key: cord-0281824-1pirzel8
authors: Shi, Yueyuan; Jin, Xin; Wu, Shuang; Liu, Junye; Zhang, Hongpeng; Cai, Xuefei; Yang, Yuan; Zhang, Xiang; Wei, Jie; Peng, Hui; Luo, Miao; Zhou, Hua; Zhou, Huihao; Huang, Ailong; Wang, Deqiang
title: HBV enveloped particle secretion is positively regulated by GRP78 through direct interaction with preS1
date: 2021-12-09
journal: bioRxiv
DOI: 10.1101/2021.12.08.471876
sha: 2421ac041cbb88df805e61a2e722b57d25de72a6
doc_id: 281824
cord_uid: 1pirzel8

Hepatitis B virus (HBV) infection is a common cause of liver diseases worldwide. Existing drugs do not effectively eliminate HBV from infected hepatocytes; thus, novel curative therapies are needed. Enveloped-particle secretion is a key but poorly studied aspect of the viral life cycle. Here, we report that GRP78 positively regulates HBV enveloped-particle secretion. GRP78 is the specific target of preS1 binding; HBV can upregulate GRP78 in liver cell lines and sera from chronic hepatitis B patients. GRP78 promoted intact HBV-particle secretion in liver cell lines and an HBV transgenic-mouse model. Some peptides screened from preS1 via phage display could inhibit viral-particle secretion by interacting with GRP78 via hydrogen bonds and hydrophobic interactions, thereby disturbing the interaction with HBV particles. These results provide insight into enveloped-particle secretion in the HBV life cycle. GRP78 might be a potential target for HBV-infection treatment via restricting GRP78–preS1 interactions to block viral-particle secretion. IMPORTANCE HBV is a major human pathogen. The virus More than 250 million n individuals globally are chronically infected with HBV and can result in the infected personsabout 800,000 die for HBV-related disease annually. The mature HBV enveloped particles containing HBs and the relaxed circular (RC) DNA genome could secreted extracellularly as virions. And these enveloped virions serves as the infectious HBV particles for initiating a complete life cycle of the virus. Here, we show the GRP78 is the specific target of preS1 binding, and it could positive regulate HBV enveloped particle secretion in cell and HBV transgenic mice model. Furthermore, several peptides screened from preS1 and Phage screening could inhibit viral particle secretion by interacting with GRP78 and disturbing its interaction with HBV, and then block the life cycle of the virus. This has important implications for our understanding of the mechanisms of antivirals that target intact HBV particles secretion.

show the GRP78 is the specific target of preS1 binding, and it could positive regulate 51 HBV enveloped particle secretion in cell and HBV transgenic mice model. 52 Furthermore, several peptides screened from preS1 and Phage screening could inhibit envelope enclosing an inner capsid with or without viral nucleic acids (6,7). In 78 particular, a complete HBV particle contains an outer envelope enclosing an inner 79 capsid with RC DNA genome content, which serves as the infectious HBV particle for 80 initiating a complete viral life cycle. Consequently, the processes of assembly and 81 transport in the cytoplasm and particle secretion are potential targets for antiviral 82 drugs (8, 9) . Host factors can interact with HBc and envelope proteins to participate in 83 5 HBV-particle secretion. Vps4 and the molecular endosomal sorting complexes 84 required for transport (ESCRT) machinery could cooperate in the secretion of the 85 HBV virion (10,11). Cellular kinases can regulate virion formation and promote 86 intracellular capsid accumulation by modifying capsid phosphorylation (12) (13) (14) . 87 However, the exact nature of complete viral-particle secretion remains unclear. 88 Glucose-regulated protein 78 (GRP78) is an important endoplasmic reticulum (ER) 89 chaperone that functions as a vital component in many cellular processes, including 90 protein assembly, folding, and translocation across the ER membrane. In the present study, preS1 was used as the bait protein to screen for its binding 105 protein by mass spectrometry analysis and GRP78 was identified as the corresponding or screening by phage display, could restrict viral particle secretion by interacting 114 with the host protein to disturb the interaction between GRP78 and HBV particles.

Our research provides new ideas for more in-depth study of HBV-particle secretion 116 and for the use of GRP78 as a novel target for the development of antiviral therapy.

