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Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus Xiaodong Wu,*,†,‡ Xian Qi,§ Mifang Liang,储 Chuan Li,储 Carol J. Cardona,¶ Dexin Li,储 and Zheng Xing*,†,‡,¶,1 *Medical School, †State Key Laboratory of Pharmaceutical Biotechnology, and ‡Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, China; §Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China; 储China Center for Disease Prevention and Control, Beijing, China; and ¶Veterinary and Biomedical Sciences, University of Minnesota–Twin Cities, St. Paul, Minnesota, USA Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins Gn and Gc, nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFN␤-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs

ABSTRACT

Abbreviations: dsRNA, double-stranded RNA; EGFP, enhanced green fluorescence protein; FITC, fluorescein isothiocyanate; GCN, Golgi complex network; HA, hemagglutinin; HEK, human embryionic kidney; hpi, hours postinfection; IB, inclusion body; IFN-␤, interferon ␤; IFA, indirect fluorescence assay; LD, lipid droplet; MOI, multiplicity of infection; mRNP, messenger ribonucleoprotein complex; NP, nucleoprotein; NSm, nonstructural M segment; NSs, nonstructural S segment; RER, rough endoplasmic reticulum; RNC, RNA-NSs complex; RT, reverse transcription; RVFV, Rift Valley fever virus, SFTS, severe fever with thrombocytopenia syndrome; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SFTSV, severe fever with thrombocytopenia syndrome virus; siRNA, small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate; VLS, viroplasm-like structure; VRC, vanadyl-ribonucleoside complex 2504

in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses.—Wu, X., Qi, X., Liang, M., Li, C., Cardona, C. J., Li, D., Xing, Z. Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus. FASEB J. 28, 2504 –2516 (2014). www.fasebj.org Key Words: nucleoprotein 䡠 viral replication 䡠 bunyavirus Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness in humans caused by a novel phlebovirus in the family Bunyaviridae (1). It has been found mainly in Chinese villagers who have experienced tick bites and has spread quickly in the past several years, becoming endemic in a dozen provinces in China (1, 2). Patients experience high fever and drastic losses of leukocytes and platelets, subsequent gastrointestinal symptoms, and hemorrhaging, leading to disseminated intravascular coagulation and multiorgan failure, with a high fatality rate in severe cases. The viral RNA of the SFTS virus (SFTSV) can be found in patients’ plasma or sera and appears to be quantitatively correlated to proinflammatory cytokine levels in the serum and to disease severity (3, 4). Significantly, 2 patients with similar symptoms have been diagnosed since 2010 in Heartland, Missouri, USA. A phlebovirus termed Heartland virus (5), which is closely related to SFTSV, has been isolated from these patients, demonstrating that these novel bunyaviruses, which form a new group in the genus Phlebovirus, have global public health significance. 1 Correspondence: 300D Veterinary Science Bldg., University of Minnesota at Twin Cities, 1971 Commonwealth Ave., St. Paul, MN 55108, USA. E-mail: [email protected] doi: 10.1096/fj.13-243857

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SFTSV is a single-stranded negative-sense RNA virus with 3 genomic segments, L, M, and S (1). The L segment encodes viral RNA polymerase, and the M segment encodes the two viral envelope glycoproteins Gn and Gc. The S segment is an ambisense RNA of 1744 bases in length encoding a nucleoprotein (NP) and an nonstructural S segment (NSs) protein with open reading frames in sense and complementary RNA, respectively. While the NP of all known members of the Bunyaviridae is associated with viral genomic RNA and is critical to viral replication, NSs proteins of variable sizes in the genera Bunyavirus, Phlebovirus, and Tospovirus of family Bunyaviridae (6, 7) appear to have roles in modulating host responses using various mechanisms. In the genus Phlebovirus, NSs protein of the Rift Valley fever virus (RVFV) is distributed both in the cytoplasm and nucleus and forms fibrillar structures in the nuclei of infected cells (8), but NSs protein of the Uukuniemi virus is distributed throughout the cytoplasm of infected cells (9). Studies have shown that NSs of RVFV targets cellular TFIIH transcription factor (10), blocks interferon production (11–13), and contributes to viral pathogenesis (11, 14, 15). The NSs protein of SFTSV suppresses interferon ␤ (IFN-␤) and nuclear factor ␬B (NF-␬B) promoter activities in in vitro reporter assays when NSs is overexpressed (16). In this article, we show that NSs protein of SFTSV formed viroplasm-like structures (VLSs) in infected cells. NSs protein of SFTSV is distributed mainly in the cytoplasm and differs from RVFV NSs protein, which has a nuclear and cytoplasmic distribution and forms a fibrillar structure in the nucleus, suggesting that NSs protein of SFTSV may function differently. NSs protein interacts with itself and also interacts with NP. Interestingly, NP could also be colocalized in VLSs, probably through an NSs-NP interaction. Our data further demonstrate that NSs-formed VLSs colocalized with lipid droplets (LDs), and inhibitors of fatty acid biosynthesis affect VLS formation and viral replication. We also found that NSs protein was associated with viral RNA, and viral double-stranded RNA (dsRNA) was colocalized in VLSs. We hypothesize that NSs protein may be involved in viral RNA replication by acting as a scaffolding protein, forming a platform, which may be critical in host-virus interaction and viral replication.

