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Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate
Accepted Manuscript Title: Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate Authors: Mrigendra K.S. Rajput, Karim Abdelsalam, Mahmoud F. Darweesh, Lyle J Braun, Jason Kerkvliet, Adam D. Hoppe, Christopher C.L. Chase PII: DOI: Reference:
S0165-2427(17)30097-1 https://doi.org/10.1016/j.vetimm.2017.09.006 VETIMM 9671
To appear in:
VETIMM
Received date: Revised date: Accepted date:
18-2-2017 22-9-2017 25-9-2017
Please cite this article as: Rajput, Mrigendra K.S., Abdelsalam, Karim, Darweesh, Mahmoud F., Braun, Lyle J, Kerkvliet, Jason, Hoppe, Adam D., Chase, Christopher C.L., Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate.Veterinary Immunology and Immunopathology https://doi.org/10.1016/j.vetimm.2017.09.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title
Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate Mrigendra K.S. Rajputa*, Karim Abdelsalama*, Mahmoud F. Darweesha, Lyle J Brauna, Jason Kerkvlietb, Adam D. Hoppeb, Christopher C.L. Chasea a
Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD
57007, USA; b
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007,
USA;
Corresponding author email: [email protected]
The current address of Mrigendra K.S. Rajput is Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lancing, MI,48824. * Authors have equal contribution
Highlights 1. Both cytopathic and non-cytopathic BVDV induce autophagy in immortal as well as primary cells. 2. Immortal cells had higher autophagy as compare to primary cells. 3. Autophagy inhibiting drug, 3-methyladenine significantly reduced autophagy and viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy and viral replication. 4.
Co-localization study showed that BVDV replicates in close associated with autophagosomes.
5.
There was no significant difference between cp or ncp strains of BVDV in autophagosome formation
Abstract Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect
of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. Results showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p <0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.
Keywords: Autophagy, cytopathic Bovine viral diarrhea virus , non-cytopathic Bovine viral
35
diarrhea virus, Rapamycin, 3-Methyladenine
Introduction Autophagy is a normal cellular physiological process, where cytoplasmic components are sequestered, enzymatically digested and recycled to maintain cellular homeostasis (Mizushima, 2007). This selfdigestion not only provides nutrients to maintain vital cellular functions during fasting but also a pathway to eliminate superfluous or damaged organelles, misfolded proteins, and invading microorganisms (Levine and Kroemer, 2008). Autophagy occurs at low basal levels in almost all cells. It is rapidly upregulated when cells need to generate intracellular nutrients and energy during starvation or high bioenergetic demands. Autophagy is also upregulated during oxidative stress, infection or protein accumulation (Kiffin et al., 2006; Dreux and Chisari, 2009; Dreux et al., 2009; Liu et al., 2010). Autophagy is important in innate and adaptive immune response against a variety of viral and bacterial pathogens (Deretic, 2005; Deretic and Levine, 2009). The interaction of single-stranded RNA with toll-like receptor 7 was found as the most potent effectors in autophagy induction (Deretic, 2011). In adaptive immunity, autophagy increases the major histocompatibility complex II antigen presentation with enhanced T cell proliferation (Schmid et al., 2007). In mice, knockdown of the autophagy related gene 5 resulted in the failure of efficient proliferation of CD4+ and CD8+ T cells after T cell receptor stimulation (Pua et al., 2007). Autophagy also controls the proliferation and survival of T and B cells (Miller et al., 2008). Despite the effective role of autophagy in immune response, many viruses utilize the autophagosomes for their efficient replication. The flaviviridae use autophagosome in two different ways. A few members of flaviviridae family such as, Hepatitis C virus (Ait-Goughoulte et al., 2008; Sir et al., 2008; Dreux and Chisari, 2009), dengue virus (Panyasrivanit et al., 2009; Heaton et al., 2010; McLean et al., 2011) and Japanese encephalitis virus (Li et al., 2012) induce autophagy for their replication while West Nile virus induces autophagy for cell survival (Beatman et al., 2012). Similarly, classical swine fever virus (CSFV) triggered autophagy and used autophagy machinery for its replication and release (Pie et al., 2014). The autophagy inducing drug, rapamycin facilitated cellular
proliferation after CSFV infection while autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) leaded to cell apoptosis (Pie et al., 2016). Bovine viral diarrhea virus (BVDV) is a member of the flavivirus family and the pestivirus virus group. BVDV causes great economic loss in cattle industry through its immunosuppression and persistent infection. A unique characteristic of BVDV is the ability of the virus to have two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp). Over 90- 95% of all field isolates are ncp and are responsible for the myriad of clinical outcomes including persistent infection (PI). PI is the result of ncp BVDV fetal infection during their first trimester. Such calves remain immunotolerant to BVDV and continuously act as a source of infection to other animals (Chase et al., 2015). The cp strains are the result of well-characterized genotypic changes that takes place in a fatal form of BVDV, mucosal disease, which occurs in PI animal where the ncp virus mutates to cp (Darweesh et al., 2014). The presence of this “pair”, ncp strain and the cp mutant strain, results in mucosal disease. Interestingly, the most studied and common vaccine strains are the cp mutants. Previous studies using a cp type 1a BVDV vaccine strain, NADL, showed that BVDV induced autophagy. The autophagy inducing drug, rapamycin, increased autophagy as well as BVDV titer while the autophagy inhibiting drug, 3-methyladenine or wortmannin, reduced autophagy and BVDV titer (Fu et al., 2014a; Fu et al., 2014b; Fu et al., 2014c; Fu et al., 2015). A study using cp type 1a NADL and ncp1b NY1 demonstrated that cpBVDV induced autophagy-like vacuolization in cp BVDV-infected cells but not in ncp BVDV-infected cells Birk et al., 2008. However, a recent study using cytopathic HJ1 (BVDV1b) and non-cytopathic New York1 (NY1) (BVDV1b) strains showed that both cp and ncp BVDV induce autopay with similar rate (Zhou et al., 2017). However, these viruses were not a pair. A further consideration is the difference in cp or ncp BVDV autophagy induction in an established cell as well as primary cells. This study examined a homologous virus pair [i.e., cytopathic
(cp) BVDV1b Tifton GeorgiA Cytopathic (TGAC) and noncytopathic (ncp) BVDV1b Tifton GeorgiA Noncytopathic (TGAN), that were isolated from a clinical BVDV mucosal disease to determine the effect of BVDV phenotypes on autophagy induction in both primary bovine turbinate cells and the MDBK cell line.
Materials and Methods Virus and cells The homologous pair of ncp and cp type 1b viruses (TGAN and TGAC) recovered from an animal that died of mucosal disease was used in the study (Ridpath et al., 1991). These viruses were provided by Dr. Julia Ridpath. The BVDV free Madin Darby bovine kidney cells (MDBK cells) (passage 95-110) and bovine turbinate cells (Bt cells) (passage 10-15) were used in the study. The cells were grown in complete minimal essential medium (CMEM). The CMEM contained minimal essential medium (MEM) (Gibco BRL, Grand Island, NY, USA), supplemented with 10% BVDV free fetal bovine serum (FBS) (PPA, Pasching, Austria), penicillin (100 U /ml) and streptomycin (100 μg /ml) (Sigma, St. Louis, MO, USA).
LC3- GFP stabilized cell line The autophagy induction was visualized through stably expressing MDBK- GFP-LC3 cells or Bt- GFP-LC3 cells. These cells were created through GFP-LC3 -pseudolentivirus transduction. Upon autophagy induction, these cells showed dots like structures of GFP-LC3II, which localized at autophagic vacuole membrane (Ait-Goughoulte et al., 2008). To create GFP-LC3 pseudolentivirus, the GFP-LC3 gene was cut between Ndel and BamH1 restriction sites from pEGFP-LC3-11546 plasmid (Addgene, Cambridge, MA, USA) and inserted in to lentivirus vector pLenti CMV GFP Puro-17448 (Addgene, Cambridge, MA, USA). Briefly, both plasmid (e.g. pEGFP-LC3-11546 or pLenti CMV GFP Puro-1154) were digested through Ndel and BamH1 restriction enzymes (Sigma, St. Louis, MO, USA) for 2 hours. After digestion, products were
separated using 1% agarose gel (Sigma, St. Louis, MO, USA). The 1538 base pair (bp) fragment of GFPLC3 from pEGFP-LC3-11546 plasmid and, 8197 bp fragment of pLenti CMV GFP Puro-1154 plasmid were isolated through QIAquick Gel Extraction Kit (QIAGEN, Germantown, MD, USA). Both fragments were ligated together using T4 ligase (Sigma, St. Louis, MO, USA) to get pLenti CMV GFP Puro-LC3 plasmid. The pLenti CMV GFP Puro-LC3 plasmid was transformed and amplified in DH5α strain of E coli (Life Technologies, Grand Island, NY, USA). After amplification, pLenti CMV GFP Puro-LC3 plasmid was confirmed for LC3 insertion through restriction digestion using Ndel and BamH1, which yielded 1538 bp fragment of GFP-LC3 and 8197 bp fragment of pLenti CMV GFP Puro vector. To make GFP-LC3pseudolentivirus, the 293T cells were co-transfected with (1) pLenti CMV GFP Puro-LC3 containing GFPLC3 gene, (2) packaging plasmid pCMVR8.74-22036 (Addgene, Cambridge, MA, USA) and, (3) a plasmid containing vesicular stomatitis virus G glycoprotein pVSV.G-14888 (Addgene, Cambridge, MA, USA) using X-treme GENE HP DNA Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA). After 48 h post co-transfection, the supernatant from 293T cells, containing GFP-LC3-pseudolentivirus was collected. The GFP-LC3-pseudolentivirus was used to transduce MDBK or Bt cells. Briefly, MDBK or Bt cells were seeded in 6 well plates with concentration of 5x105 cell/well. After overnight attachment, the cell supernatant was replaced with 0.5 ml GFP-LC3-pseudolentivirus containing 5µg/ml polybrene (kindly provided by Adam Hoppe, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD, USA) and incubated at 37 ºC for 1 h adsorption. After 1 h, non-adsorbed virus was removed and cells were supplemented with 3 ml CMEM. The LC3-GFP transduction in MDBK- GFP-LC3 cells or Bt-GFP-LC3 cells were confirmed after 4 days post transduction through fluorescent microscope (Olympus, PA, USA) at 395 nm wavelength. LC3-GFP transduced cells were further selected using puromycin (10µg/ml) for 4 days. The positively expressing LC3-GFP selected cells (>95% LC3-GFP transduced cells) were used to determine the BVDV induced autophagy in MDBK or Bt cells. To further confirm our results,
another set of MDBK- GFP-LC3 cells or Bt- GFP-LC3 were created using commercially available recombinant lentivirus or LentiBrite control virus (EMD Millipore Corporation, Billerica, MA, USA). Both set of cells (using lab made lentivirus and commercially purchased lentivirus with >90% transduced) gave similar results, which were averaged and analyzed for significant difference using student One-way ANOVA with post-hoc Tukey HSD test at 95% level of significance using SAS version 8.0.