Screening host proteins interacting with preS1. The preS1 domain is localized on 119 the outer membrane of HBV, and it interacts with host proteins, such as NTCP, which 120 is the receptor of HBV (26). Therefore, the recombinant preS1-GST protein was 121 prepared, and the fused GST tag with the bait protein facilitated subsequent affinity 122 mass spectrometry experiments. The experimental group combined the biotin-labeled 123 HepG2 hepatocyte lysate with the preS1-GST protein, and the control group was 124 7 treated with biotin-labeled lysates of HEK293 cells and TM-GST irrelevant proteins 125 to remove nonspecific signals. The results showed that some specific proteins with a 126 molecular weight of 97-116 kDa were present in the HepG2 cell lysate (Fig. S1 ). Four 127 proteins were identified in the preS1-GST protein captured from the HepG2 cell 128 lysate by mass spectrometry, including keratin type II skeleton 1, type I skeleton 9, 129 LanC-like protein 1 (LanCL1), and GRP78 (Fig. S1 ). The two skeleton proteins were 130 present in relatively low quantities, and their presence was considered to be caused by (19, 20, 30) . Confocal immunofluorescence detection 138 revealed that both GRP78 and preS1 were spatially colocalized and mainly distributed 139 in the cytoplasm (Fig. 1A) . Co-immunoprecipitation revealed that GRP78 and preS1 140 could capture each other in HepG2 cells (Fig. 1B) . To further verify the interaction 141 between GRP78 and preS1, three GST-preS1 truncated proteins, including preS1-p1 142 (residues 1-65), preS1-p2 (residues 25-90) and preS1-p3 (residues 66-119), were 143 expressed and purified, and these proteins were found to interact with GRP78 directly (Table S1 ). The experiments showed that the average 157 GRP78 concentration (125.1 mg/mL) of chronic HBV carriers (n = 92) was higher 158 than that in the serum of healthy individuals (92.31 mg/mL, n = 79) ( Fig 1E) . five mutants exhibited a much lower ability to promote HBV-particle secretion 249 compared to wild-type GRP78. In particular, T38A, F451A, I463A, and R492A 250 almost lost their ability to promote viral particle secretion, while the T229A mutant 251 retained 50% of the effect (Fig. 6D) . The above data suggest that the SBD and NBD 252 might be involved in collaboratively promoting viral particle secretion, implying that 253 inhibitors of GRP78 might obstruct HBV secretion. 

Compared with the control, pep145 inhibited 80% of the activity of GRP78, thereby 263 positively regulating viral particle secretion (Fig. 7D) . To further explore whether the 264 effect of GRP78 on HBV is based on its binding to HBV preS1, hydrophobicity 265 analysis of the amino acid sequence of preS1 was carried out using bioinformatics. were found to be too hydrophobic to be synthesized (Table S3 ). The MTS cell (Table   301 S4); meanwhile, ITC experiments showed that there was little interaction between 302 GRP78 and GBP67. Consequently, the inhibitory activity of the other five peptides on 303 HBV-particle secretion was determined by particle gel and HBV DNA assays. GBP68 304 exhibited a much stronger inhibitory effect than GBP63, GBP64, GBP65, and GBP66 305 in the supernatant of HepG2.2.15 cells ( Fig. 7A and 7B) . Furthermore, GBP68 306 inhibited HBV-particle secretion both in HepG2.2.15 and HepAD38 cells but 307 exhibited little effect on viral particles in the cytoplasm and core particle excretion 308 ( Fig. 7C and S8 ). The inhibitory effect of HBV-particle secretion by GBP68 was 309 dose-dependent (Fig. 7D) . Figure 7E represents the best-scoring binding poses of 310 GBP68 in complex with GRP78 (docking score of -12.008 kcal/M), as well as the key 311 16 interacting residues at the binding pose. It was observed that the GBP68 generated 312 nine hydrogen bonds within the range of 3.50 Å with GRP78, and the key residue, 313 Met6, inserted into a hydrophobic bag formed by Phe451, Phe461, Val499 and Val511.

There was also a strong hydrophobic effect between GBP68 and GRP78. Taken 315 together, the above experiments further demonstrated that the interaction of peptides 316 with GRP78 could inhibit HBV secretion (Fig. S9) .

The HBV life cycle includes a phase that occurs within the hepatocyte nucleus, 319 wherein HBV DNA is converted to cccDNA, which is a highly stable double-stranded conversely, GRP78 mainly acts on HBV virion secretion and promotes HBV-particle 329 secretion in a nonreplicating manner. Moreover, the hydrophobic peptide preS1 330 interacts with GRP78 with strong affinity and inhibits HBV enveloped-particle 331 17 secretion. Additionally, peptide screening by phage display showed that GBP68 could 332 also interact with recombinant GRP78 and significantly inhibit viral secretion. To the 333 best of our knowledge, this is the first study to provide new avenues for more in-depth 334 study of HBV enveloped-particle secretion and new targets for antiviral drugs. Consequently, the host protein that directly interacts with preS1 plays a key role in 365 HBV-particle secretion. As such, we screened the preS1 binding protein in human These results suggest that GRP78 could positively regulate HBV-particle secretion 380 both in vitro and in vivo. Consequently, GRP78 may provide a target for antiviral 381 drugs to inhibit HBV-particle secretion.

