key: cord-0842959-275wut5u
authors: Liang, Wulong; Zheng, Minping; Bao, Changlei; Zhang, Yanming
title: CSFV proliferation is associated with GBF1 and Rab2
date: 2016-12-29
journal: J Biosci
DOI: 10.1007/s12038-016-9659-0
sha: 4c9356e8feead0ecd3d707cd2d52f5ededc5646f
doc_id: 842959
cord_uid: 275wut5u

The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. In this study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi was disturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA), and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particle generation after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected with GBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using a lentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate in CSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlying CSFV replication.

Classical swine fever (CSF) is a highly contagious disease that affects domestic and wild pigs; it is listed as one of the notifiable swine diseases by the Office International Des Epizooties (OIE) (Lin et al. 2014) . Classical swine fever virus (CSFV), which belongs to the genus Pestivirus of the Flaviviridae family, is a single-stranded positive-sense RNA virus encoding four structural proteins (C, E rns , E1 and E2) and eight non-structural proteins (N pro , P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Lamp et al. 2011) . These non-structural proteins participate in self-maturation and replication complex assembly (Sheng et al. 2012) .

Plus-stranded RNA viruses replicate in the cytoplasm, and their synthesis is associated with cellular membrane structures. Specifically, the endoplasmic reticulum (ER) and Golgi apparatus as well as their resident proteins are used by many viruses to complete one or more stages in their life cycle (Ravindran et al. 2016; Wang et al. 2016) . Anterograde and retrograde trafficking of cargo proteins between the ER and Golgi are mediated by the cytosolic coat protein complex II (COP II) and COP I transport systems, respectively. These systems are regulated by viruses to facilitate their multiplication (Rust et al. 2001; Richards et al. 2002; Yamayoshi et al. 2008) . Viruses also regulate two important targets, i.e. Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) and Rab2 GTPases, which are primarily involved in Golgi-to-ER retrograde trafficking (Raj 1961; Wessels et al. 2006) .

Increasing evidence has shown that CSFV replication relies on cellular membrane structures. CSFV can utilize the membranes of autophagosome-like vesicles to facilitate selfreplication (Pei et al. 2013) . CSFV p7, a viroporin protein in the ER and plasma membrane, modifies membrane permeability (Guo et al. 2013; Largo et al. 2014) . According to our previous study, CSFV NS2 and NS5A proteins localize to the ER and regulate cellular oxidative stress to promote CSFV replication (Tang et al. 2010; He et al. 2012) . However, whether Golgi function and its resident proteins are involved in CSFV replication remains unknown.

In this study, chemical inhibitors, shRNA and lentiviral systems were used to regulate the function of Golgi-resident proteins. The results of these analyses indicated that GBF1 and Rab2 play important roles in CSFV replication.

Swine testicle (ST) cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, UK) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA). The CSFV Shimen strain was purchased from the Control Institute of Veterinary Bio-products and Pharmaceuticals (China) and grown in ST cells. The MOI of CSFV infection in this experiment was set at 10 TCID 50 .

The CSFV polyclonal antibody was collected from Hog Cholera Lapinized Virus (HCLV) inoculated pigs and stored at −20°C. The anti-pig IgG-FITC antibody was purchased from Sigma Aldrich (F1638; St. Louis, MO, USA). Goat anti-GBF1 polyclonal antibody (C-16, sc-27940) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Rab2 polyclonal antibody (ab154729) was purchased from Abcam (Cambridge, UK). Mouse anti-GAPDH antibody was obtained from GenScript (Piscataway, NJ, USA). The secondary antibodies for Western blotting were HRP-labelled goat anti-rabbit IgG, goat anti-mouse IgG, and rabbit antigoat IgG (Beyotime, Haimen, China). Brefeldin A (BFA) and golgicide A (GCA) were purchased from Selleck Chemicals (Houston, USA).

An MTT assay was used to test the cytotoxicity of BFA and GCA. Various concentrations of BFA and GCA were added to ST cells, which were plated in 96-well plates at a confluence of about 60%. The cells were then maintained in DMEM with 1% FBS at 37°C in 5% CO 2 . After 72 h of incubation, the supernatant was removed. Each well was supplemented with 50 μL of MTT (2 μg/ mL) and incubated at 37°C for 4 h. MTT was then removed, and 200 μL of DMSO was added to dissolve the formazan crystals. A microplate reader (Thermo, Waltham, MA, USA) was used to determine light absorbance for each well at 490 nm. Untreated ST cells were used as controls.

