Rab27a promotes degradation of West Nile virus E protein in the lysosome

Summary Rab27a, a Rab family small GTPases, plays an important role in the trafficking and secretion of the intracellular proteins and has been reported to promote various viral multiplication. However, whether Rab27a is involved in West Nile virus (WNV) multiplication is unknown. This study examined the ability of Rab27a to suppress WNV multiplication. The inhibition of Rab27a expression increased viral multiplication and the intracellular levels of WNV structural proteins, E and prM proteins. Rab27a partially colocalized with E protein, mainly in the perinuclear region, while inhibition of Rab27a expression resulted in diffuse subcellular localization of E protein. In addition, some of the perinuclear E protein colocalized with the lysosomal marker LAMP1, and inhibition of lysosomal acidification increased intracellular levels of Rab27a and E proteins. These observations suggested that Rab27a inhibits WNV multiplication by inducing the degradation of viral protein in lysosomes.


INTRODUCTION
West Nile virus (WNV) is a positive-sense, single-stranded RNA virus that belongs to the family Flaviviridae, genus Orthoflavivirus, species Orthoflavivirus nilense. 1 In nature, WNV is maintained in a cycle between mosquitoes and birds and is transmitted to other animals by mosquitoes. 2 Humans and horses are considered to be incidental dead-end hosts and suffer serious disease, such as encephalitis and neurological complications. 2 WNV is widespread, with evidence of its activity obtained on all continents except Antarctica. 3 Human cases of WNV infection have been increasing in recent years, especially in Europe, and pose a risk to global health. 4Although vaccines are available for use in horses, no vaccine has yet been approved for use in humans, due to the requirement for multiple primary doses and an annual booster. 4Furthermore, no specific treatments of WNV disease are presently available.Therefore, investigations of the mechanism of viral replication and the pathogenesis of WNV infection are needed to develop vaccines and effective treatments for use in human.
WNV is internalized into mammalian host cells through receptor-mediated endocytosis. 5,6The decrease in pH in the endosome triggers viral and host membrane fusion to release the viral RNA. 5,6The viral polyprotein is synthesized using viral RNA as a template at the endoplasmic reticulum (ER) and is cleaved by host and viral proteases into the structural proteins [capsid (C), precursor membrane (prM), and envelope (E)], and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). 7Viral genome replication occurs in replication organelles consisting of viral nonstructural proteins, host proteins, and the ER membrane. 8Immature virions are assembled in the ER and transported through the host secretory pathway. 8Virion maturation occurs in the trans-Golgi network by furin-mediated cleavage of the prM to M, and mature virions are released by exocytosis. 8ab proteins are small GTPases belonging to the Ras GTPase superfamily.They are involved in regulating membrane traffic, such as vesicle formation, motility, fusion with target membranes, and exosome release. 9There are more than 60 Rab proteins in humans, mutations or dysfunctions of which are related to neurological diseases, such as Charcot-Marie-Tooth Type 2B, Parkinson's disease, and Alzheimer's disease. 10,11Rab proteins are also involved in infection by various viruses.Previous studies showed that Rab5 was required for cellular entry of flaviviruses, such as Dengue virus, Japanese encephalitis virus, and WNV. 12,13Rab14 was shown to support Ebola virus infection by transporting VP40 protein to the plasma membrane, and to be involved in Classical Swine Fever virus infection by promoting virus assembly and maturation. 14,15 previous study using small interfering RNA (siRNA) screening to identify Rab proteins involved in the release of WNV particles showed that Rab8b was related to the transportation of WNV particles from recycling endosomes to the plasma membrane.16 Furthermore, downregulation of Rab27a promoted WNV particle release, but the detailed underlying mechanisms were unclear.16 Rab27a has several functions, including docking of multivesicular bodies to the plasma membrane, inhibition of phagocytosis, secretion of insulin, and trafficking and secretion of several lysosome-related organelles, including melanosomes.[17][18][19][20] Hepatitis C virus (HCV), Hepatitis E virus, and Enterovirus A71 were reported to release their virions via a Rab27a-dependent pathway.[21][22][23] Rab27a was shown to promote assembly of human immunodeficiency virus (HIV) and human cytomegalovirus, 24,25 and to support HCV genome replication by interacting with HCV core protein around lipid droplets.26 Our previous findings on the role of Rab27a in WNV infection stand in contrast to those that implicated Rab27a in facilitating the replication of several viruses.Therefore, determination of the negative effect of Rab27a in WNV infection is needed to understand the multifaceted involvement of Rab27a in viral replication processes.
8][29] Moreover, Rab27a was shown to play an important role in the regulation of the tumor microenvironment as an essential protein for vesicle exocytosis and exosome release, while Rab27a expression levels correlate positively with a poor prognosis in cancer patients. 30,31Because Rab27a is associated with viral infection, investigation of its function may provide insights into the pathogenesis of the resulting diseases and lead to novel treatment.
In this study, the roles of Rab27a in WNV infection were analyzed by using an siRNA-mediated knockdown approach.The mechanisms underlying Rab27a-mediated inhibition of WNV particle release were analyzed using Rab27a-downregulated WNV-infected cells.This study demonstrated that Rab27a is involved in the degradation of WNV E protein in lysosomes.