Drugs and virus production The drug rapamycin (Thermo scientific, Wilmington, DE, USA) an autophagy inducer with a concentration of 50 nM in CMEM while 3-methyladenine (3 MA) (Thermo scientific, Wilmington, DE, USA) with concentration of 10 mM in CMEM was used as autophagy inhibitor. The dose of rapamycin or 3MA were chosen following their dose titration on BVDV production, autophagy induction in GFP-LC3 transduced MDBK cells and viability (Suppl. Fig. 2 and Suppl. Fig. 3). Cell viability were examined through trypan blue exclusion assay following trypsinization. Out of 10 nM/ml, 50 nM/ml and 100 nM/ml, rapamycin with 50 nM/ml and, out of 0.5µm/ml, 5.0µm/ml and 10 Mm, 3-MA with 10 Mm/ml were chosen for highest effect on autophagy induction and 100% cell viability. The MDBK cells or Bt cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with 6 Multiplicity of infection (MOI). The 6 MOI was chosen to ensure that all cells become infected with BVDV. While mock-infected cells were used as negative controls. To measure the virus production, cell supernatant was collected at 1 h, 6 h, 12 h, 24 h and 48 h p.i. and analyzed for BVDV titer as per the method described earlier (Reed and Munch, 1938). Each experiment was done in three replicates and reproduced three times. The data was analyzed using one-way ANOVA with post-hoc Tukey HSD Test at 95% level of significance using SAS version 8.0.
Autophagy measurement through GFP-LC3 transduced cells To determine the effect of BVDV biotypes on autophagy induction, the stable expressing MDBK-GFP-LC3 cells or Bt-GFP-LC3 cells were cultured in 4 well chamber slide (Vector Laboratories, Burlingame, CA, USA). The cells were infected with either cp BVDVb1 TGAC or ncp BVDVb1TGAN with MOI of 6. The mock-infected cells were used as control. After 1 hour of incubation non-adsorbed virus was removed. The cells were supplemented with medium containing rapamycin (50 nM) or 3 MA (10 mM) or none. The cells were fixed with 4% paraformaldehyde at 12 h, 24 h and 48 h p.i. and mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA). The cells showing green fluorescence (LC3 dot formation/green punctated structure) at each time point was counted using a fluorescent microscope (Olympus, PA, USA). One hundred (100) cells on each slide using four replicates were counted at each time point. All experiments were reproduced three times. The percentage of autophagosome positive cells were calculated and one-way ANOVA with post-hoc Tukey HSD Test at 95% level of significance sing SAS version 8.0.
Autophagy measurement in MDBK cells The 5.0 x105 MDBK cells were seeded in each well of 6 well plates. After overnight attachment, cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with 6 MOI with or without autophagy inducing drug, rapamycin (50 nM) or inhibiting drug, 3MA (10 mM), while mock infected non-drug treated cells were used as control. After 48 hours, cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer) (Sigma, St. Louis, MO, USA) containing a proteinase inhibitor (ULTRA tablet) (Roch Diagnostic, GmbH, Sandhofen, Germany) and examined for LCI and LCII protein using Western blot analysis. Briefly, 40µg cell lysate were run in each well of SDS (sodium dodecyl sulfate) gel, after running, protein were transfected to nitrocellulose membrane (Whatman GmbH, Germany). Membranes
were blocked with 5% gelatin in PBS for 1 h at room temperature followed by incubation with anti-LC3 antibody (1:500) (Abcam, Cambridge, MA) overnight at 4ºC. Membranes were washed as incubated with goat anti-rabbit HRP conjugated (1:2000) and developed using Pierce ECL Western Blotting Substrate (Thermo scientific, Wilmington, DE, USA). Band Intensities in image were measured through ImageJ software.
Co-localization of autophagosome and BVDV proteins To determine the topological relationship between autophagosomes and BVDV replication site, the colocalization of BVDV proteins and autophagy marker was carried out. The MDBK-GFP-LC3 cells were cultured in 4 well chamber slides. The MDBK-GFP-LC3 were infected with ncp TGAN with MOI of 6. The mock-infected cells were used as control. The cells were fixed with 4% paraformaldehyde at 48 h p.i. The fixed cells were permeabilized using 0.3% triton in phosphate buffered saline (PBS) containing 3% bovine serum albumin. The permeabilized MDBK-GFP-LC3 cells were stained with rabbit polyclonal anti-NS5A, Erns or E1 antibodies (produced in our laboratory using a recombinant BVDV NS5A, Erns or E1 proteins as an immunogen) with concentration of 1:1000 in PBS followed by staining with anti-rabbit antibody conjugated with rhodamine (Sigma, St. Louis, MO, USA) with concentration of 1:1000 in PBS. The stained cells were mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined for co-localization of BVDV proteins (red) and autophagosomes (green punctated structure: GFP-LC3) using florescent microscope (Olympus, PA, USA).
Results Both biotypes of BVDV induced autophagy
The BVDV infected MDBK-GFP-LC3 cells or Bt-GFP-LC3 cells showed that both cp BVDV1b TGAC (Fig. 1C) and ncp BVDV1b TGAN (Fig. 1B) induced autophagy in MDBK as well as Bt cells (figure not shown) as indicated by the punctate vesicles. However, there was no significant change in cellular morphology in cells infected with either cp or ncp BVDV within the first 48 h of infection. There was no significant difference in autophagy induction between both biotypes in either cell types. However, TGAN significantly enhanced autophagy in MDBK as well as Bt cells at 12 h, 24 h and 48 h post infection as compared to its time point control (p <0.05). (Fig. 2B and Fig. 3B). Similarly, TGAC significantly enhanced autophagy in MDBK at 12 h, 24 h and 48 h post infection (Fig. 2A) while 24 h and 48 hr post infection in Bt cell as compared to its time point control (p <0.05) (Fig. 3A). Autophagy inhibiting drug, 3MA, significantly reduced autophagy induction in MDBK cells as compared to cells infected with TGAC, TGAN, TGAC supplemented with rapamycin or TGAN supplemented with rapamycin at 12h, 24 h or 48 h post infection (p <0.05) (Fig. 2A and Fig. 2B). Similarly in Bt cells, 3MA significantly reduced autophagy induction as compared to cells infected with TGAC supplemented with rapamycin or TGAN supplemented with rapamycin at 12h, 24 h or 48 h post infection (p <0.05) (Fig.3A and Fig. 3B). Autophagy induction was significantly higher in Bt cells supplemented with rapamycin as compared to TGAC or TGAN infected cells alone at 12h, 24 h or 48 h post infection (p <0.05) (Fig.3A and Fig. 3B). While comparing different cell types, MDBK cells showed significantly higher autophagy as compared to Bt cells, either infected with ncp TGAN (Fig. 2B and, 3B), cp TGAC (Fig. 2A, and 2B) or in the mock infection (p <0.05). The higher autophagy activity in MDBK cells may be due to the faster growth and doubling time of MDBK cells compared to Bt cells. The autophagy also increased over the course of infection in both cell types. The MDBK cells infected with ncp TGAN resulted in autophagy at levels of 28.00±3.26%, 72.66±5.03%, 92.66±1.15% 12 h, 24 h and 48 h p.i. (Fig. 2B) while cp TGAC infection resulted in autophagy at levels of 26.66±2.30%, 74.66±4.04%, and 89.33±1.15% at 12 h, 24 h and 48 h p.i.
(Fig. 2A), while non-BVDV infected MDBK cells, treated with rapamycin or 3MA showed the autophagy as 60.0±8.80 or 23.16±1.16 at 48 hr p.i. respectively. Similarly, Bt cells infected with ncp TGAN or cp TGAC resulted in autophagy as 13.33±0.57%, 23.25±2.87%, 32.0±2.64% and 15.0±3.0%, 23.25±2.06%, 25.66±1.52% at 12 h, 24 h and 48 h p.i. respectively (Fig. 3B and 3A), while non-BVDV infected Bt cells, treated with rapamycin or 3MA showed the autophagy as 25.4±2.4 or 22.4±1.67 at 48 hr p.i. respectively. The phosphoinositide 3- kinase (PI-3 kinase) inhibiting drug, 3-methyladenine (3MA) that suppresses autophagy, significantly reduced autophagy 10-60% in TGAN or TGAC BVDV infected MDBK cells (p <0.05) (Fig. 2B and 2A respectively). Autophagy was reduced 4-12% by 3MA in ncp or cp BVDV infected Bt cells (p˂0.05) (Fig. 3B and 3A). The autophagy inducing drug, rapamycin, resulted in no to a slight increase (0-10%) in autophagy that was not significant in ncp TGAN or cp TGAC infected MDBK cells (p <0.05) (Fig. 2B and Fig. 2A) respectively. In contrast, autophagy was significantly increased (10-18%) in TGAN or TGAC infected Bt cells (p <0.05) (Fig. 3B and Fig. 3A) respectively. To avoid any interference of overexpression on autophagy induction, native MDBK cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with or without autophagy inducing drug, rapamycin or autophagy inhibiting drug, 3MA, while mock infected non-drug treated cells were used as control. After 48 hours, cells were lysed and examined for LCI and LCII protein using Western blot analysis. Autophagy induction in MDBK cells were analyzed through LC3II/LC3I ratio using ImageJ software. Rapamycin treatment enhanced LC3II expression. MDBK cells infected with TGAC or TGAN and treated with rapamycin had a LC3II/LC3I ratio of 0.72±0.12 and 0.81±0.06 respectively as compared with TGAC (0.65±0.12) or TGAN (0.65±0.02) alone (Suppl. Fig. 1). Autophagy in MDBK cells infected with TGAC or TGAN and treated with 3MA had a LC3II/LC3I ratio of 0.52±0.12 and 0.54±0.26 respectively. Mock infected control cells autophagy was 0.51±0.00 (Suppl. Fig. 1).