The pGFP-V-RS shRNA plasmid (OriGene Co., Rockville, MD, USA) was used to construct Rab2 and GBF1 shRNA interference vectors. For each gene, three pairs of interference fragments were used (table 1) , which were synthesized and sequenced by BGI Shenzhen (China). ST cells were transfected with the interference vector when cell confluence reached 70-90%. The transfection operation followed the protocol for the jetPRIME transfection reagent (Polyplustransfection, Illkirch, France). At 48 h post-transfection, 5 μg/mL puromycin was added to continuously screen positive cells for about 20 days. Scrambled shRNA was constructed and transfected using the same methods. (table 2) were used to determine the expression levels of different genes. The reaction was performed with SYBR Premix Ex Taq II (TaKaRa) using the Bio-Rad iQ5 system under the following conditions: pre-degeneration at 95°C for 10 min, followed by 40 cycles of degeneration at 95°C for 10 s, and annealing and extension at 60°C for 30 s. RNA expression in the samples was normalized to that of β-actin. The 2 −ΔΔCT method was used to analyse the data. The ΔC T values were calculated as the C T values of the target gene minus the C T values of βactin. GBF1 and Rab2 gene expression levels in ST cells or CSFV RNA in ST cells at the earliest time point were selected as comparative references, and so the average ΔC T values were set as the standard reference for the −ΔΔC T calculation. Then the 2 −ΔΔCT values were obtained and histograms were obtained in which the height of the bar for each sample represented the average 2 −ΔΔCT value.

CSFV-infected cells at 48 hpi were observed by indirect immunofluorescence assay (IFA). Cell monolayers were fixed with 4% paraformaldehyde for 20 min, following cell penetration with 1% Triton X100 for 10 min. Subsequently, 5% skim milk was added to the cell monolayer at 4°C for 6 h. The CSFV polyclonal antibody was diluted by 100-fold with PBS and incubated with the cell monolayer at 4°C for 8 h. The FITC anti-pig antibody diluted 200-fold was used for coloration at room temperature for 1 h. The nuclei were stained with DAPI at room temperature for 5 min. Images were observed under an inverted fluorescence microscope (Nikon, Tokyo, Japan). CSFV-infected cell supernatants Table 2 . Primers used in this study

were collected for viral titration. The supernatants were serially diluted from 10 −1.0 to 10 −10.0 and added to ST cells in 96-well plates. At 72 hpi, CSFV infection was observed by IFA. The proportionate distance (PD) and the log lower dilution were calculated according to the Reed-Muench method as follows:

. % next above 50% positive ð Þ -% next below 50% positive ð Þ

Log lower dilution ¼ dilution in which position is next above 50% positive lgTCID 50 ¼ PD Â the difference of logarithmic dilution degrees þ Log lower dilution

Cells were collected and incubated in RIPA lysis buffer (Beyotime) containing the proteinase inhibitor PMSF (Beyotime) for 30 min on ice. A BCA Protein Assay Kit (Beyotime) was used to determine protein concentrations. Equivalent amounts of protein samples were separated by 12% SDS-PAGE and transferred to 0.45μm PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in TBST containing 5% skim milk powder for 2 h at room temperature. Then, membranes were incubated with a mouse anti-GAPDH antibody or the primary antibodies at the appropriate dilutions according to the instructions overnight at 4°C. Afterwards, the HRP-conjugated secondary antibodies diluted at 1:2000 were added and incubated for 2 h at room temperature. After washing with TBST, immunoreactive bands were detected using luminol chemiluminescence reagents (Pierce) and analysed by Tanon-410 automatically gel imaging system (Shanghai Tianneng, China).

The primers used for GBF1 and Rab2 gene amplification contained EcoRI and NotI restriction sites. The l e n t i v e c t o r p C D H -C M V -M C S -E F 1 -G r e e n P u r o (CD513B-1) (SBI, Mountain View, CA, USA) was used to construct the over-expression vectors pCDH-GBF1 and pCDH-Rab2. The overexpression vector was transfected into HEK-293T cells with three auxiliary plasmids (pGag/Pol, pRev, and pVSV-G). 

Differences among groups were examined for statistical significance using Student's t-tests. P<0.05 was considered statistically significant.