Rab27a inhibits WNV multiplication
WNV is a neurotropic virus.Thus, to examine the effects of Rab27a on WNV multiplication, two different siRNAs for Rab27a were introduced into human neuroblastoma SH-SY5Y cells, and the growth of WNV was analyzed in these cells.The expression of Rab27a was decreased by siRNA treatment (Figure 1A).The viral titer of siRNA No.1-pretreated cells was significantly increased at both 12 and 24 h post-infection (hpi), and that of siRNA No.2-pretreated cells showed a significant increase at 24 hpi compared to controls (Figure 1B).Then, cells stably expressing Rab27a were produced and viral growth was analyzed (Figures 1C and 1D).The viral titers at both 12 and 48 hpi were significantly decreased in the cells stably expressing Rab27a compared to mock transfected controls (Figure 1D).These results indicated that Rab27a inhibited WNV multiplication.

Rab27a decreases the levels of intracellular WNV E protein
Rab27a was shown previously to be related to the release of WNV particles. 16The effects of inhibition of Rab27a on the release of WNV particles were confirmed by examining the production of virus-like particles (VLPs).SH-SY5Y cells treated with siRNA for Rab27a were transfected with plasmids for the production of VLPs expressing luciferase in infected cells.The supernatants of transfected cells were collected at 48 h post-transfection (hpt) and inoculated into Vero cells; luciferase activity was measured at 48 h.The luciferase activity was significantly higher in the cells inoculated with the supernatant from Rab27a knockdown (KD) cells than controls (Figure 2A).Next, self-replicating WNV replicon RNA encoding luciferase was introduced into Rab27a KD cells to assess the effects of Rab27a on WNV genome replication.There were no significant differences in luciferase activity between control and Rab27a KD cells at 6, 12, or 24 hpt (Figure 2B).Finally, Rab27a KD cells were transfected with plasmid containing prME to analyze the effects of Rab27a on intracellular WNV structural protein levels.The intracellular levels of prM protein and E protein increased in Rab27a KD cells (Figure 2C).Taken together, these results suggested that Rab27a decreased the intracellular WNV structural protein levels.

Intracellular localization of E protein is affected by Rab27a
To examine the roles of Rab27a in E protein expression, we analyzed the intracellular localization of Rab27a and E protein in WNV-infected cells.The fluorescence intensity of Rab27a in WNV-infected cells was significantly stronger than in mock-infected cells (Figures 3A and 3B), and Rab27a partially colocalized with E protein (Figure 3A).Next, the effects of Rab27a inhibition on E protein localization were examined.Diffusion of granular E protein throughout the cytoplasm was observed (Figure 3C), and the fluorescence intensity of E protein was significantly increased in Rab27a KD cells compared with controls (Figure 3D).The intracellular level of E protein was increased in Rab27a KD cells (Figure 3E).These results suggested that Rab27a was induced upon WNV infection and affected the intracellular level and localization of E protein.