The effect of autophagy on BVDV replication To determine the relationship between autophagy and BVDV replication, the cp TGAC or ncp TGAN infected MDBK cells or Bt cells were treated with either the autophagy inducing drug, rapamycin or autophagy inhibiting drug, 3MA and analyzed for BVDV production. The controls were cells infected with BVDV (TGAN or TGAC) with no drug or mock infection with no drug treatment. There was a correlation between autophagy and BVDV production. Rapamycin-treatment of BVDV-infected MDBK cells significantly increased cp TGAC as well as ncp TGAN virus production at 12 h p.i. (Fig. 4A and, 4B) (p <0.05). Rapamycin increased the ncp TGAN titer at 12 h p.i. from 0 log10/ml in the untreated infected cells to 1.00±0.00 log10/ml rapamycin-treated cells (Fig. 4B). In MDBK cells infected with cp TGAC, the titer also increased from 0.50±0.57 log10/ml in the untreated infected cells to 1.50 ±0.57 log10/ml (Fig. 4A) (p <0.05). Overtime, the rapamycin-treated MDBK cells had ncp TGAN titers that were consistently 0.5 log10 higher (3.50±0.70 log10/ml at 24 h and 5.00±0.00 log10/ml at 48 h). This was not significantly different at (p <0.05)] as compared to untreated ncp TGAN infected MDBK cells (3.00±0.00 log10/ml at 24 h and 4.50±0.57 log10/ml at 48 h) (Fig. 4B). The rapamycin treatment of cp TGAC-infected MDBK cells did not increase the virus production at 24 h p.i. as compared to untreated infected cells (3.00±0.00 log10/ml in both groups). In contrast at 48 h, rapamycin treatment significantly (p <0.05) increased the titer one log as compared to the untreated infected cells (5.00±0.00 log10/ml in untreated vs. 6.00±0.00 log10/ml in rapamycin-treated cells) (Fig. 4A). Like MDBK cells, there appeared to be an increase in viral titers in rapamycin-treated Bt cells over time. The rapamycin treatment significantly increased (p <0.05) both the ncp TGAN and cp TGAC titers at 24 h by 1 log10 (1.00±0.00 log10/ml in untreated infected vs. 2.00±0.00 log10/ml in rapamycin-treated Bt cells infected with TGAN; 2.00±0.00 log10/ml in untreated infected Bt cells vs. 3.00±0.00 log10/ml in rapamycin-treated Bt cells infected with TGAC (Fig. 5B and 5A, respectively). The increased viral titer was also observed at 48 h post infection for both virus biotypes. The
rapamycin-treated Bt cells infected with ncp TGAN had a titer of 3.00±0.00 log10/ml as compared to 2.50±0.57 log10/ml from untreated infected Bt cells (p <0.05) (Fig. 5B). Rapamycin treated Bt cells infected with cp TGAC had viral titers of 4.66±0.57 log10/ml vs. 3.50±0.57 log10/ml in the untreated infected Bt cells (Fig. 5A). The autophagy inhibiting drug, 3MA, significantly suppressed cp TGAC as well as ncp TGAN production (p <0.05) at 12 h, 24 h and 48 h p.i.in both MDBK and Bt cells (Fig. 4A, 4B, 5A and, 5B). There was no BVDV production in 3MA-treated MDBK cell or Bt cells until 24 h p.i. BVDVinfected MDBK cells produced virus titers of 1.00±0.00 log10/ml for both cp TGAC and ncp TGAN at 48 h p.i. (Fig. 4A and, 4B). Similarly, 3MA treated BVDV-infected Bt cells produced ncp TGAN titers of 1.00±0.00 log10/ml and cp TGAC titers of 0.50±0.57 log10/ml at 48 h p.i. (Fig. 5A and, 5B). Taken together, the increased production with autophagosome agonist and decreased production with an autophagosome inhibitor supported the importance of autophagosomes for the production of BVDV regardless of biotype.
Co-localization of autophagy with BVDV proteins To determine whether BVDV replicated in association with autophagosomes, a co-localization study of autophagy marker, LC3 and BVDV proteins NS5A, Erns and E1 was carried out. The MDBK-GFP-LC3 cells were infected with ncp BVDV1b TGAN at a 6 MOI. Confirmation that all cells were infected with virus, was done using immunohistochemistry (IHC) with anti-BVDV 16C6 antibody (IDEXX Laboratories, Westbrook, ME, USA) (Rajput, 2013). The ncp BVDV1b TGAN or mock-infected cells were fixed with 4% paraformaldehyde at 48 h p.i. and stained for BVDV NS5A, Erns or E1 proteins using rabbit polyclonal anti-NS5A, Erns or E1 antibodies followed by anti-rabbit antibody conjugated with rhodamine. Stained cells were mounted and examined for co-localization of BVDV proteins (red) and autophagosomes (green punctate structure). The ncp TGAN infected MDBK-GFP-LC3 cells expressed the BVDV proteins, Erns
(Fig. 6B), E1 (Fig. 6E) and NS5A (Fig. 6H) with red color while mock-infected cells did not express any viral proteins such as Erns (Fig. 6K), E1 or NS5A (Fig. not shown). The ncp BVDV1b TGAN infected MDBK-GFP-LC3 induced the formation of autophagosomes as distinct punctate vesicles (Fig. 6A, 6D and 6G) as compared to mock-infected cells (Fig. 6J). The co-localization of autophagosomes and structural Erns (Fig. 6C), E1 (Fig. 6F)) and non-structural proteins NS5A (Fig. 6I) indicating that BVDV replicates in close association with autophagosomes regardless of biotype or cell type. However, a future confocal microscopy study is needed to determine the specific location of virus replication in relation to autophagosomes.
Discussion The current study was carried out to determine effect of cp and ncp strain of BVDV on autophagy induction in immortal as well as primary cell line. Immortal MDBK-GFP-LC3 cells or primary Bt-GFP-LC3 cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN. Such infected cells were supplemented either with autophagy inducing drug, rapamycin or autophagy inhibiting drug 3MA or no drug. The study revealed that both ncp and cp biotypes of BVDV induced autophagy in the primary Bt cells as well as the MDBK cell line, which was significantly higher than mock-infected control cells (p <0.05). There was no significant difference between cp or ncp BVDV infection and autophagy induction (p <0.05). The autophagy inducing drug, rapamycin increased autophagy as well as virus production, while the autophagy inhibiting drug, 3MA, significantly suppressed autophagy and BVDV replication in both cell types. The MDBK cell line had significantly higher autophagosomes as compared to Bt cells, whether infected with ncp TGAN, cp TGAC or mock infected (p <0.05). The higher autophagy activity in MDBK cells may be due to its faster growth characteristics and higher metabolic activity than Bt cells.
