNPC1 domain A, C, I and BmNPV gp64). Gp64-attached protein G agarose beads through mouse anti-gp64 141 antibody were incubated with BmNPC1 domain-specific proteins (domain A, C and I, respectively) for 142 immunoprecipitation. The proteins that bound to the beads, i.e. the co-immunoprecipitation samples were then analyzed 143 by Western blot. As shown in Fig 5A, gp64 proteins could be detected in the immune pellets by gp64 monoclonal 144 antibody. The same immune pellets were subsequently probed with anti-HA antibody, and a specific band 145 corresponding to the NPC1 domain A, C and I in each individual blot was shown in Fig 5B, and the band 146 corresponding to NPC1-C displaying the strongest signal. Our results suggest that gp64 is able to interact with distinct 147 domains (A, C and I) of the NPC1 protein, with the strongest binding affinity for NPC1-C. Next, we performed the 148 reciprocal co-immunoprecipitation experiments, in which NPC1 domain specific protein-attached beads were used to 149 immune-precipitate gp64 proteins. The presence of NPC1-A, C or I in the retrieved beads was confirmed with anti-HA 150 antibody (Fig 5C), however, the band corresponding to gp64 was only present in the NPC1-C-attached immune pellets 151 ( Fig 5D). Our results suggest that domain C of BmNPC1 is sufficient for gp64 binding, while in comparison, the 152 binding affinity of BmNPC1-A or BmNPC1-I to gp64 is relatively low. 153 To further confirm these interactions, we employed a yeast two-hybrid system (Y2H). Full-length gp64 was cloned 154 into the pGBKT7 vector as bait, and BmNPC1-A, BmNPC1-C, BmNPC1-I were used as the prey. These Y2H screens 155 revealed that BmNPC1-C interacted with full-length gp64, whereas BmNPC1-A or BmNPC1-I bound to gp64 with a 156 weak affinity ( Fig S4). Collectively, these results demonstrate a specific interaction between BmNPC1 domain C and 157 BmNPV gp64. 158 Next, we investigated whether the interaction between BmNPV gp64 and BmNPC1 domain C was pH dependent. 159 For this purpose, the Co-IP between BmNPV gp64 and BmNPC1-C was performed at pH 5 or 8. NPC1-C and gp64 160 proteins were expressed with His tag in vitro, and then the protein mixtures were incubated with protein G beads that 161 had been conjugated with mouse monoclonal antibody to gp64. Anti-His antibody was used to detect both gp64 and 162 NPC1-C at the same blot for more accurate comparison. As shown in Fig 5E, we found a stronger BmNPC1-C signal at pH 5 compared to that at pH 8, indicating that BmNPV gp64 binding affinity to BmNPC1-C was enhanced at a lower 164 pH environment. 165 To further confirm which domain of BmNPC1 was most critical for BmNPV infection, we assessed the 166 susceptibility of BmNPV infection in the presence of BmNPC1 domain specific antibodies. Specific antibodies 167 targeting extracellular loops A, C and I of BmNPC1 were produced and incubated with BmE cells for 2 h prior to virus 168 infection. Compared to cells treated with a negative IgG control antibody, BmNPV infection was significantly reduced 169 in BmE cells treated with antibodies specific for BmNPC1-C, but not BmNPC1-A or BmNPC1-I as measured by GFP 170 positive cells and viral gene expression by qPCR (Fig 5F and 5G). These data suggest that the interaction between 171 BmNPC1 domain C and BmNPV gp64 is essential for BmNPV infection in BmE cells. 172

DISCUSSION 174
Baculoviruses have been widely used as important biological agents for controlling insect populations, and 175 powerful biological tools for gene delivery and expression; a better understanding of the molecular mechanisms and 176 host factors involved in baculovirus virus entry is of great significance in bioscience and biotechnology. In this study, 177 we used the well-studied baculovirus BmNPV as a tool to investigate the host factor requirements for baculovirus 178 infection, and found that BmNPC1, the hNPC1 homolog in insect cells, is indispensable for BmNPV infection in insect 179 cells. Our results, together with previous work identifying a role for hNPC1 as an intracellular receptor for Ebola virus 180 entry, have revealed that the conserved host protein NPC1, essential for cholesterol homeostasis, has been exploited by 181 a group of divergent viruses for entry. 