Rab27a localizes E protein to lysosomes
As it affected the intracellular level and localization of E protein, Rab27a was considered to be involved in the degradation of E protein.Therefore, the effects of Rab27a inhibition on the localization of E protein in the lysosome were examined.Immunofluorescence analysis showed that lysosomal-associated membrane protein (LAMP1) was partially colocalized with E protein in WNV-infected cells treated with control siRNA (Figure 4A).However, the colocalization of E protein and LAMP1 was decreased in WNV-infected cells treated with Rab27a siRNA compared to control siRNA (Figures 4A and 4B).These results suggested that Rab27a induced localization of E protein to the lysosome.
Next, the effects of lysosome inhibition on the localization of Rab27a and E protein were analyzed using bafilomycin A1 (Baf), which inhibits protein degradation by preventing acidification of lysosomes.Baf treatment increased the fluorescence intensity of E protein, which was colocalized with Rab27a and LAMP1 (Figure 5A).Colocalization of E protein, Rab27a, and LAMP1 was significantly increased in Baf-treated cells compared to untreated controls (Figure 5B).Next, the effects of lysosome inhibition on the levels of intracellular Rab27a and E protein were examined.The levels of intracellular Rab27a and E protein were significantly increased in WNV-infected cells treated with Baf-treatment compared to untreated controls (Figure 5C).These results suggested that E protein was degraded in lysosomes along with Rab27a.

DISCUSSION
A previous study showed that inhibition of Rab27a resulted in an increase in release of WNV VLPs. 16The present study was performed to examine the relation between Rab27a and WNV infection in greater detail.This study showed that inhibition of Rab27a promoted WNV multiplication and increased the levels of intracellular E protein, and that inhibition of lysosomal degradation resulted in increase in E protein and Rab27a levels.The main function of Rab proteins is to regulate vesicle transport in cells. 9Rab27a plays a role in the fusion of multivesicular bodies to the plasma membrane and in exosome secretion. 25,324][35] These findings suggested a novel role for Rab27a in inhibition of viral multiplication by transporting WNV viral proteins to lysosomes for degradation.
Rab proteins are localized on the membrane surface of vesicles and regulate internal protein transport. 36Various Rab proteins, including Rab5, Rab8, and Rab11, have been shown to mediate vesicular transport of flavivirus particles. 12,13,16,37The simultaneous expression of flavivirus prM and E proteins in mammalian cells leads to the production of sub-viral particles, which have biological properties similar to authentic virions. 38,39After assembly in the ER, WNV virions, in the form of particles, are transported to the plasma membrane by vesicle transport. 8In this study, Rab27a inhibition was shown to result in an increase in the level and granular intracellular distribution of E protein in WNVinfected cells.In addition, Rab27a inhibition increased the expression levels of prM and E protein in the cells.These finding suggested that Rab27a transports these viral proteins into the lysosomal compartment in the form of particles.After assembly in the ER, flavivirus virions are transported to the plasma membrane via the Golgi apparatus. 8Identification of the original organelles of the vesicles transported by Rab27a to the lysosome provided insight into the detailed function of Rab27a in vesicular transport.
The Rab27a fluorescence signal was increased and a portion of Rab27a was colocalized with E protein in cells infected with WNV.It has been reported that upregulation of Rab27a contributes to the progression and malignancy of cancer cells. 40,41Increased expression of Rab27a in colon cancer cells in response to the NF-kB related inflammatory signaling pathway was shown to promote tumorigenesis. 42As this signaling pathway is activated in the cells infected with WNV, 43 expression of Rab27a may be increased as an antiviral inflammatory response.
Rab27a was not related to WNV genome replication despite suppressing viral multiplication.However, Rab27a was reported to promote replication of the HCV genome, a member of the Flaviviridae family. 262][23][24][25] On the other hand, foot-and-mouth disease virus was shown to be negatively regulated by promotion of the exosome-mediated antiviral immune response by Rab27a. 44The findings of the present study and these previous studies suggested that Rab27a has various functions in cells infected with different viruses.The inhibitory effect of Rab27a on WNV multiplication observed in this study seemed to be the result of several factors.
This study showed that Rab27a inhibited WNV infection by mediating viral protein degradation in lysosomes.Investigation of the late stage of WNV infection, including viral transport, may be useful for the development of novel antiviral therapies.6][47][48][49] Further investigations and elucidation of functions of Rab proteins in virus infection may facilitate the development of novel therapies for various diseases.