To rule out the effect of LC3 overexpression on autophagy induction, native MDBK cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN, supplanted with autophagy inducing drug, rapamycin or autophagy inhibiting drug 3MA or no drug. Cells were lysed and examined for LC3II expression through western blot. Results showed that both cp BVDV1b TGAC and ncp BVDV1b TGAN induced autophagy in MDBK cells with similar rate. The co-localization study using BVDV nonstructural protein NS5A or structural proteins Erns and E1 with the autophagosome marker, LC3, indicated that BVDV replicated in close association with autophagosomes. However a detail study is needed using confocal microscopy to determine the specific location of virus replication compared to autophagosome localization. These results are similar to those observed in electron microscopy studies with cp BVDV, which found the BVDV induced autophagy and BVDV particles were present in autophagosomes (Fu et. al., 2014a; Fu et. al., 2014b; Fu et. al., 2014c; Fu et al., 2015). However, all of these studies used the cp type 1a vaccine strain NADL in MDBK cells. One of the enigmas of BVDV is the distinct in vitro phenotype differences in cell culture between ncp and cp BVDV. The cellular-viral protein interactions responsible for the different phenotypes have not been fully defined. The cp BVDV strains are isolated in nature, predominately from mucosal disease, where the cp strain has mutated from its homologous ncp strain (Brownlie et al., 1984; Ridpath et al., 1991). Cp BVDV is unable to establish chronic infection or cause persistent infection in the host. Previous autophagy-BVDV studies used the NADL strain (Fu et. al., 2014a; Fu et. al., 2014b; Fu et. al., 2014c; Fu et al., 2015). Although the NADL is a vaccine strain and has been an effective immunogen, NADL BVDV infections are self-limiting unlike their ncp counterparts. Therefore, cp BVDV “illustrates a case of viral emergence to extinction – irrelevant for BVDV evolution, but fatal for the persistently infection host” (Peterhans et al., 2010). On the other hand, the ncp BVDV biotypes are economically important viruses, affecting the cattle industry worldwide and the major cause of immune suppression and
persistent infection. The infection of the bovine fetus with ncp BVDV during the first trimester results in persistently infected (PI) calves that remain immunotolerant to BVDV and continuously act as a source of infection to other animals (Chase et al., 2015). The ncp BVDV inhibits double-stranded RNA-induced apoptosis and interferon synthesis in tissue culture while cp strains induce interferon and cause apoptosis (Schweizer and Peterhans, 2001). There are two studies that compared the effect of cp and ncp BVDV on autophagy induction (Birk et al., 2008; Zhou et al., 2017). However, the findings of these two studies contradict each other. Birk et al. (2008) used cp type 1a NADL and ncp1b NY1 and showed that cpBVDV infection induced vacuolization in infected cells. Those vacuoles have all structural characteristics of autophagy and their formation was inhibited with 3MA treatment. Vacuolization increased in cp BVDV infected MDBK cells in a time dependent manner; however, ncp BVDV infected cells had no vacuolization (Birk et al., 2006; Birk et al., 2008). In contrast, Zhou et al. (2017) using cp BVDV1b HJ1or ncp BVDV1b NY1, showed that that there is no significant difference in autophagy induction between cp and ncp BVDV. However, Birk et al. (2008) used BVDV strains from two subgenotypes (BVDV1a and BVDV1b) while Zhou et al. (2017) used two strains from same subgenotypes (BVDV1b) but not paired viruses. To precisely characterize the effect of cp and ncp BVDV on autophagy induction, a true virus pair arising from the same animal from a naturally occurring infection was used. The cp TGAC and ncp TGAN viruses used in this study were isolated at the National Animal Disease Center, Ames, IA from a mucosal disease (MD) case. The MD occurred spontaneously in the unvaccinated animal and was not induced experimentally. Following isolation, the virus pairs antigenic properties and genomic sequence were determined. The TGAC and TGAC differed in their binding of three of 10 BVDV MAbs specific for gp53. TGAC and TGAN reacted similarly to BVDV conserved cDNA probes while they were differentiated by oligomer probes (3289-3309) and (11081-11101) (Ridpath et al., 1991). Later, the authors successfully produced MD in animals using the virus pair, TGAC/TGAN. (Ridpath et al., 1991). Further sequencing of
TGAN and TGAC was reported in 1992 found that cp and ncp pairs were not identical. The reason for variation was concluded as there was no cellular RNA insertion or duplication in p125 region of ncp virus while its counterpart, cp virus had a p80 gene duplication as well as a cellular ubiquitin RNA insertion (Qi et. al., 1992). Additionally a sequence comparison done between TGAN and TGAC in an approximately 270 base pair region of the 5' UTR of each virus and showed a 97% homology (J. Ridpath, personal communication). Therefore, it can be concluded that the TGAN is the parent with the larger TGAC containing the entire TGAN RNA backbone nucleotide along with a duplication of TGAN NS3 gene (p80) and a cellular ubiquitin RNA insertion (Qi et. al., 1992; Quadros et al., 2006). Cleavage of the NS23 region (Kümmerer et al., 1998), duplication of NS3 (p80) (Fritzemeier et al., 1995) or cellular ubiquitin RNA insertion (Qi et. al., 1992) have been shown to generate cp strain of BVDV. Using these closely related viruses, we did not find any significant difference in cp and ncp BVDV infection in autophagy induction or formation. Flaviviruses such as West Nile virus (WNV) (Tonry et al., 2005; Appler et al., 2010) or BVDV (Brock et al., 1998) often resulted in persistent infection. WNV infects CNS to escape immune system while BVDV persistent infected animals fail to distinguish BVDV as foreign. Both viruses exploit persistently infected cells for their survival and maintenance. There is no evidence indicating that flaviviruses establish persistent infection by autophagy (Codogno and Meijer, 2005; Ogata et al., 2006). A study with vesicular stomatitis virus revealed that the conjugated autophagy gene Atg5–Atg12 negatively regulated type I IFN production (Jounai et al., 2007). Similarly, the absence of autophagy gene Atg 5, resulted in ROS-dependent amplification of RLR (RIG-I-like receptors) signaling and increased type I IFNs (Tal et al., 2009). It is possible then that reduced type I IFN production may result in enhanced viral replication as seen in the current study with increased autophagy. Autophagy may also have a potential role in establishing the ncp BVDV persistent infection, which do not produce sufficient type 1 IFN (Charleston et al., 2001). Several
hypotheses have been proposed suggesting that autophagy regulates the cell assembly or metabolism and facilitates virus replication. The double membrane autophagosomes provide a scaffold for anchoring and concentrating the replication complexes for virus, as well as preventing the immune response triggered by dsRNA intermediates (Wileman, 2006; Miller and Locker, 2008). Autophagy regulated the cellular metabolism through autophagy-dependent processing of lipid droplets and triglycerides to release free fatty acids. This resulted in an increase intracellular beta-oxidation and ATP generation. The energy released by beta-oxidation may be utilized for efficient viral replication (Heaton and Randall, 2010). In the current study, using BVDV NS5A, Erns and E1 proteins with autophagosomes marker LC3 also revealed that BVDV replicate in close association with autophagosomes, which was similar to the previous studies, which found BVDV particles in or in close association with autophagosomes (Fu et al., 2014a; Fu et al., 2014b; Fu et al., 2014c). The scaffold for anchoring the replication complexes with energy provided by autophagosomes maybe the reason for higher BVDV titers seen following treatment with an autophagy agonist in these experiments. A similar association with autophagosomes was reported in HCV replication (Dreux et al., 2009). Other positive strain RNA viruses including, poliovirus (Jackson et al., 2005; Taylor and Kirkegaard, 2007), coxsackieviruses (Wong et al., 2008; Yoon et al., 2008) and flavivirus (AitGoughoulte et al., 2008; Sir et al., 2008; Dreux and Chisari, 2009) have used autophagy machinery to facilitate their replication. Another flavivirus, WNV induced autophagy and virus replication was independent of autophagy. While WNV induced autophagy was reduced by class III phosphatidylinositol 3kinase (PI3K) inhibiting drug such as 3MA or knockdown of Atg 5 gene, the effect of 3MA on virus replication may be due to association of other cellular mechanism involving PI3K pathways, independent of autophagy (Beatman et al., 2012). Another study showed that the expression of NS4B gene of HCV alone is able to induce autophagy via interactions with both the early endosome-associated GTPase Rab5 and a class
III phosphoinositide 3-kinase, Vps34 (Su et. al., 2011). In future studies, it would be interesting to know which BVDV viral protein(s) is/are responsible for autophagosome induction.
Conclusions This study indicated that both cp and ncp biotypes BVDV induced autophagy in infected primary as well as immortal cell lines. There was no significant difference between cp or ncp BVDV biotypes in autophagy induction. The autophagy inducing drug, rapamycin enhanced autophagy while autophagy inhibiting drug, 3MA suppressed autophagy as well as the virus production in both the biotypes. The colocalization of BVDV structural as well as non-structural proteins such as Erns, E1 and NS5As with the autophagosome marker, LC3 revealed that BVDV replicated in close association with autophagosomes. These findings further supported the importance of the presence of autophagosomes for the production of BVDV which need to be studied in detail for better understanding of BVDV pathophysiology. Conflict of interest None of the author in manuscript has any conflict of interest related to publication of this manuscript.
Acknowledgments The authors thank the Department of Veterinary and Biomedical Sciences/Animal Disease Research and Diagnostic Laboratory, South Dakota State University (SDSU), Brookings, SD, USA; SDSU Experimental Station and the Center for Biological Control and Analysis by Applied Photonics, Department of Chemistry and Biochemistry, SDSU, Brookings SD, USA, for providing funding to conduct the research. Authors are also thankful to Dr. Julia F Ridpath and Dr. John Neill (National Animal Disease Center, Ames, IA) for proving BVDV strains and sequence comparison between TGAN and TGAC.
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Figure Captions Figure 1. Autophagy induction in MDBK cells after ncp BVDV1BtGAN or cp BVDV1BtGAC infection. The stable expressing MDBK-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (B) or cp BVDV1BtGAC (C) with multiplicity of infection (MOI) 6, while mock infected cells were used as negative control (A). The cells were fixed with 4% paraformaldehyde (PFA) at 48 h post infection. Autophagy was determined through green punctated structure having GFP-LC3 on the membrane of autophagosomes through florescent microscope (White square: Zoomed in at upper panel).
Figure 2. The percentage of autophagy induction in MDBK cells following BVDV infection. The MDBK-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (A) or cp BVDV1BtGAC (B) with MOI of 6. The mock infected cells were used as control. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The percentage of autophagy induction was calculated at 12 h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 3. The percentage of autophagy induction in Bt cells following BVDV infection. The Bt-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (A) or cp BVDV1BtGAC (B) with MOI of 6. The mock infected cells were used as control. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The percentage of autophagy induction was calculated at 12 h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 4. Effect of autophagy on BVDV production in MDBK cells. The MDBK cells were infected with either cp BVDV1BtGAC (A) or ncp BVDV1BtGAN (B) with MOI of 6. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The virus titer in cell supernatants were evaluated at 1 h, 6 h, 12h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 6. Effect of autophagy on BVDV production in Bt cells. The Bt cells were infected with either cp BVDV1BtGAC (A) or ncp BVDV1BtGAN (B) with MOI of 6. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The virus titer in cell supernatants were evaluated at 1 h, 6 h, 12h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 6. The co-localization of BVDV Erns, E1 or NS5A protein with autophagosomes. The MDBK-GFP-LC3 cells were infected with ncp BVDV1BtGAN with MOI of 6 for 48 h and fixed with 4% paraformaldehyde. The fixed cells were permeabilized using 0. 3% triton in PBS containing 3% BSA and stained with rabbit polyclonal anti- Erns, E1 or NS5A antibodies followed by anti-rabbit antibody conjugated with rhodamine. Autophagosome induction was observed by green punctated structure in MDBK cells following ncp BVDV1BtGAN infection (Fig.6A, 6D, 6G respectively). BVDV Erns, E1 and NS5A proteins were detectable in infected cells (Fig. 6B, 6E, 6H respectively) but not in mock cells stained with BVDV Erns (Fig.6K) as well as mock infected cells stained with E1 or NS5A (figure not shown). Co-localization of LC3 with BVDV Erns, E1 and NS5A proteins (Fig. 6C, 6F, 6I, 6L).