The safe concentration of BFA in ST cells was tested using an MTT assay. The inhibitors were co-cultured with ST cells for 72 h. The concentration of BFA used in the antiviral experiment was 100 nM (figure 1A). After 1 h of CSFV infection, the unbound viruses were removed with the supernatant, and the cells were maintained in media with or without BFA. The cell monolayers were harvested for real-time PCR, and the supernatants were collected for viral titration after 24, 48 and 72 hpi. Cellular CSFV RNA loads decreased by about 2-6 times after BFA treatment (figure 1B). The TCID 50 of CSFV exhibited a titer reduction of approximately 1.5-2.5 in the BFA treatment group compared with the untreated group ( figure 1C ). CSFV proliferation with BFA was evaluated by IFA. The CSFV plaques were smaller and less abundant than the untreated ST cells at all three test points ( figure 1D ).

GCA is a specific inhibitor of GBF1. Before the anti-CSFV test, the cytotoxicity of GCA was detected and, 400 nM was considered the maximum safe concentration for ST cells. This concentration was used in subsequent experiments ( figure 2A ). GCA cell treatment and the collection of cell monolayers and supernatants were performed using the same methods as those for BFA.

Compared with the untreated ST cells, cellular CSFV RNA declined significantly when GCA was added during viral proliferation ( figure 2B) . Moreover, the viral titer and the percentage of infected ST cells were lower in GCA-treated cells than control cells (figure 2C, D).

Three shRNA vectors for the swine GBF1 gene and a scrambled shRNA vector were constructed and transfected into ST cells (figures 3A). After puromycin screening, the down-regulation of GBF1 in the transfected cells was tested. GBF1 was knocked down using three shRNA vectors. shRNA-3 was the most effective and was therefore selected for further analysis. Moreover, the transcription levels of the GBF1 were not significantly different between the untransfected cells and scrambled shRNA-transfected cells ( figure 3B, C) .

shRNA -transfected cells, and scrambled shRNAtransfected cells were then evaluated. CSFV was incubated with cell monolayers at 37°C for 1 h, and unbound particles were removed with the supernatants. Infected cell monolayers were collected for CSFV RNA quantitative detection, and the supernatants were collected for viral titration at 24, 48 and 72 hpi. The detection of cellular CSFV RNA and viral titers showed that the CSFV proliferation dynamics in GBF1 shRNAtransfected cells were slower than those in ST cells and scrambled shRNA-transfected cells ( figure 4A, B) . CSFV proliferation in ST cells, GBF1 shRNA-transfected cells, and scrambled shRNA-transfected cells was observed by IFA. The percentage of CSFV-infected GBF1 knockdown cells was significantly lower than those of the infected ST cells and scrambled shRNA-transfected cells, especially at 48 and 72 hpi. GFP expression in shRNAtransfected cells was quenched after fixation with 4% paraformaldehyde, which had no effect on the observation of FITC on anti-pig IgG (figure 4C). 

shRNA vectors and transfected cells specific to the swine Rab2 gene were constructed and selected using the same methods used for the GBF1 gene. Rab2 shRNA-1 demonstrated the optimal down-regulation of expression (figure 5B, C), and the corresponding transfected cells were used for the anti-CSFV test. Cellular CSFV RNA tested by real-time PCR at three time points showed that the down-regulation of the Rab2 gene significantly slowed CSFV RNA replication. Cellular CSFV RNA in Rab2 knockdown cells declined by about 10 times relative to the levels in ST cells and scrambled shRNA-transfected cells ( figure 6A ). In addition, viral titration showed that the production of CSFV virions declined by about 1-2.5 titers by Rab2 knock-down ( figure 6B ). Furthermore, CSFV-infected Rab2 interference cells were less abundant than cells of the control groups at the three testing points ( figure 6C ).

3.5 GBF1 and Rab2 overexpression facilitated CSFV proliferation GBF1 and Rab2 were overexpressed using the lentiviral vector system. GBF1 and Rab2 expression increased at both the mRNA (figure 7B, C) and protein levels (figure 7D, E). CSFV proliferation was higher after the upregulation of 3.6 GBF1 and Rab2 regulated CSFV RNA replication Cellular CSFV RNA in the first 12 h was measured by realtime PCR to evaluate whether GBF1 and Rab2 regulate CSFV RNA replication. CSFV RNA replication was suppressed when GBF1 and Rab2 were disturbed by inhibitors or shRNA ( figure 9A, B) . Conversely, overexpression of GBF1 and Rab2 could accelerate CSFV RNA replication ( figure 9C ).