Limitations of the study
This study demonstrated the novel role of Rab27a in inhibiting WNV multiplication by mediating viral protein degradation in lysosomes.However, elucidation of the involvement of Rab27a in the pathogenesis of West Nile encephalitis in terms of drug development remains challenging.Further analyses using Rab27a knockout mice and drugs that regulate Rab27a expression will lead to a better understanding of the importance of Rab27a in the pathogenesis of encephalitis in vivo.Another limitation was the difficulty in identifying the pathway and original organelle of the vesicles containing viral proteins, due to the lack of appropriate equipment and materials.As experiments using live WNV must be performed at a BSL3 facility, further development of genetic manipulations of WNV is necessary to investigate the intracellular dynamics of WNV in detail.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We would like to thank Dr. Hiroyuki Miyoshi for providing CSII-CMV-MCS-IRES2-Bsd, pCAG-HIVgp, and pCMV-VSV-G-Rev, and all members of the Sawa and Kariwa Laboratories for helpful discussion.We are especially grateful for the experimental assistance of Ms. Sato.This work introduced into CSII-CMV-MCS-IRES2-Bsd using an In-Fusion HD Cloning Kit (Takara Bio Inc.); the resultant construct was designated CSII-CMV-Rab27a-IRES2-Bsd.
The plasmids were transfected into cells by using Lipofectamine 3000 Transfection Reagent (Invitrogen, Waltham, MA, USA).Lipofectamine RNAiMax Transfection Reagent (Invitrogen) and Lipofectamine Messenger MAX Reagent (Invitrogen) were used for siRNA or RNA transfection, respectively.

Immunoblotting
Cells were washed once with PBS and lysed in RIPA buffer consisting of 50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100 containing protease inhibitor (Nacalai Tesque, Kyoto, Japan) for 10 min on ice.The cell lysates were centrifuged at 17700 3 g for 15 min at 4 C.The supernatants were mixed with an equal volume of 23 sample buffer consisting of 3% SDS, 10% glycerol, 100 mM Tris-HCl (pH 6.8), 0.1% bromophenol blue, and 10% 2-mercaptoethanol, and denatured at 95 C for 5 min.The cell lysates were separated by 12% SDS-PAGE and the proteins transferred onto PVDF membranes (Millipore).The membranes were blocked with 5% skim milk in TBS-T consisting of 0.1% Triton X-100, 25 mM Tris-HCl (pH 7.4), 132 mM NaCl, and 2.7 mM KCl, and incubated overnight at 4 C with the appropriate antibody.Bound antibodies were detected using HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).The bands were visualized using the chemiluminescent HRP substrate (Millipore) and ChemiDoc XRS+ Imager (Bio-Rad, Hercules, USA), and the obtained images were analyzed using Image Lab Software (Bio-Rad).

Viral growth assay
SH-SY5Y cells transfected with siRNA for Rab27a or stably expressing Rab27a were infected with WNV at 1 or 0.5 plaque-forming units (pfu)/ cell, respectively.The supernatants of WNV-infected cells were collected at 12 and 24 hpi and stored at À80 C until use.The diluted supernatants were inoculated into Vero cells.After 1-h incubation at 37 C with rocking, the inoculum was removed and the cells were incubated with overlay medium consisting of MEM containing 5% FBS and 1.25% methyl cellulose for 4 days.Plaques visualized using crystal violet solution (0.1% crystal violet, 70% ethanol, 10% formalin) were counted.

VLP release assay
For production of VLPs, SH-SY5Y cells into which siRNA had been introduced were transfected with pCXSN-WNC, pCMV-WNV-prME, and pCMV-WNrep-NLuc(sec)2A.The culture supernatants were collected at 48 hpt and inoculated into Vero cells.After 48 h, the cells were treated with Nano Glo luciferase assay substrate (Promega) for 3 min and luminescence was measured using GloMax (Promega).