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S0165-2427(17)30097-1 https://doi.org/10.1016/j.vetimm.2017.09.006 VETIMM 9671
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Please cite this article as: Rajput, Mrigendra K.S., Abdelsalam, Karim, Darweesh, Mahmoud F., Braun, Lyle J, Kerkvliet, Jason, Hoppe, Adam D., Chase, Christopher C.L., Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate.Veterinary Immunology and Immunopathology https://doi.org/10.1016/j.vetimm.2017.09.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title
Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate Mrigendra K.S. Rajputa*, Karim Abdelsalama*, Mahmoud F. Darweesha, Lyle J Brauna, Jason Kerkvlietb, Adam D. Hoppeb, Christopher C.L. Chasea a
Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD
57007, USA; b
Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007,
USA;
Corresponding author email: [email protected]
The current address of Mrigendra K.S. Rajput is Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lancing, MI,48824. * Authors have equal contribution
Highlights 1. Both cytopathic and non-cytopathic BVDV induce autophagy in immortal as well as primary cells. 2. Immortal cells had higher autophagy as compare to primary cells. 3. Autophagy inhibiting drug, 3-methyladenine significantly reduced autophagy and viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy and viral replication. 4.
Co-localization study showed that BVDV replicates in close associated with autophagosomes.
5.
There was no significant difference between cp or ncp strains of BVDV in autophagosome formation
Abstract Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect
of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. Results showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p <0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.
Keywords: Autophagy, cytopathic Bovine viral diarrhea virus , non-cytopathic Bovine viral
35
diarrhea virus, Rapamycin, 3-Methyladenine
Introduction Autophagy is a normal cellular physiological process, where cytoplasmic components are sequestered, enzymatically digested and recycled to maintain cellular homeostasis (Mizushima, 2007). This selfdigestion not only provides nutrients to maintain vital cellular functions during fasting but also a pathway to eliminate superfluous or damaged organelles, misfolded proteins, and invading microorganisms (Levine and Kroemer, 2008). Autophagy occurs at low basal levels in almost all cells. It is rapidly upregulated when cells need to generate intracellular nutrients and energy during starvation or high bioenergetic demands. Autophagy is also upregulated during oxidative stress, infection or protein accumulation (Kiffin et al., 2006; Dreux and Chisari, 2009; Dreux et al., 2009; Liu et al., 2010). Autophagy is important in innate and adaptive immune response against a variety of viral and bacterial pathogens (Deretic, 2005; Deretic and Levine, 2009). The interaction of single-stranded RNA with toll-like receptor 7 was found as the most potent effectors in autophagy induction (Deretic, 2011). In adaptive immunity, autophagy increases the major histocompatibility complex II antigen presentation with enhanced T cell proliferation (Schmid et al., 2007). In mice, knockdown of the autophagy related gene 5 resulted in the failure of efficient proliferation of CD4+ and CD8+ T cells after T cell receptor stimulation (Pua et al., 2007). Autophagy also controls the proliferation and survival of T and B cells (Miller et al., 2008). Despite the effective role of autophagy in immune response, many viruses utilize the autophagosomes for their efficient replication. The flaviviridae use autophagosome in two different ways. A few members of flaviviridae family such as, Hepatitis C virus (Ait-Goughoulte et al., 2008; Sir et al., 2008; Dreux and Chisari, 2009), dengue virus (Panyasrivanit et al., 2009; Heaton et al., 2010; McLean et al., 2011) and Japanese encephalitis virus (Li et al., 2012) induce autophagy for their replication while West Nile virus induces autophagy for cell survival (Beatman et al., 2012). Similarly, classical swine fever virus (CSFV) triggered autophagy and used autophagy machinery for its replication and release (Pie et al., 2014). The autophagy inducing drug, rapamycin facilitated cellular
proliferation after CSFV infection while autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) leaded to cell apoptosis (Pie et al., 2016). Bovine viral diarrhea virus (BVDV) is a member of the flavivirus family and the pestivirus virus group. BVDV causes great economic loss in cattle industry through its immunosuppression and persistent infection. A unique characteristic of BVDV is the ability of the virus to have two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp). Over 90- 95% of all field isolates are ncp and are responsible for the myriad of clinical outcomes including persistent infection (PI). PI is the result of ncp BVDV fetal infection during their first trimester. Such calves remain immunotolerant to BVDV and continuously act as a source of infection to other animals (Chase et al., 2015). The cp strains are the result of well-characterized genotypic changes that takes place in a fatal form of BVDV, mucosal disease, which occurs in PI animal where the ncp virus mutates to cp (Darweesh et al., 2014). The presence of this “pair”, ncp strain and the cp mutant strain, results in mucosal disease. Interestingly, the most studied and common vaccine strains are the cp mutants. Previous studies using a cp type 1a BVDV vaccine strain, NADL, showed that BVDV induced autophagy. The autophagy inducing drug, rapamycin, increased autophagy as well as BVDV titer while the autophagy inhibiting drug, 3-methyladenine or wortmannin, reduced autophagy and BVDV titer (Fu et al., 2014a; Fu et al., 2014b; Fu et al., 2014c; Fu et al., 2015). A study using cp type 1a NADL and ncp1b NY1 demonstrated that cpBVDV induced autophagy-like vacuolization in cp BVDV-infected cells but not in ncp BVDV-infected cells Birk et al., 2008. However, a recent study using cytopathic HJ1 (BVDV1b) and non-cytopathic New York1 (NY1) (BVDV1b) strains showed that both cp and ncp BVDV induce autopay with similar rate (Zhou et al., 2017). However, these viruses were not a pair. A further consideration is the difference in cp or ncp BVDV autophagy induction in an established cell as well as primary cells. This study examined a homologous virus pair [i.e., cytopathic
(cp) BVDV1b Tifton GeorgiA Cytopathic (TGAC) and noncytopathic (ncp) BVDV1b Tifton GeorgiA Noncytopathic (TGAN), that were isolated from a clinical BVDV mucosal disease to determine the effect of BVDV phenotypes on autophagy induction in both primary bovine turbinate cells and the MDBK cell line.
Materials and Methods Virus and cells The homologous pair of ncp and cp type 1b viruses (TGAN and TGAC) recovered from an animal that died of mucosal disease was used in the study (Ridpath et al., 1991). These viruses were provided by Dr. Julia Ridpath. The BVDV free Madin Darby bovine kidney cells (MDBK cells) (passage 95-110) and bovine turbinate cells (Bt cells) (passage 10-15) were used in the study. The cells were grown in complete minimal essential medium (CMEM). The CMEM contained minimal essential medium (MEM) (Gibco BRL, Grand Island, NY, USA), supplemented with 10% BVDV free fetal bovine serum (FBS) (PPA, Pasching, Austria), penicillin (100 U /ml) and streptomycin (100 μg /ml) (Sigma, St. Louis, MO, USA).
LC3- GFP stabilized cell line The autophagy induction was visualized through stably expressing MDBK- GFP-LC3 cells or Bt- GFP-LC3 cells. These cells were created through GFP-LC3 -pseudolentivirus transduction. Upon autophagy induction, these cells showed dots like structures of GFP-LC3II, which localized at autophagic vacuole membrane (Ait-Goughoulte et al., 2008). To create GFP-LC3 pseudolentivirus, the GFP-LC3 gene was cut between Ndel and BamH1 restriction sites from pEGFP-LC3-11546 plasmid (Addgene, Cambridge, MA, USA) and inserted in to lentivirus vector pLenti CMV GFP Puro-17448 (Addgene, Cambridge, MA, USA). Briefly, both plasmid (e.g. pEGFP-LC3-11546 or pLenti CMV GFP Puro-1154) were digested through Ndel and BamH1 restriction enzymes (Sigma, St. Louis, MO, USA) for 2 hours. After digestion, products were
separated using 1% agarose gel (Sigma, St. Louis, MO, USA). The 1538 base pair (bp) fragment of GFPLC3 from pEGFP-LC3-11546 plasmid and, 8197 bp fragment of pLenti CMV GFP Puro-1154 plasmid were isolated through QIAquick Gel Extraction Kit (QIAGEN, Germantown, MD, USA). Both fragments were ligated together using T4 ligase (Sigma, St. Louis, MO, USA) to get pLenti CMV GFP Puro-LC3 plasmid. The pLenti CMV GFP Puro-LC3 plasmid was transformed and amplified in DH5α strain of E coli (Life Technologies, Grand Island, NY, USA). After amplification, pLenti CMV GFP Puro-LC3 plasmid was confirmed for LC3 insertion through restriction digestion using Ndel and BamH1, which yielded 1538 bp fragment of GFP-LC3 and 8197 bp fragment of pLenti CMV GFP Puro vector. To make GFP-LC3pseudolentivirus, the 293T cells were co-transfected with (1) pLenti CMV GFP Puro-LC3 containing GFPLC3 gene, (2) packaging plasmid pCMVR8.74-22036 (Addgene, Cambridge, MA, USA) and, (3) a plasmid containing vesicular stomatitis virus G glycoprotein pVSV.G-14888 (Addgene, Cambridge, MA, USA) using X-treme GENE HP DNA Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA). After 48 h post co-transfection, the supernatant from 293T cells, containing GFP-LC3-pseudolentivirus was collected. The GFP-LC3-pseudolentivirus was used to transduce MDBK or Bt cells. Briefly, MDBK or Bt cells were seeded in 6 well plates with concentration of 5x105 cell/well. After overnight attachment, the cell supernatant was replaced with 0.5 ml GFP-LC3-pseudolentivirus containing 5µg/ml polybrene (kindly provided by Adam Hoppe, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD, USA) and incubated at 37 ºC for 1 h adsorption. After 1 h, non-adsorbed virus was removed and cells were supplemented with 3 ml CMEM. The LC3-GFP transduction in MDBK- GFP-LC3 cells or Bt-GFP-LC3 cells were confirmed after 4 days post transduction through fluorescent microscope (Olympus, PA, USA) at 395 nm wavelength. LC3-GFP transduced cells were further selected using puromycin (10µg/ml) for 4 days. The positively expressing LC3-GFP selected cells (>95% LC3-GFP transduced cells) were used to determine the BVDV induced autophagy in MDBK or Bt cells. To further confirm our results,
another set of MDBK- GFP-LC3 cells or Bt- GFP-LC3 were created using commercially available recombinant lentivirus or LentiBrite control virus (EMD Millipore Corporation, Billerica, MA, USA). Both set of cells (using lab made lentivirus and commercially purchased lentivirus with >90% transduced) gave similar results, which were averaged and analyzed for significant difference using student One-way ANOVA with post-hoc Tukey HSD test at 95% level of significance using SAS version 8.0.