The functions of CSFV non-structural proteins have been studied extensively in recent years (Lamp et al. 2011; Sheng et al. 2012) ; nevertheless, the CSFV replication mechanism and the subcellular location of the replication complex remain unclear. Studies on the replication membranes of positive-stranded viruses demonstrated that they can induce the alteration of intracellular membranes to facilitate the replication cycle (Miller and Krijnse-Locker 2008) . CSFV NS2 and NS5A are located in the ER (Tang et al. 2010; He et al. 2012) . NS5B, which is an RNA-dependent RNA polymerase of CSFV, is located in the cytoplasm (Zhao et al. 2006) . The locations of these CSFV replication-associated proteins suggest that the complex is located in the cytoplasm, and the ER may support CSFV replication. However, it is not clear whether other intracytoplasmic membrane structures and their resident proteins are involved in CSFV replication.

In this study, we found that CSFV replication is sensitive to BFA. Both CSFV RNA replication and virus particle generation were inhibited by BFA treatment. BFA inhibited protein transport between the ER and the Golgi by suppressing the formation of COP I-mediated transport vesicles. The proliferation of viruses, such as hepatitis C virus (HCV) (Goueslain et al. 2010) and dengue virus (Carpp et al. 2014) , is sensitive to BFA treatment owing to the inhibitory effect of BFA on GBF1 and the consequent inhibition of COP I vesicle formation. However, three BFA-sensitive large Arf-GEFs (GBF1, BIG1 and BIG2) are present in mammalian cells . Therefore, GCA and RNA interference were used for further tests. GCA is a highly specific inhibitor of GBF1 and had the same inhibitory effect on CSFV proliferation as BFA. Furthermore, CSFV replication in GBF1 shRNA-transfected cells was significantly inhibited compared with that in GBF1-undisturbed cells. Conversely, CSFV multiplication was increased in cells overexpressing GBF1. Therefore, GBF1 participated in CSFV multiplication.

GBF1 participates in the retrograde Golgi-to-ER transportation by activating the ADP-ribosylation factor (ARF) to regulate cytosolic COP I recruitment (Zhao et al. 2006 ; (Carpp et al. 2014) and influenza virus (Watanabe et al. 2014) . One of the mechanisms by which GBF1 regulates viral multiplication involves the GBF1-ARF1-COP I pathway. HCV replication is suppressed when the functions of GBF1 and ARF1 are disturbed (Goueslain et al. 2010; Matto et al. 2011) . Dengue virus uses the GBF1-ARF1/ ARF4-COP I pathway to form capsids around lipid droplets (Iglesias et al. 2015) . GBF1-mediated ARF1 activation is crucial for mouse hepatitis coronavirus (MHV) RNA replication (Verheije et al. 2008) , Ebola virus virion formation (Yamayoshi et al. 2010) , and diverse families of negativestranded RNA viruses (Panda et al. 2011) . Other metabolic pathways are also regulated by GBF1 and are involved in virus proliferation. GBF1 simultaneously regulates ARF4 and ARF5 to modulate HCV replication (Farhat et al. 2016) . GBF1 has other functions in HCV replication in BFA-resistant hepatoma-derived cells (Farhat et al. 2012) .

Here, we discovered that both CSFV RNA replication and virus particle formation were inhibited by the disturbance of GBF1 function. However, the CSFV proliferation dynamics in ARF1 shRNA-transfected cells were not different from those in control cells (data not shown). Therefore, GBF1 may regulate some metabolic pathways without ARF1 to modulate CSFV proliferation. The detailed function of GBF1 in CSFV proliferation requires further investigation.

Rab2 is a component of pre-Golgi intermediates that mainly regulates retrograde trafficking from the ERGIC (ER-Golgi intermediate compartment) to the ER (Ortiz Sandoval and Simmen 2012). Moreover, Rab2 is essential for Golgi organization (Haas et al. 2007 ) and can regulate COP I-mediated retrograde transport to intervene with the release of HCV particles (Raj 1961 ). In the present study, CSFV replication was suppressed when Rab2 was downregulated. The overexpression of Rab2 can accelerate CSFV proliferation. The mechanisms by which Rab2 influences CSFV proliferation must be further investigated.