Viral genome replication assay
pWNrep-Nluc(sec)2A was linearized with SalI and purified with phenol, isopropanol, and ethanol.The linearized plasmid was used to produce replicon RNA using an mMESSANGE mMACHINE SP6 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.The replicon RNA was introduced into Rab27a-inhibited SH-SY5Y cells usign Lipofectamine Messenger MAX Reagent (Invitrogen).Luciferase activity was performed at 6, 12, or 24 hpt was measured using Nano Glo luciferase assay substrate (Promega).

Immunocytochemistry
The cells were fixed with 4% paraformaldehyde for 10 min at room temperature.Cells were permeabilized with PBST (PBS +0.1% Tween 20) for 10 min, blocked with PBS containing 1% BSA for 30 min, and stained with each antibody for overnight at 4 C.The cells were then washed with PBS and incubated with Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific) or Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific) at room temperature for 1 h.The cells were observed by confocal microscopy (Zeiss LSM 800; Carl Zeiss, Jena, Germany), and the obtained images were analyzed using Zen software (Carl Zeiss) or Fiji software (http://fiji.sc/Fiji).More than three fields were observed in each experiment.The fluorescence intensity of Rab27a and E protein in each cell was measured.The pixel number of E protein or Rab27a colocalized with LAMP1 and that of whole E protein in each cell was measured and the colocalization ratio was calculated.

Lysosome inhibition assay
SH-SY5Y cells were infected with WNV at 1 pfu/cell and treated with 50 or 100 nM bafilomycin A1 for 3 h at 45 hpi.The cells were collected for immunocytochemistry or immunoblotting analysis.For immunocytochemistry, the cells were stained with mouse anti-WNV E protein antibody overnight at 4 C, followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG for 1 h at room temperature.The cells were stained with rabbit anti-Rab27a antibody labeled with FlexAble CoraLite Plus 555 antibody (Proteintech) and rabbit ani-LAMP1 antibody labeled with FlexAble CoraLite Plus 405 antibody (Proteintech) for overnight at 4 C.

Figure 1 .
Figure 1.Effects of Rab27a on WNV multiplication (A) SH-SY5Y cells were transfected with siRNA for Rab27a, and Rab27a expression was assessed by immunoblotting at 1 dpt.The graph shows the relative band intensity of Rab27a/b-actin compared with the siRNA control.Bar: mean, circle: value from three independent experiments (**p < 0.01, by Mann-Whitney U test).(B) siRNA-transfected cells were infected with WNV at 1 pfu/cell for 12 or 24 h.Viral titers were measured by plaque assay.Bar: mean, circle: value from three independent experiments (*p < 0.05, by Tukey-Kramer test).(C) The Rab27a expression of SH-SY5Y cells stably expressing Rab27a without WNV infection was measured by immunoblotting.The graph shows the relative band intensity of Rab27a/b-actin compared with the siRNA control.bar; mean, circle; value of three independent experiments (*p < 0.05 by Scheffe's F test).(D) Cells stably expressing Rab27a or control cells were infected with WNV at 0.5 pfu/cell for 12 or 24 h.Viral titers were determined by plaque assay.Bar: mean, circle: value from three independent experiments (*p < 0.05, **p < 0.01, by Student's t test).

Figure 2 .
Figure 2. Analysis of WNV particle production in Rab27a-inhibited cells (A) SH-SY5Y cells were transfected with siRNA for Rab27a (si-1) or the control.After 1 day, these cells were transfected with plasmids expressing C protein, prME protein, and replicon.The supernatants were harvested at 48 h and inoculated into Vero cells.Luciferase activity was measured at 48 h.Bar: mean, circle: value from eight independent experiments (**p < 0.01, by Mann-Whitney U test).(B) siRNA-introduced cells were transfected with WNV replicon RNA and the luciferase activity was measured as relative light units (RLUs) at 6, 12, and 24 h by luciferase assay.Line: mean, circle and cross; value from three independent experiments (n.s. = not significant by Student's t test).(C) siRNA introduced cells were transfected with plasmid expressing prME and harvested at 48 hpt.Intracellular levels of E or prM protein were analyzed by immunoblotting.The graph showed the relative band intensity of E or prM protein/b-actin compared with siRNA control.Bar: mean, circle: value from seven independent experiments (**p < 0.01, by Mann-Whitney U test).