MATERIALS AND METHODS Cells, viruses, and reagents African green monkey kidney Vero cells, HeLa cells, human embryonic kidney (HEK) 293T cells, and human liver hepatocellular carcinoma HepG2 cells, all from American Type Culture Collection (ATCC; Manassas, VA, USA), were grown in Dulbecco’s modified Eagle’s medium (Gibco; Invitrogen, Carlsbad, CA, USA) supplemented with 8% fetal bovine serum (HyClone; Thermo Scientific, Logan, UT, USA), 1 mM sodium pyruvate (HyClone), and 1% antibiotic-antimycotic solution (Gibco). Cells were cultured at 37°C with 5% CO2. A previously described SFTSV strain, JS-2010-014 (16), was used SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

in this study and propagated in cell culture as described previously (16). All viral aliquots were stored at ⫺80°C. Antibodies for fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse, and rhodamine [tetramethylrhodamine isothiocyanate (TRITC)]-conjugated goat anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Other secondary antibodies used for immunoblots, confocal microscopy, and immunoprecipitation were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Abcam (Cambridge, MA, USA). Anti-Flag M2 antibody, vanadyl-ribonucleoside complex (VRC), Nile Red, and DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-␤-actin antibody and anti-hemagglutinin (HA) antibody were purchased from Santa Cruz Biotechnology. Anti-dsRNA antibody (J2-1201) was purchased from English & Scientific Consulting (Szirak, Hungary). Rabbit anti-NSs and anti-NP antibodies were obtained by immunizing rabbits with purified recombinant NSs proetein and NP of SFTSV. Protein G Plus/Protein A-Agarose beads were purchased from Millipore (Billerica, MA, USA). RiboLock RNase inhibitor and proteinase K were obtained from Thermo Scientific (Pittsburgh, PA, USA). Small interfering RNA (siRNA) of 22 bases specific to human IFN-␤ or scramble control were chemically synthesized at Shanghai GenePharma Co. (Shanghai, China), and the sequences were as follows: IFN-␤, GCAAUUUUCAGUGUCAGAATT (forward) and UUCUGACACUGAAAAUUGCTT (reverse); scramble, GAAUAUGCUUGACGAUACUTT (forward) and AGUAUCGUCAAGCAUAUUCTT (reverse). PrimeScript (R036A; Takara, Shiga, Japan) was used as reverse transcriptase for reverse transcription (RT). Trizol reagent and Lipofactamine 2000 reagents were purchased from Invitrogen (Carlsbad, CA, USA). Construction of plasmids Plasmids pRK5-NSs and pRK5-NP were described previously (16). Briefly, NSs and NP cDNA were synthesized by PCR from the RT product with indicated primers and subcloned into pRK5 with either a flag or HA tag for mammalian cell expression under a cytomegalovirus (CMV) promoter. The 5=-primer (EcoRI) and 3=-primer (SalI) were used for HAtagged pRK5 plasmid construction, while 5=-primer (BglII) and 3=-primer (SalI) were used for Flag-tagged pRK5 plasmid construction. The primer sequences were as follows: 5= primer (EcoRI), 5=-CGGAATTCCATGTCACTGAGCAAATGCT; 5=-primer (BglII), 5=-GAAGATCTCATGTCACTGAGCAAATGCT; and 3=primer (SalI), 5=-GCGTCGACGACAAAATTAGACCTCCTTC. NSs and NP cDNA were also subcloned into phosphorylated enhanced green fluorescence protein (pEGFP)-N3 and pDsRed-N1 for expression of EGFP-NSs and DsRed-NP fusion proteins, respectively. NSs truncated mutants were prepared by PCR cloning using synthetic primers, which resulted in cDNA fragments of NSs1-66, NSs66-160, NSs66-205, and NSs206-293, respectively, and cloned into tagged pRK5. HeLa cells or HEK293T cells were transfected with plasmids with NSs, NSs truncated mutants, or NP cDNA with Lipofactamine 2000 reagents for expression of respective proteins. Immunoprecipitation and immunoblot analysis Cell lysates of transfected or cotransfected cells were incubated with specific or control antibodies at 4°C for 2 h and then precipitated with protein A/G beads. After 2 h incubation, the beads were washed 4 times with lysis buffer (25 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 5% glycerol; 1 mM DTT; 1 mM PMSF; 2 mM NaF; 1 mM Na3VO4; and 1 ␮g of aprotinin/ml). The immunoprecipitates were subjected to sodium dodecyl sulfate–polyacrylamide gel 2505