182 As an essential component of cellular membranes, cholesterol is one of the most important lipids for maintaining 183 cell viability, cell signaling and physiology (28,29). Two major proteins that function in cholesterol transport are 184 NPC1 and NPC2. NPC2 is the central shuttle in a unidirectional transfer pathway that mobilizes cholesterol to NPC1, 185 leading to NPC1 export of cholesterol from late endosomes (16,23,24,30 binding between hNPC1 and GPcl was hypothesized to facilitate fusion between vial membrane and host endosomal 206 membranes (23). In contrast, the BmNPV glycoprotein gp64 is a Class III fusion protein, and viral fusion is triggered 207 by a caustic pH without proteolytic cleavage. We show here that BmNPC1 proteins are located at both the cell plasma 208 membrane and intracellular compartments, and that BmNPC1 is capable of binding to gp64 at a neutral pH, indicating 209 that BmNPC1 may serve as a host factor to facilitate virus attachment to the cell surface. The enhanced binding 210 between BmNPC1 and gp64 at a lower pH as demonstrated in our study suggests that low pH triggered-conformational 211 changes in the gp64 protein may expose certain epitopes or domains to allow better accessibility by BmNPC1, and the 212 specific binding between gp64 and BmNPC1 may ultimately enable gp64 to maintain a certain structure which is 213 essential for targeting its fusion domain into the endosomal membranes. However, the detailed mechanism of BmNPC1 214 protein in baculovirus entry will rely on future crystallographic examination of the BmNPC1 and gp64 complex. Such 215 studies will ultimately shed light on how a wide range of enveloped viruses utilize the shared host factor NPC1 as part 216 of fusion triggers for intracellular entry.
Baculovirus can transduce a broad range of cells including both vertebrate and invertebrate cells, suggesting that 218 conserved host factors may be involved in viral entry. In our study, we have only examined the role of NPC1 in viral 219 entry in insect cells; it will be of great interest in the future to assess whether NPC1 is involved in viral entry in 220 mammalian cells. Furthermore, the cell surface localization of BmNPC1 in insect cells raises the possibility that 221 BmNPC1 was PCR amplified with specific primer (Table S1) from a BmE cell cDNA library and then cloned into the 246 pMD19-T vector for sequencing. The sequences of BmNPC1 extracellular loop A (31 to 264 amino acids) or I (881 to 247 1152 amino acids) were amplified with primer pairs BmNPC1-A-PE and BmNPC1-I-PE (Table S1) (Table S1), and were subsequently 260 cloned into the RNAi vector pSL1180 with A3 intron as a spacer (3) (Fig 3A), generating RNAi vectors 261 psl-BmNPC1-a and psl-BmNPC1-b. Unmodified pSL1180 vector designated as psl-null was used as a negative control 262 for RNAi. 263 BmE cells (7 × 10 5 cells/well) grown in six-well culture plates were transfected with 2 μg of psl-BmNPC1-a or 264 psl-BmNPC1-b, or negative control psl-null siRNA, using 2 μl of X-treme GENE HP DNA tranfection reagent (Roche, 265 Germany) following the manufacturer's protocol for 48h before cells were subjected to viral infection. The knockdown 266 effect of RNAi transfection was confirmed by mRNA expression via qPCR with primer set BmNPC1-q (Table S1). 267

Reverse transcriptase PCR and Quantitative real-time PCR (qPCR). Total RNA from BmE cell was extracted 284