Drugs and virus production The drug rapamycin (Thermo scientific, Wilmington, DE, USA) an autophagy inducer with a concentration of 50 nM in CMEM while 3-methyladenine (3 MA) (Thermo scientific, Wilmington, DE, USA) with concentration of 10 mM in CMEM was used as autophagy inhibitor. The dose of rapamycin or 3MA were chosen following their dose titration on BVDV production, autophagy induction in GFP-LC3 transduced MDBK cells and viability (Suppl. Fig. 2 and Suppl. Fig. 3). Cell viability were examined through trypan blue exclusion assay following trypsinization. Out of 10 nM/ml, 50 nM/ml and 100 nM/ml, rapamycin with 50 nM/ml and, out of 0.5µm/ml, 5.0µm/ml and 10 Mm, 3-MA with 10 Mm/ml were chosen for highest effect on autophagy induction and 100% cell viability. The MDBK cells or Bt cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with 6 Multiplicity of infection (MOI). The 6 MOI was chosen to ensure that all cells become infected with BVDV. While mock-infected cells were used as negative controls. To measure the virus production, cell supernatant was collected at 1 h, 6 h, 12 h, 24 h and 48 h p.i. and analyzed for BVDV titer as per the method described earlier (Reed and Munch, 1938). Each experiment was done in three replicates and reproduced three times. The data was analyzed using one-way ANOVA with post-hoc Tukey HSD Test at 95% level of significance using SAS version 8.0.
Autophagy measurement through GFP-LC3 transduced cells To determine the effect of BVDV biotypes on autophagy induction, the stable expressing MDBK-GFP-LC3 cells or Bt-GFP-LC3 cells were cultured in 4 well chamber slide (Vector Laboratories, Burlingame, CA, USA). The cells were infected with either cp BVDVb1 TGAC or ncp BVDVb1TGAN with MOI of 6. The mock-infected cells were used as control. After 1 hour of incubation non-adsorbed virus was removed. The cells were supplemented with medium containing rapamycin (50 nM) or 3 MA (10 mM) or none. The cells were fixed with 4% paraformaldehyde at 12 h, 24 h and 48 h p.i. and mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA). The cells showing green fluorescence (LC3 dot formation/green punctated structure) at each time point was counted using a fluorescent microscope (Olympus, PA, USA). One hundred (100) cells on each slide using four replicates were counted at each time point. All experiments were reproduced three times. The percentage of autophagosome positive cells were calculated and one-way ANOVA with post-hoc Tukey HSD Test at 95% level of significance sing SAS version 8.0.
Autophagy measurement in MDBK cells The 5.0 x105 MDBK cells were seeded in each well of 6 well plates. After overnight attachment, cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with 6 MOI with or without autophagy inducing drug, rapamycin (50 nM) or inhibiting drug, 3MA (10 mM), while mock infected non-drug treated cells were used as control. After 48 hours, cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer) (Sigma, St. Louis, MO, USA) containing a proteinase inhibitor (ULTRA tablet) (Roch Diagnostic, GmbH, Sandhofen, Germany) and examined for LCI and LCII protein using Western blot analysis. Briefly, 40µg cell lysate were run in each well of SDS (sodium dodecyl sulfate) gel, after running, protein were transfected to nitrocellulose membrane (Whatman GmbH, Germany). Membranes
were blocked with 5% gelatin in PBS for 1 h at room temperature followed by incubation with anti-LC3 antibody (1:500) (Abcam, Cambridge, MA) overnight at 4ºC. Membranes were washed as incubated with goat anti-rabbit HRP conjugated (1:2000) and developed using Pierce ECL Western Blotting Substrate (Thermo scientific, Wilmington, DE, USA). Band Intensities in image were measured through ImageJ software.
Co-localization of autophagosome and BVDV proteins To determine the topological relationship between autophagosomes and BVDV replication site, the colocalization of BVDV proteins and autophagy marker was carried out. The MDBK-GFP-LC3 cells were cultured in 4 well chamber slides. The MDBK-GFP-LC3 were infected with ncp TGAN with MOI of 6. The mock-infected cells were used as control. The cells were fixed with 4% paraformaldehyde at 48 h p.i. The fixed cells were permeabilized using 0.3% triton in phosphate buffered saline (PBS) containing 3% bovine serum albumin. The permeabilized MDBK-GFP-LC3 cells were stained with rabbit polyclonal anti-NS5A, Erns or E1 antibodies (produced in our laboratory using a recombinant BVDV NS5A, Erns or E1 proteins as an immunogen) with concentration of 1:1000 in PBS followed by staining with anti-rabbit antibody conjugated with rhodamine (Sigma, St. Louis, MO, USA) with concentration of 1:1000 in PBS. The stained cells were mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA) and examined for co-localization of BVDV proteins (red) and autophagosomes (green punctated structure: GFP-LC3) using florescent microscope (Olympus, PA, USA).
Results Both biotypes of BVDV induced autophagy
The BVDV infected MDBK-GFP-LC3 cells or Bt-GFP-LC3 cells showed that both cp BVDV1b TGAC (Fig. 1C) and ncp BVDV1b TGAN (Fig. 1B) induced autophagy in MDBK as well as Bt cells (figure not shown) as indicated by the punctate vesicles. However, there was no significant change in cellular morphology in cells infected with either cp or ncp BVDV within the first 48 h of infection. There was no significant difference in autophagy induction between both biotypes in either cell types. However, TGAN significantly enhanced autophagy in MDBK as well as Bt cells at 12 h, 24 h and 48 h post infection as compared to its time point control (p <0.05). (Fig. 2B and Fig. 3B). Similarly, TGAC significantly enhanced autophagy in MDBK at 12 h, 24 h and 48 h post infection (Fig. 2A) while 24 h and 48 hr post infection in Bt cell as compared to its time point control (p <0.05) (Fig. 3A). Autophagy inhibiting drug, 3MA, significantly reduced autophagy induction in MDBK cells as compared to cells infected with TGAC, TGAN, TGAC supplemented with rapamycin or TGAN supplemented with rapamycin at 12h, 24 h or 48 h post infection (p <0.05) (Fig. 2A and Fig. 2B). Similarly in Bt cells, 3MA significantly reduced autophagy induction as compared to cells infected with TGAC supplemented with rapamycin or TGAN supplemented with rapamycin at 12h, 24 h or 48 h post infection (p <0.05) (Fig.3A and Fig. 3B). Autophagy induction was significantly higher in Bt cells supplemented with rapamycin as compared to TGAC or TGAN infected cells alone at 12h, 24 h or 48 h post infection (p <0.05) (Fig.3A and Fig. 3B). While comparing different cell types, MDBK cells showed significantly higher autophagy as compared to Bt cells, either infected with ncp TGAN (Fig. 2B and, 3B), cp TGAC (Fig. 2A, and 2B) or in the mock infection (p <0.05). The higher autophagy activity in MDBK cells may be due to the faster growth and doubling time of MDBK cells compared to Bt cells. The autophagy also increased over the course of infection in both cell types. The MDBK cells infected with ncp TGAN resulted in autophagy at levels of 28.00±3.26%, 72.66±5.03%, 92.66±1.15% 12 h, 24 h and 48 h p.i. (Fig. 2B) while cp TGAC infection resulted in autophagy at levels of 26.66±2.30%, 74.66±4.04%, and 89.33±1.15% at 12 h, 24 h and 48 h p.i.
(Fig. 2A), while non-BVDV infected MDBK cells, treated with rapamycin or 3MA showed the autophagy as 60.0±8.80 or 23.16±1.16 at 48 hr p.i. respectively. Similarly, Bt cells infected with ncp TGAN or cp TGAC resulted in autophagy as 13.33±0.57%, 23.25±2.87%, 32.0±2.64% and 15.0±3.0%, 23.25±2.06%, 25.66±1.52% at 12 h, 24 h and 48 h p.i. respectively (Fig. 3B and 3A), while non-BVDV infected Bt cells, treated with rapamycin or 3MA showed the autophagy as 25.4±2.4 or 22.4±1.67 at 48 hr p.i. respectively. The phosphoinositide 3- kinase (PI-3 kinase) inhibiting drug, 3-methyladenine (3MA) that suppresses autophagy, significantly reduced autophagy 10-60% in TGAN or TGAC BVDV infected MDBK cells (p <0.05) (Fig. 2B and 2A respectively). Autophagy was reduced 4-12% by 3MA in ncp or cp BVDV infected Bt cells (p˂0.05) (Fig. 3B and 3A). The autophagy inducing drug, rapamycin, resulted in no to a slight increase (0-10%) in autophagy that was not significant in ncp TGAN or cp TGAC infected MDBK cells (p <0.05) (Fig. 2B and Fig. 2A) respectively. In contrast, autophagy was significantly increased (10-18%) in TGAN or TGAC infected Bt cells (p <0.05) (Fig. 3B and Fig. 3A) respectively. To avoid any interference of overexpression on autophagy induction, native MDBK cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN with or without autophagy inducing drug, rapamycin or autophagy inhibiting drug, 3MA, while mock infected non-drug treated cells were used as control. After 48 hours, cells were lysed and examined for LCI and LCII protein using Western blot analysis. Autophagy induction in MDBK cells were analyzed through LC3II/LC3I ratio using ImageJ software. Rapamycin treatment enhanced LC3II expression. MDBK cells infected with TGAC or TGAN and treated with rapamycin had a LC3II/LC3I ratio of 0.72±0.12 and 0.81±0.06 respectively as compared with TGAC (0.65±0.12) or TGAN (0.65±0.02) alone (Suppl. Fig. 1). Autophagy in MDBK cells infected with TGAC or TGAN and treated with 3MA had a LC3II/LC3I ratio of 0.52±0.12 and 0.54±0.26 respectively. Mock infected control cells autophagy was 0.51±0.00 (Suppl. Fig. 1).