electrophoresis (SDS-PAGE) and then transferred onto an Immunoblot PVDF (Millipore) membrane for primary antibody incubation overnight. Alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies were used for further incubation with the membranes for 90 min, and signals on blots were developed by BCIP/ NBT or ECL reagents (Invitrogen). To prepare soluble and insoluble fractions of cell lysates (17), HEK293T cells were cultured in 6-well plates for 16 h, followed by transfection with 2 ␮g of pRK5-F-NSs or/and pRK5-F-NP using Lipofectamine 2000 reagents for the expression of NSs protein and NP. At 24 to 32 h post-transfection, the cells were rinsed with PBS and harvested by gentle scraping into 500 ␮l of ice-cold 1⫻ Nonidet P-40 lysis buffer containing 1 mM DTT, 1 mM PMSF, 2 mM NaF, 1 mM Na3VO4, and 1 ␮g of aprotinin/ml. Cell lysates were incubated at ⫺80°C for 15 min, followed by centrifugation (19,000 g for 10 min) at 4°C for separation into 2 fractions: Nonidet P-40-soluble (supernatant) and Nonidet P-40-insoluble (pellet). The pellet was resuspended in a buffer containing 1% SDS and 10 mM Tris-EDTA (pH 7.5). Proteins from both fractions were quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) before loading for SDS-PAGE analysis. Association of NSs protein and viral RNA by immunoprecipitation Immunoprecipitation procedures were performed to detect association of NSs protein and viral RNA in infected cells as described previously (16). We used a protocol designed for performing messenger ribonucleoprotein (mRNP) complex immunoprecipitation to prepare viral RNA-NSs complex (RNC) with modifications (18). Briefly, Vero cells infected with SFTSV for 72 h were lysed with polysome lysis buffer (100 mM KCl; 5 mM MgCl2; 10 mM HEPES, pH 7.0; 0.5% Nonidet P-40; 1 mM DTT; 100 U/ml RNase Out; 400 ␮M VRC; 1 mM PMSF; 2 mM NaF; 1 mM Na3VO4; and 1 ␮g of aprotinin/ml) and NT2 buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM MgCl2; and 0.05% Nonidet P-40). The lysates were then incubated with anti-NSs antibody-coated protein A/G beads in a solution containing NT2 buffer, RNase inhibitor, VRC, DTT, and EDTA at 4°C overnight; washed 4 –5 times with ice-cold NT2 buffer; resuspended with NT2 buffer supplemented with proteinase K; and incubated for 30 min at 55°C. Then, the RNC components were released, and RNA was isolated from the immunoprecipitated pellet with Trizol reagent (Invitrogen). The RNA was reverse transcribed with RT for cDNA synthesis and PCR.