As the cargo sorting station within the cell, the Golgi apparatus participates in protein and vesicular transport (Mironov et al. 2013; Guo et al. 2014) . Additionally, the secretory pathways involved in the Golgi apparatus are always utilized by viruses for egress on infected cells. Transport from early endosomes to the trans-Golgi network is important for vaccinia virus membrane wrapping and egress (Sivan et al. 2016) . Human herpesvirus-6 (HHV-6) can egress via the exosomal release pathway by budding at trans-Golgi network vacuoles and inducing multivesicular body formation (Mori et al. 2008) . Furthermore, the hepatitis E virus and HCV also utilize the exosome release pathway for egress (Tamai et al. 2011; Nagashima et al. 2014 ). However, viral egress is a complicated process that involves many pathways and related proteins. Reports have shown that HCV can be released from Huh7 cells via a trans-Golgi network-to-endosome pathway (Jamel et al. 2016) . Additionally, HCV particles are found in endosomal compartments and released via a non-canonical secretory route that is unrelated to the Golgi-associated secretory route and may be an undescribed cellular secretion route (Bayer et al. 2016) . Viral egress has a close connection with the virus particle formation process. CSFV is a single-stranded positive-sense RNA virus that has no cytopathic effect in infected cells. The CSFV assembly process is still unclear and mysterious because its core protein is dispensable for CSFV assembly (Riedel et al. 2012) . Additional experiments are urgently needed to determine the mechanisms of CSFV assembly and egress.

In summary, we demonstrated that GBF1 and Rab2 play important roles in CSFV proliferation. Golgi and pre-Golgi intermediates are also important in CSFV replication. Nevertheless, the effect of COP I-mediated retrograde trafficking on CSFV replication must be further investigated.

Hepatitis c virus is released via a non-canonical secretory route

Quantitative proteomic analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 (GBF1) in dengue infection

A Kinomewide small interfering RNA screen identifies proviral and antiviral host factors in severe acute respiratory syndrome coronavirus replication, including double-stranded RNA-activated protein kinase and early secretory pathway proteins

Hepatitis C virus replication and Golgi function in brefeldin a-resistant hepatomaderived cells

Identification of class II ADP-ribosylation factors as cellular factors required for hepatitis C virus replication

Identification of GBF1 as a cellular factor required for hepatitis C virus RNA replication

Viroporin activity and membrane topology of classic swine fever virus p7 protein

Protein sorting at the trans-golgi network

Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells

Classical swine fever virus NS5A protein localizes to endoplasmic reticulum and induces oxidative stress in vascular endothelial cells

Dengue virus uses a noncanonical function of the host GBF1-Arf-COPI system for capsid protein accumulation on lipid droplets

Release of infectious hepatitis c virus from huh7 cells occurs via atrans-golgi network-toendosome pathway independent of very-low-density lipoprotein secretion

Biosynthesis of Classical Swine Fever Virus nonstructural proteins

Poreforming activity of pestivirus p7 in a minimal model system supports genus-specific viroporin function

BIG1, a brefeldin A-inhibited guanine nucleotideexchange factor, is required for GABA-Gated Cl -influx through regulation of GABA a receptor trafficking

Classical swine fever virus and p7 protein induce secretion of IL-1β in macrophages

The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the Trans-Golgi Network (TGN)

Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication

Modification of intracellular membrane structures for virus replication

Golgi's way: a long path toward the new paradigm of the intra-golgi transport

Human herpesvirus-6 induces mvb formation, and virus egress occurs by an exosomal release pathway

Hepatitis e virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies

Rab proteins of the endoplasmic reticulum: functions and interactors

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

Research article: autophagy enhances the replication of classical swine fever virus in vitro

Biological roles of cellular glyceraldehyde-3-phosphate dehydrogenase in the hepatitis C virus life cycle

Opportunistic intruders: how viruses orchestrate ER functions to infect cells

Inhibitors of COP-mediated transport and cholera toxin action inhibit simian virus 40 infection

The core protein of classical swine fever virus is dispensable for virus propagation in vitro

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex

Classical swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation by binding to NS5A

Retrograde transport from early endosomes to the trans-golgi network enables membrane wrapping and egress of vaccinia virus virions

Regulation of hepatitis c virus secretion by the hrs-dependent exosomal pathway

Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress

Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation

A KDEL retrieval system for ER-golgi transport of japanese encephalitis viral particles

Influenza virushost interactome screen as a platform for antiviral drug development

A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1

Ebola virus matrix protein VP40 uses the COPII transport system for its intracellular transport

Role of the GTPase Rab1b in ebolavirus particle formation

GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic

Corresponding editor: VEENA K PARNAIK

This work was supported by the National Natural Science Foundation of China (No. 31472210).