According to our experimental results, GRP78 could bind to both preS1 and HBV by 383 its SBD at the carboxyl terminus. Thus, we hypothesized that if a peptide interacts 384 with GRP78, it should be able to block or interfere with its binding to the viral 385 envelope protein, thereby blocking or inhibiting viral secretion. Indeed, we identified 386 fragments of preS1 that could interact with GRP78 directly, thereby inhibiting binding to the MERS-CoV spike protein and enhances viral entry into host cells (19) .

Data derived mainly from the study of other coronaviruses provide sufficient evidence 398 to discuss the putative role of CD147 and GRP78 as entry receptors for SARS-CoV-2. Instead, GRP78 is involved in viral particle secretion, which provides some new 408 perspectives for clarifying the function of GRP78 in the viral life cycle.

In the present study, GRP78 was found to interact with preS1 directly, and it 410 positively regulated HBV-particle secretion. The peptide inhibited enveloped-particle 411 secretion by disturbing the interaction between GRP78 and preS1. These findings 412 provide a basis for more in-depth study of HBV enveloped-particle secretion, and The HBV virion was identified by the particle gel assay (Ran et al., 2017) . 435 HBV-producing cells were cultured, and the cell culture medium was harvested. The 436 cell supernatant was added to 35% PEG8000 and rotated at 4 °C for more than 2 h. 437 The viral particles were dissolved in TNE buffer and incubated overnight at 4 °C. The 48-well plates were washed three times with precooled phosphate-buffered saline, 473 fixed with 4% paraformaldehyde for 10 min, and permeabilized for 10 min at RT with 474 0.5% Triton X-100. After incubation for 1 h with 3% bovine serum albumin to block 475 nonspecific binding, primary antibodies were added and incubated for 1 h at 37 °C. 476 The bound antibodies were visualized by incubation with secondary antibodies (Alexa

Fluor 488 donkey anti-mouse IgG or Alexa Fluor 594 anti-mouse IgG). Images were 478 acquired using a fluorescence microscope.

Co-immunoprecipitation 480 Total protein lysates were extracted from hepatoma cells using the IP lysate buffer. 481 The lysate was mixed with 40 µL protein G agarose (Millipore, United States) to Isothermal titration calorimetry (ITC) 487 Affinity constants under equilibrium (Ka) were obtained using a Nano ITC instrument. the best energy score was selected for subsequent analysis. The images for clarifying 531 the interactions between GRP78 and the peptide were generated using Pymol 532 (https://pymol.org/2/).

Microscale thermophoresis (MST) 534 The interaction between GRP78 and preS1 was confirmed by microscale 535 thermophoresis (48). Experiments were performed with an MST power of 60%, LED 536 power of 40%, and capillaries with a hydrophobic coating under standard conditions. 537 Purified GRP78-His and preS1-GST (preS1-p1-GST, preS1-p2-GST, and 538 preS1-p3-GST) was buffer exchanged into phosphate-buffered saline buffer (pH 7.4), 539 and its concentration was adjusted to 10 µM using UV absorbance. GRP78-His was We declare no competing interests. 

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APH-2 and Suppresses Viral Replication

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GRP78: A cell's response to stress

HSPA5 is an essential host factor for Ebola virus infection

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Figure 1. Direct interaction between preS1 of hepatitis B virus (HBV)

Immunofluorescence confocal detection showed colocalization of GRP78 and 724 preS1. Rabbit anti-GRP78 and mouse anti-preS1 were used to label two proteins in 725

HepG2.2.15 cells that could stably express HBV, and these two proteins were mainly 726 distributed in the cytoplasm

Immunoprecipitation was performed using agarose beads, and western blot 728 detection confirmed that GRP78 was a specific binding target of preS1

The purified GRP78 protein could interact with HBV particles and adenovirus 730 according to isothermal titration calorimetry (ITC). HBV and adenovirus particles 731 were collected in the HepAD38 and 293 cell lines

HBV upregulated the expression of GRP78 in the HepG2, HepAD 38, and 733 HepG2-NTCP cell lines