The effect of autophagy on BVDV replication To determine the relationship between autophagy and BVDV replication, the cp TGAC or ncp TGAN infected MDBK cells or Bt cells were treated with either the autophagy inducing drug, rapamycin or autophagy inhibiting drug, 3MA and analyzed for BVDV production. The controls were cells infected with BVDV (TGAN or TGAC) with no drug or mock infection with no drug treatment. There was a correlation between autophagy and BVDV production. Rapamycin-treatment of BVDV-infected MDBK cells significantly increased cp TGAC as well as ncp TGAN virus production at 12 h p.i. (Fig. 4A and, 4B) (p <0.05). Rapamycin increased the ncp TGAN titer at 12 h p.i. from 0 log10/ml in the untreated infected cells to 1.00±0.00 log10/ml rapamycin-treated cells (Fig. 4B). In MDBK cells infected with cp TGAC, the titer also increased from 0.50±0.57 log10/ml in the untreated infected cells to 1.50 ±0.57 log10/ml (Fig. 4A) (p <0.05). Overtime, the rapamycin-treated MDBK cells had ncp TGAN titers that were consistently 0.5 log10 higher (3.50±0.70 log10/ml at 24 h and 5.00±0.00 log10/ml at 48 h). This was not significantly different at (p <0.05)] as compared to untreated ncp TGAN infected MDBK cells (3.00±0.00 log10/ml at 24 h and 4.50±0.57 log10/ml at 48 h) (Fig. 4B). The rapamycin treatment of cp TGAC-infected MDBK cells did not increase the virus production at 24 h p.i. as compared to untreated infected cells (3.00±0.00 log10/ml in both groups). In contrast at 48 h, rapamycin treatment significantly (p <0.05) increased the titer one log as compared to the untreated infected cells (5.00±0.00 log10/ml in untreated vs. 6.00±0.00 log10/ml in rapamycin-treated cells) (Fig. 4A). Like MDBK cells, there appeared to be an increase in viral titers in rapamycin-treated Bt cells over time. The rapamycin treatment significantly increased (p <0.05) both the ncp TGAN and cp TGAC titers at 24 h by 1 log10 (1.00±0.00 log10/ml in untreated infected vs. 2.00±0.00 log10/ml in rapamycin-treated Bt cells infected with TGAN; 2.00±0.00 log10/ml in untreated infected Bt cells vs. 3.00±0.00 log10/ml in rapamycin-treated Bt cells infected with TGAC (Fig. 5B and 5A, respectively). The increased viral titer was also observed at 48 h post infection for both virus biotypes. The
rapamycin-treated Bt cells infected with ncp TGAN had a titer of 3.00±0.00 log10/ml as compared to 2.50±0.57 log10/ml from untreated infected Bt cells (p <0.05) (Fig. 5B). Rapamycin treated Bt cells infected with cp TGAC had viral titers of 4.66±0.57 log10/ml vs. 3.50±0.57 log10/ml in the untreated infected Bt cells (Fig. 5A). The autophagy inhibiting drug, 3MA, significantly suppressed cp TGAC as well as ncp TGAN production (p <0.05) at 12 h, 24 h and 48 h p.i.in both MDBK and Bt cells (Fig. 4A, 4B, 5A and, 5B). There was no BVDV production in 3MA-treated MDBK cell or Bt cells until 24 h p.i. BVDVinfected MDBK cells produced virus titers of 1.00±0.00 log10/ml for both cp TGAC and ncp TGAN at 48 h p.i. (Fig. 4A and, 4B). Similarly, 3MA treated BVDV-infected Bt cells produced ncp TGAN titers of 1.00±0.00 log10/ml and cp TGAC titers of 0.50±0.57 log10/ml at 48 h p.i. (Fig. 5A and, 5B). Taken together, the increased production with autophagosome agonist and decreased production with an autophagosome inhibitor supported the importance of autophagosomes for the production of BVDV regardless of biotype.
Co-localization of autophagy with BVDV proteins To determine whether BVDV replicated in association with autophagosomes, a co-localization study of autophagy marker, LC3 and BVDV proteins NS5A, Erns and E1 was carried out. The MDBK-GFP-LC3 cells were infected with ncp BVDV1b TGAN at a 6 MOI. Confirmation that all cells were infected with virus, was done using immunohistochemistry (IHC) with anti-BVDV 16C6 antibody (IDEXX Laboratories, Westbrook, ME, USA) (Rajput, 2013). The ncp BVDV1b TGAN or mock-infected cells were fixed with 4% paraformaldehyde at 48 h p.i. and stained for BVDV NS5A, Erns or E1 proteins using rabbit polyclonal anti-NS5A, Erns or E1 antibodies followed by anti-rabbit antibody conjugated with rhodamine. Stained cells were mounted and examined for co-localization of BVDV proteins (red) and autophagosomes (green punctate structure). The ncp TGAN infected MDBK-GFP-LC3 cells expressed the BVDV proteins, Erns
(Fig. 6B), E1 (Fig. 6E) and NS5A (Fig. 6H) with red color while mock-infected cells did not express any viral proteins such as Erns (Fig. 6K), E1 or NS5A (Fig. not shown). The ncp BVDV1b TGAN infected MDBK-GFP-LC3 induced the formation of autophagosomes as distinct punctate vesicles (Fig. 6A, 6D and 6G) as compared to mock-infected cells (Fig. 6J). The co-localization of autophagosomes and structural Erns (Fig. 6C), E1 (Fig. 6F)) and non-structural proteins NS5A (Fig. 6I) indicating that BVDV replicates in close association with autophagosomes regardless of biotype or cell type. However, a future confocal microscopy study is needed to determine the specific location of virus replication in relation to autophagosomes.
Discussion The current study was carried out to determine effect of cp and ncp strain of BVDV on autophagy induction in immortal as well as primary cell line. Immortal MDBK-GFP-LC3 cells or primary Bt-GFP-LC3 cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN. Such infected cells were supplemented either with autophagy inducing drug, rapamycin or autophagy inhibiting drug 3MA or no drug. The study revealed that both ncp and cp biotypes of BVDV induced autophagy in the primary Bt cells as well as the MDBK cell line, which was significantly higher than mock-infected control cells (p <0.05). There was no significant difference between cp or ncp BVDV infection and autophagy induction (p <0.05). The autophagy inducing drug, rapamycin increased autophagy as well as virus production, while the autophagy inhibiting drug, 3MA, significantly suppressed autophagy and BVDV replication in both cell types. The MDBK cell line had significantly higher autophagosomes as compared to Bt cells, whether infected with ncp TGAN, cp TGAC or mock infected (p <0.05). The higher autophagy activity in MDBK cells may be due to its faster growth characteristics and higher metabolic activity than Bt cells.