Figure 3 .
Figure 3. Analysis of intracellular localization of E protein and Rab27a (A) SH-SY5Y cells were infected with WNV at 1 pfu/cell.At 48 hpi, cells were fixed, permeabilized, and stained for Rab27a (magenta), WNV E protein (green), and nuclei (blue).Yellow arrowheads show colocalization of Rab27a and WNV E protein (scale bar: 10 mm).(B) The fluorescence intensity of Rab27a in each cell in mock and WNV-infected cells were analyzed using Fiji software (http://fiji.sc/Fiji).Bar: mean, circle: value from six independent experiments (*p < 0.05, by Mann-Whitney U test).(C) Control cells or siRNA-transfected cells were infected with WNV at 1 pfu/cell and cultured for 48 h.The cells were fixed, permeabilized, and stained for E protein (green) and nuclei (blue) (scale bar: 10 mm).(D) The fluorescence intensity of E protein in each cell was analyzed using Fiji software (http://fiji.sc/Fiji).Bar: mean, circle: value from seven independent experiments (**p < 0.01, by Mann-Whitney U test).(E) siRNA-introduced cells were infected with WNV at 1 pfu/cell and harvested at 48 hpi.The intracellular level of E protein was analyzed by immunoblotting.The graph showed the relative band intensity of E protein/b-actin compared to siRNA control.Bar: mean, circle: value from four independent experiments (*p < 0.05, by Mann-Whitney U test).

Figure 4 .
Figure 4. Analysis of relationships among Rab27a, E protein, and LAMP1 in WNV-infected cells (A) Control or Rab27a siRNA-transfected cells were infected with WNV at 1 pfu/cell and cultured for 48 h.The cells were fixed, permeabilized, and stained for LAMP1 (magenta), E protein (green), and nuclei (blue).Yellow arrowheads show the colocalization of E protein and LAMP1 (scale bar: 10 mm).(B) Quantification of colocalization of E protein and lysosomes.The pixel number of colocalized E protein and LAMP1 to the whole pixel number of E protein was quantified using Fiji software (http://fiji.sc/Fiji).Bar: mean, circle: value from six independent experiments (*p < 0.05, by Mann-Whitney U test).

Figure 5 .
Figure 5. Effects of lysosome inhibition on intracellular localization and expression of Rab27a, E protein, and LAMP1 in WNV-infected cells (A) SH-SY5Y cells were infected with WNV at 1 pfu/cell.After 45 h, the cells were treated with 50 nM bafilomycin A1 (Baf) for 3 h followed by fixation and permeabilization.The results showed colocalization of Rab27a, E protein, and LAMP1.The cells were stained for Rab27a (magenta), E protein (blue), and LAMP1 (green).Yellow arrowheads indicate the colocalization of Rab27a, E protein, and LAMP1 (scale bar: 10 mm).(B) Quantification of colocalization of Rab27a, E protein, and LAMP1.The pixel number of colocalized E protein to the whole pixel number of E protein was quantified using Fiji software (http://fiji.sc/Fiji).Bar: mean, circle: value from three independent experiments (*p < 0.05, by Student's t test).(C) SH-SY5Y cells were infected with WNV at 1 pfu/cell.After 45 h, the cells were treated with 100 nM Baf for 3 h and harvested.Rab27a, E protein, and LAMP1 expression was analyzed by immunoblotting.Graphs showed relative band intensities of Rab27a or E protein/b-actin compared to Baf (À) cells.Bar: mean, circle: value from eight independent experiments (**p < 0.05, by Mann-Whitney U test).

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell lines B Virus d METHODS DETAILS B Plasmid constructions and transfections B Generation of SH-SY5Y cells stably expressing Rab27a B Immunoblotting