The GRP78-specific ELISA kit was used for quantitative detection, and unpaired 735 t-test was used for statistical analysis

Figure 2. Overexpression of GRP78 promoted HBV-particle secretion

Particle gel assay to detect the HBV particles (core particle and intact viral particle) 738 in the supernatant and cytoplasm of HepG2.2.15 cells infected by pAd-GRP78

Overexpressing GRP78 promoted HBV-particle secretion in HepG2.2.15 cells 740 infected with GRP78Ad. HBV DNA was subjected to real-time quantitative 741 polymerase chain reaction (RT-qPCR) and Southern blotting. The HBV particles were 742 detected by viral particle gel experiments

The relative level of HBV e antigen (HBeAg) and HBV surface antigen (HBsAg) 744 in cellular supernatant of HepG2.2.15 treated with GRP78Ad were subjected to 745 ELISA

Overexpressing GRP78 promoted HBV secretion extracted from HepAD38 cells 747 infected with GRP78Ad. HBV DNA were subjected to RT-qPCR and Southern 748 blotting. The HBV particles were detected by viral particle gel experiments with 749 preS1 antibody

The relative levels of HBeAg and HBsAg in the supernatant of HepAD38 cells 751 treated with GRP78Ad were observed via ELISA

Figure 3. GRP78 knockdown in HepG2.2.15 and HepAD38 cells disturbed 753 HBV-particle secretion

A. GRP78 knockdown inhibited HBV-particle secretion in HepG2.2.15 cells infected

HBV DNA was subjected to real-time quantitative polymerase 756 chain reaction (RT-qPCR) and Southern blotting. The HBV particles were detected by 757 viral particle gel experiments with preS1 antibody

The relative levels of HBeAg and HBsAg in the cellular supernatant of

HepG2.2.15 cells treated with GRP78 siRNA were observed using ELISA

knockdown promoted HBV secretion in HepAD38 cells infected with 761 GRP78 siRNA. HBV DNA was subjected to RT-qPCR and Southern blotting. The 762 HBV particles were detected by viral particle gel experiments with preS1 antibody

The relative levels of HBeAg and HBsAg in cellular supernatant of HepAD38 cells 764 treated with GRP78 siRNA were observed using ELISA

Figure 4. GRP78 overexpression in HBV transgenic mice (TGM) increased viral 766 particle secretion

The mice (4-6 weeks) were randomly allocated to two groups

We dissolved 7×10 9 GFU GRP78Ad (n1-4) or GFPAd (n5-8) in 0.3 mL 0.9% normal 769 saline and injected it into mice through the tail vein. The mice were sacrificed at three 770 weeks after injection, and the liver tissue and serum were collected

GRP78 expression in liver tissue lysates was tested by western blotting with 772 GAPDH as an internal control. The HBV particles from serum were detected by viral

particle gel experiments with preS1 antibody. The serum of the mice was collected to 774 detect the copy number of HBV DNA and the secretion level of HBV virions

Immunohistochemistry analysis detected less HBc in sectioned liver samples in the 776 presence of TGM treated with GRP78Ad or GFPAd at 1 dpi

The amount of HBeAg, HBsAg and HBV DNA before and after adenovirus 778 injection and TGM serum extraction was analyzed by ELISA or qPCR

Figure 5. Domains of GRP78 participate in HBV-particle secretion

Schematic showing the GRP78 sequence and domains. The signal sequence

ATPase domain, and substrate binding domain are highlighted in yellow, blue, and 782 aubergine boxes, respectively

The HBV particles in HepG2.2.15 cells treated with pAd-GFP, pAd-GRP78, and 784 pAd-GFP-△ss were detected with preS1 antibody

The HBV particles in HepG2.2.15 cells treated with pAd-GFP, pAd-GRP78, 786 pAd-NBD, and pAd-SBD were detected with preS1 antibody

HBV particles in HepG2.2.15 cells treated with Ad-GFP, Ad-GRP78-T38A

Ad-GRP78-F451A, Ad-GRP78-I463A, and Ad-GRP78-R492A

A, B. Interaction between recombinant GRP78 and peptide (pep56 and pep59) 792 according to ITC assay. 793 C. The (Table S2 ).

F. The model of GRP78 regulates HBV-particle secretion. GRP78 promotes intact 814 HBV-particle secretion by directly interacting with preS1 located at the viral envelope.

Some peptides could inhibit HBV particle excretion by binding to GRP78 with high 816 affinity, which in turn reduced the inhibitory interaction between GRP78 and 817 preS1/HBV particles.