To rule out the effect of LC3 overexpression on autophagy induction, native MDBK cells were infected with either cp BVDV1b TGAC or ncp BVDV1b TGAN, supplanted with autophagy inducing drug, rapamycin or autophagy inhibiting drug 3MA or no drug. Cells were lysed and examined for LC3II expression through western blot. Results showed that both cp BVDV1b TGAC and ncp BVDV1b TGAN induced autophagy in MDBK cells with similar rate. The co-localization study using BVDV nonstructural protein NS5A or structural proteins Erns and E1 with the autophagosome marker, LC3, indicated that BVDV replicated in close association with autophagosomes. However a detail study is needed using confocal microscopy to determine the specific location of virus replication compared to autophagosome localization. These results are similar to those observed in electron microscopy studies with cp BVDV, which found the BVDV induced autophagy and BVDV particles were present in autophagosomes (Fu et. al., 2014a; Fu et. al., 2014b; Fu et. al., 2014c; Fu et al., 2015). However, all of these studies used the cp type 1a vaccine strain NADL in MDBK cells. One of the enigmas of BVDV is the distinct in vitro phenotype differences in cell culture between ncp and cp BVDV. The cellular-viral protein interactions responsible for the different phenotypes have not been fully defined. The cp BVDV strains are isolated in nature, predominately from mucosal disease, where the cp strain has mutated from its homologous ncp strain (Brownlie et al., 1984; Ridpath et al., 1991). Cp BVDV is unable to establish chronic infection or cause persistent infection in the host. Previous autophagy-BVDV studies used the NADL strain (Fu et. al., 2014a; Fu et. al., 2014b; Fu et. al., 2014c; Fu et al., 2015). Although the NADL is a vaccine strain and has been an effective immunogen, NADL BVDV infections are self-limiting unlike their ncp counterparts. Therefore, cp BVDV “illustrates a case of viral emergence to extinction – irrelevant for BVDV evolution, but fatal for the persistently infection host” (Peterhans et al., 2010). On the other hand, the ncp BVDV biotypes are economically important viruses, affecting the cattle industry worldwide and the major cause of immune suppression and
persistent infection. The infection of the bovine fetus with ncp BVDV during the first trimester results in persistently infected (PI) calves that remain immunotolerant to BVDV and continuously act as a source of infection to other animals (Chase et al., 2015). The ncp BVDV inhibits double-stranded RNA-induced apoptosis and interferon synthesis in tissue culture while cp strains induce interferon and cause apoptosis (Schweizer and Peterhans, 2001). There are two studies that compared the effect of cp and ncp BVDV on autophagy induction (Birk et al., 2008; Zhou et al., 2017). However, the findings of these two studies contradict each other. Birk et al. (2008) used cp type 1a NADL and ncp1b NY1 and showed that cpBVDV infection induced vacuolization in infected cells. Those vacuoles have all structural characteristics of autophagy and their formation was inhibited with 3MA treatment. Vacuolization increased in cp BVDV infected MDBK cells in a time dependent manner; however, ncp BVDV infected cells had no vacuolization (Birk et al., 2006; Birk et al., 2008). In contrast, Zhou et al. (2017) using cp BVDV1b HJ1or ncp BVDV1b NY1, showed that that there is no significant difference in autophagy induction between cp and ncp BVDV. However, Birk et al. (2008) used BVDV strains from two subgenotypes (BVDV1a and BVDV1b) while Zhou et al. (2017) used two strains from same subgenotypes (BVDV1b) but not paired viruses. To precisely characterize the effect of cp and ncp BVDV on autophagy induction, a true virus pair arising from the same animal from a naturally occurring infection was used. The cp TGAC and ncp TGAN viruses used in this study were isolated at the National Animal Disease Center, Ames, IA from a mucosal disease (MD) case. The MD occurred spontaneously in the unvaccinated animal and was not induced experimentally. Following isolation, the virus pairs antigenic properties and genomic sequence were determined. The TGAC and TGAC differed in their binding of three of 10 BVDV MAbs specific for gp53. TGAC and TGAN reacted similarly to BVDV conserved cDNA probes while they were differentiated by oligomer probes (3289-3309) and (11081-11101) (Ridpath et al., 1991). Later, the authors successfully produced MD in animals using the virus pair, TGAC/TGAN. (Ridpath et al., 1991). Further sequencing of
TGAN and TGAC was reported in 1992 found that cp and ncp pairs were not identical. The reason for variation was concluded as there was no cellular RNA insertion or duplication in p125 region of ncp virus while its counterpart, cp virus had a p80 gene duplication as well as a cellular ubiquitin RNA insertion (Qi et. al., 1992). Additionally a sequence comparison done between TGAN and TGAC in an approximately 270 base pair region of the 5' UTR of each virus and showed a 97% homology (J. Ridpath, personal communication). Therefore, it can be concluded that the TGAN is the parent with the larger TGAC containing the entire TGAN RNA backbone nucleotide along with a duplication of TGAN NS3 gene (p80) and a cellular ubiquitin RNA insertion (Qi et. al., 1992; Quadros et al., 2006). Cleavage of the NS23 region (Kümmerer et al., 1998), duplication of NS3 (p80) (Fritzemeier et al., 1995) or cellular ubiquitin RNA insertion (Qi et. al., 1992) have been shown to generate cp strain of BVDV. Using these closely related viruses, we did not find any significant difference in cp and ncp BVDV infection in autophagy induction or formation. Flaviviruses such as West Nile virus (WNV) (Tonry et al., 2005; Appler et al., 2010) or BVDV (Brock et al., 1998) often resulted in persistent infection. WNV infects CNS to escape immune system while BVDV persistent infected animals fail to distinguish BVDV as foreign. Both viruses exploit persistently infected cells for their survival and maintenance. There is no evidence indicating that flaviviruses establish persistent infection by autophagy (Codogno and Meijer, 2005; Ogata et al., 2006). A study with vesicular stomatitis virus revealed that the conjugated autophagy gene Atg5–Atg12 negatively regulated type I IFN production (Jounai et al., 2007). Similarly, the absence of autophagy gene Atg 5, resulted in ROS-dependent amplification of RLR (RIG-I-like receptors) signaling and increased type I IFNs (Tal et al., 2009). It is possible then that reduced type I IFN production may result in enhanced viral replication as seen in the current study with increased autophagy. Autophagy may also have a potential role in establishing the ncp BVDV persistent infection, which do not produce sufficient type 1 IFN (Charleston et al., 2001). Several
hypotheses have been proposed suggesting that autophagy regulates the cell assembly or metabolism and facilitates virus replication. The double membrane autophagosomes provide a scaffold for anchoring and concentrating the replication complexes for virus, as well as preventing the immune response triggered by dsRNA intermediates (Wileman, 2006; Miller and Locker, 2008). Autophagy regulated the cellular metabolism through autophagy-dependent processing of lipid droplets and triglycerides to release free fatty acids. This resulted in an increase intracellular beta-oxidation and ATP generation. The energy released by beta-oxidation may be utilized for efficient viral replication (Heaton and Randall, 2010). In the current study, using BVDV NS5A, Erns and E1 proteins with autophagosomes marker LC3 also revealed that BVDV replicate in close association with autophagosomes, which was similar to the previous studies, which found BVDV particles in or in close association with autophagosomes (Fu et al., 2014a; Fu et al., 2014b; Fu et al., 2014c). The scaffold for anchoring the replication complexes with energy provided by autophagosomes maybe the reason for higher BVDV titers seen following treatment with an autophagy agonist in these experiments. A similar association with autophagosomes was reported in HCV replication (Dreux et al., 2009). Other positive strain RNA viruses including, poliovirus (Jackson et al., 2005; Taylor and Kirkegaard, 2007), coxsackieviruses (Wong et al., 2008; Yoon et al., 2008) and flavivirus (AitGoughoulte et al., 2008; Sir et al., 2008; Dreux and Chisari, 2009) have used autophagy machinery to facilitate their replication. Another flavivirus, WNV induced autophagy and virus replication was independent of autophagy. While WNV induced autophagy was reduced by class III phosphatidylinositol 3kinase (PI3K) inhibiting drug such as 3MA or knockdown of Atg 5 gene, the effect of 3MA on virus replication may be due to association of other cellular mechanism involving PI3K pathways, independent of autophagy (Beatman et al., 2012). Another study showed that the expression of NS4B gene of HCV alone is able to induce autophagy via interactions with both the early endosome-associated GTPase Rab5 and a class
III phosphoinositide 3-kinase, Vps34 (Su et. al., 2011). In future studies, it would be interesting to know which BVDV viral protein(s) is/are responsible for autophagosome induction.
Conclusions This study indicated that both cp and ncp biotypes BVDV induced autophagy in infected primary as well as immortal cell lines. There was no significant difference between cp or ncp BVDV biotypes in autophagy induction. The autophagy inducing drug, rapamycin enhanced autophagy while autophagy inhibiting drug, 3MA suppressed autophagy as well as the virus production in both the biotypes. The colocalization of BVDV structural as well as non-structural proteins such as Erns, E1 and NS5As with the autophagosome marker, LC3 revealed that BVDV replicated in close association with autophagosomes. These findings further supported the importance of the presence of autophagosomes for the production of BVDV which need to be studied in detail for better understanding of BVDV pathophysiology. Conflict of interest None of the author in manuscript has any conflict of interest related to publication of this manuscript.
Acknowledgments The authors thank the Department of Veterinary and Biomedical Sciences/Animal Disease Research and Diagnostic Laboratory, South Dakota State University (SDSU), Brookings, SD, USA; SDSU Experimental Station and the Center for Biological Control and Analysis by Applied Photonics, Department of Chemistry and Biochemistry, SDSU, Brookings SD, USA, for providing funding to conduct the research. Authors are also thankful to Dr. Julia F Ridpath and Dr. John Neill (National Animal Disease Center, Ames, IA) for proving BVDV strains and sequence comparison between TGAN and TGAC.
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Figure Captions Figure 1. Autophagy induction in MDBK cells after ncp BVDV1BtGAN or cp BVDV1BtGAC infection. The stable expressing MDBK-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (B) or cp BVDV1BtGAC (C) with multiplicity of infection (MOI) 6, while mock infected cells were used as negative control (A). The cells were fixed with 4% paraformaldehyde (PFA) at 48 h post infection. Autophagy was determined through green punctated structure having GFP-LC3 on the membrane of autophagosomes through florescent microscope (White square: Zoomed in at upper panel).
Figure 2. The percentage of autophagy induction in MDBK cells following BVDV infection. The MDBK-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (A) or cp BVDV1BtGAC (B) with MOI of 6. The mock infected cells were used as control. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The percentage of autophagy induction was calculated at 12 h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 3. The percentage of autophagy induction in Bt cells following BVDV infection. The Bt-GFP-LC3 cells were infected with either ncp BVDV1BtGAN (A) or cp BVDV1BtGAC (B) with MOI of 6. The mock infected cells were used as control. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The percentage of autophagy induction was calculated at 12 h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 4. Effect of autophagy on BVDV production in MDBK cells. The MDBK cells were infected with either cp BVDV1BtGAC (A) or ncp BVDV1BtGAN (B) with MOI of 6. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The virus titer in cell supernatants were evaluated at 1 h, 6 h, 12h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 6. Effect of autophagy on BVDV production in Bt cells. The Bt cells were infected with either cp BVDV1BtGAC (A) or ncp BVDV1BtGAN (B) with MOI of 6. The infected cells were supplemented with either autophagy inducing drug, rapamycin (50 nM) or autophagy inhibiting drug, 3-MA (10 mM) or none. The virus titer in cell supernatants were evaluated at 1 h, 6 h, 12h, 24 h and 48 h p.i. The significant difference between different treatments are shown by asterisk sign {*}, p<0.05).
Figure 6. The co-localization of BVDV Erns, E1 or NS5A protein with autophagosomes. The MDBK-GFP-LC3 cells were infected with ncp BVDV1BtGAN with MOI of 6 for 48 h and fixed with 4% paraformaldehyde. The fixed cells were permeabilized using 0. 3% triton in PBS containing 3% BSA and stained with rabbit polyclonal anti- Erns, E1 or NS5A antibodies followed by anti-rabbit antibody conjugated with rhodamine. Autophagosome induction was observed by green punctated structure in MDBK cells following ncp BVDV1BtGAN infection (Fig.6A, 6D, 6G respectively). BVDV Erns, E1 and NS5A proteins were detectable in infected cells (Fig. 6B, 6E, 6H respectively) but not in mock cells stained with BVDV Erns (Fig.6K) as well as mock infected cells stained with E1 or NS5A (figure not shown). Co-localization of LC3 with BVDV Erns, E1 and NS5A proteins (Fig. 6C, 6F, 6I, 6L).
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