Total RNA (200 ng) precipitated from the lysates of SFTSVinfected Vero cells, described above, with either anti-NSs antibodies or control rabbit serum, or extracted directly from SFTSV- or mock-infected Vero cells, were used for RT with a Primescript reagent kit (Takara). Quantitative real-time PCR was performed with 1 ␮l of cDNA in a total volume of 10 ␮l with SYBR Premix Ex TaqII (Takara) following the manufacturer’s instructions. Relative expression values were standardized by an internal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. Fold change of the S segment was calculated following the formula 2(⌬Ct gene ⫺ ⌬Ct GAPDH). Immunofluorescence assay and virus titration HeLa cells transfected with plasmids encoding NSs protein, NSs mutants, and NP or Vero cells infected with SFTSV Vol. 28

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Inhibition of fatty acid synthesis with chemical compounds Triacsin C (Sigma-Adrich), which inhibits long-chain fatty acid acyl-CoA synthesis, C75, a novel synthetic inhibitor of fatty acid synthesis that prevents the condensation of acetyl-CoA and malonyl-CoA (Cayman Chemical, Ann Arbor, MI, USA; ref. 20), and TOFA, an potent inhibitor of acetyl-CoA carboxylase (Sigma-Aldrich; ref. 20), were used in cell cultures to inhibit lipid metabolism. Cytotoxic effects of the compounds were determined using the MTT assay (21), and the compounds were used at 15 and 1.5 ␮M, respectively for the treatment. Vero cells were incubated with the above inhibitors for 1–2 h before infection, then infected with SFTSV at a multiplicity of infection (MOI) of ⬃1. Culture medium was changed at 1 and 12 h postinfection (hpi), and inhibitors were added in the fresh medium. Statistical analysis For statistical analysis, a 2-tailed Student’s t test was used to evaluate the data by SPSS software (IBM SPSS, Armonk, NY, USA). A ␹2 analysis was used to determine significant differences of the data in ⱖ2 groups, and a value of P ⬍ 0.05 was considered statistically significant.

RESULTS Viroplasm-like structures formed by NSs protein in SFTSV-infected Vero and HepG2 cells

Quantitative real-time PCR

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(JS-2010-014) were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.1% Triton X-100 for 10 min, followed by 3 washes with PBS. The coverslips were blocked with 5% bovine serum albumin (Sigma) in PBS for 30 min at 37°C and then incubated with anti-NSs antibody at 1:50 to 1:100 dilution at 4°C overnight. After 3 washes with PBST, the cells were incubated with a 1:200 dilution of FITC-conjugated donkey anti-mouse IgG (H⫹L) or TRITC-conjugated anti-rabbit IgG (H⫹L) at 37°C for 1 h and then washed and stained for 10 min with DAPI (1 ␮g/ml). After washes, the coverslips were subjected to microscopy under an Olympus confocal microscope (Olympus, Tokyo, Japan). Cell culture medium was collected at various time points from SFTSV-infected Vero cells and titrated for infectious virus titration (TCID50) with indirect immunofluorescence as described previously (16). Infectious virus titers (TCID50/ml) were calculated based on the Reed and Muench method (19).

Vero cells were infected with SFTS bunyavirus strain JS-2010-014 (16). Infected cells were fixed at various time points postinfection and stained with anti-NSs immune serum in an indirect fluorescence assay (IFA), after which a unique structure was identified. Granulates, which turned into patches, were detected mainly in the cytoplasm, with numbers and size increasing over time. These patches, or inclusion bodies (IBs), appear similar to viroplasm or viral factories described in rotavirus or other virus infections (refs 22–25 and Fig. 1A). These VLS or IBs were not restricted to infected Vero cells. In infected liver hepatoceullar carcinoma HepG2 cells, similar granulates turning to patches at later stages of infection were observed, as well (Fig. 1B). The

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A

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Figure 1. VLSs or IBs were formed in SFTSVinfected cells. A) VLSs in infected Vero cells. Cells, uninfected, or infected with SFTSV (JS36h 2010-014) at an MOI of 1, were fixed at 12, 24, and 36 hpi and stained with rabbit anti-NSs antibodies, followed by TRITC-conjugated antirabbit IgG antibody staining. B) VLSs in HepG2 cells infected with the SFTSV. Noninfected or infected cells were fixed and subsequently stained with the anti-NSs antibodies at 12 and 24 hpi After staining, the cells were subjected to confocal fluorescence microscopy (⫻400).