using Total RNA kit II (Omega, USA) following manufacturer's protocol, and reverse transcription was carried out 285 using MLV Reverse Transcriptase (Promega, USA). These cDNA samples were used to detect transcripts of BmNPC1 286 using the primers BmNPC1-q (Table S1). The BmRPL3 (gi|112982798) amplified with primers BmRPL3-q (Table S1) 287 was used as the internal reference. Sample analysis was performed on the CFX96 TM Real-Time System (Biorad, 288 France). 289 The viral DNA loads were calculated based on qPCR of the BmNPV gp41 gene. Total DNA from each sample was 290 prepared with a Wizard Genomic DNA extraction Kit (Promega, USA) according to the manufacturer's protocol, and 291 the qPCR was performed using primers gp41-q (Table S1) targeting a 120-bp region of the BmNPV gp41 gene 292 (AAC63752.1). The BmGAPDH gene amplified by primers BmGAPHG-q (Table S1) was used as the reference. 293

Viral infection and quantification of infectivity. BmE cells or ΔNPC1-BmE cells (7 × 10 5 cells/well) seeded into 294
six-well culture plates were inoculated with BmNPV-GFP virus at an MOI of 1 for 2 h, after which cells were washed 295 three times with serum-free Grace medium followed by incubation with Grace medium supplemented with 10% FBS. following the manufacturer's protocol (Thermo Fisher Scientific, USA). A, C, and I-domains of BmNPC1 were 312 expressed with His 9 -tag and GST-tag to the N-terminus and a HA-tag to the C-terminus; gp64 protein were only 313 expressed with His 9 -tag and GST-tag to the N-terminus (not include a HA-tag). The expression of recombinant proteins 314 was confirmed by Western blot using anti-HA monoclonal antibody (Sigma-Aldrich, USA) or anti-gp64 monoclonal 315 antibody (Abcom, UK). Co-immunoprecipitation was performed in accordance with standard protocols to probe the 316 interaction between gp64 and BmNPC1 proteins. In brief, the protein G agarose beads (BioRad, USA) bearing gp64 317 protein through mouse anti-gp64 antibody were incubated with NPC1-A, NPC1-C or NPC1-I protein overnight at 4°C 318 with gentle shaking. Proteins eluted from the beads were probed with anti-HA monoclonal antibody via Western blot. 319 Co-immunoprecipitation was also performed in a reciprocal manner; in which NPC1-A, NPC1-C, and NPC1-I 320 conjugated beads were incubated with gp64 protein, with proteins eluted from the beads probed with mouse anti-gp64 321 monoclonal antibody. 322 Yeast two-hybrid assay. The yeast two-hybrid assay was used to confirm the interaction between NPC1-A, 323 NPC1-C, NPC1-I and gp64 in vivo according to the previously described (39). The bait and prey constructs pairs 324 pGBKT7-gp64/pGADT7-NPC1-A, pGBKT7-gp64/pGADT7-NPC1-C, pGBKT7-gp64/pGADT7-NPC1-I, and pGBKT7-NPC1-C /pGADT7-gp64 were transformed simultaneously into competent yeast cells to examine the protein 326 interaction. All primers used are listed in Supplemental Table S1. 327 Immunofluorescence assay (IFA). BmE cells grown on cover glasses in 12-well plates (Corning, USA) for 12h at 328 28°C, then were fixed by 4% paraformaldehyde at room temperature for 30 min followed by permeabilization with 329 0.2% Triton X-100 or without permeabiliztion. The cells were stained with PcAb-hNPC1 antibody (Abcom, UK) or 330 negative rabbit IgG for immunofluorescence microscopy as described previously (40), and imaged with an Olympus 331 confocal microscope. 332 Antibody blocking assay. BmE cells (2 × 10 5 cells/well) seeded in twelve-well culture plates were incubated with 333 one of the following antibodies: mouse anti-NPC1-A,anti-NPC1-I polyclonal antibody, rabbit anti-NPC1-C polyclonal 334 antibody, mouse or rabbit naïve serum, at a concentration of 10 μg/ml for 2h at 28˚C, after which cells were infected 335 with BmNPV-GFP at an MOI of 3. At 72 h p. i. infected cells were fixed for microscopy and the supernatants were 336 harvested for measuring viral DNA load by qPCR as described previously. 337 Statistics. Independent-samples T tests were used for statistical analysis. Significant differences are marked with * 338 at P < 0.05, ** at P < 0.01 and *** at P < 0.001; n.s, non-significant, respectively. All results are graphed as means ± 339 SD for triplicate samples. All the data presented are representative of a minimum of three independent experiments.