VLSs or IBs were not exhibited when the infected cells were stained with antibodies specific for other viral proteins, including the glycoproteins Gc and Gn (data not shown). The formation of VLSs was infection independent. When cDNA of NSs was cloned into an expression plasmid with a Flag tag and the plasmid was used to transfect HeLa cells, the fusion protein appeared posttransfection in granulates, first as tiny dots but later becoming larger patches by fusion, distributed in the cytoplasm, identical to the observed VLSs or IBs in infected cells (Fig. 2B). To confirm that NSs protein causes the formation of VLS, we transfected cells with tagged cDNA of NSs, NP, Gc, and Gn proteins, respectively. When the cells were stained with FITC-conjugated tag antibodies, only NSs protein was found to form VLS. NP was dispersed in the cytoplasm (Fig. 2A), and viral glycoproteins were distributed both in the cytoplasm and on the surface of the transfected cells (data not shown), indicating that NSs protein can singularly form VLSs in the cell. We also noted that more delicate ring-like structures could be observed on VLSs at later times with higher magnification (Fig. 2C). In SFTSV-infected Vero cells, VLS could also be observed as IBs with electron microscopy (X. J. Yu and V. Popov, Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; personal communication) and were morphologically similar to those viewed by confocal microscope. SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

NSs protein interacted with itself To understand how NSs protein forms VLSs, cDNA of NSs, tagged with either Flag or HA, was used to cotransfect HEK293T cells. The cell lysates were immunoprecipitated with either anti-Flag or anti-HA antibodies and the immunoprecipitates were electrophoresed on SDS-PAGE for Western blot analyses, using either anti-HA or antiFlag antibodies, respectively. As shown in Fig. 3A, Flag-NSs and HA-NSs were associated and bound to each other, indicating that NSs interacted with itself. We generated truncated mutants of NSs protein, all tagged with Flag (Fig. 3B), for expression in HeLa cells to examine which portion of the NSs protein drives VLS formation. It appeared that the middle portion of NSs, especially the region NSs66 –205, was essential for the formation of VLS, while both 5= and 3= termini were dispensable, based on IFA results (Fig. 3C). Interaction of NSs protein with NP and localization of NP in VLSs We examined NSs protein interactions with other viral proteins. When HEK293T cells were cotransfected with HA-tagged NSs protein and Flag-tagged NP, most NP was detected in the soluble fraction of the lysates prepared 16 h post-transfection, while most NSs protein was found in the insoluble fraction (Fig. 4A) possibly due to VLS formation. Interestingly, a small fraction of NP was also detected in the insoluble fraction. 2507

Figure 2. VLSs were formed by NSs protein specifically in the cells. Transfected cells were fixed at 6, 20, and 32 h after transfection before incubation with primary antibodies. A) HeLa cells were transfected with cDNA expressing HA-tagged NP and stained with FITC-conjugated anti-HA antibodies. B) HeLa cells were transfected with cDNA expressing Flag-tagged NSs protein and stained with FITC-conjugated anti-Flag antibodies. C) Ring-like structures of VLSs (arrowheads) were shown in NSs-transfected HeLa cells observed with higher magnification (⫻1000).

To understand why a portion of NP was in the insoluble fraction, we coimmunoprecipitated the cell lysates of the cells cotransfected with pRK5-HA-NSs and

pRK5-F-NP with either anti-HA or anti-Flag antibodies, and the immunoprecipitates were subjected to Western blot analyses. We found that HA-NSs and Flag-NP were

Figure 3. NSs protein interacted with itself and was localized in VLSs. A) NSs protein interacted with itself. HEK293 cells were transfected with Flag-NSs and HA-NSs. Flag-NSs was immunoprecipitated with anti-Flag antibodies, and the immunoprecipitates were analyzed by Western blot analysis with anti-HA antibodies (top) or in a reciprocal manner (middle); input of HA-NSs is at bottom. B) Generation of truncated NSs protein. PCR amplification with specific primers was used to generate deletion mutants with Flag tagged at the N terminus. C) HeLa cells were transfected with various truncated mutants of NSs tagged with Flag. Transfected cells were fixed at 24 h post-transfection and stained with FITC-conjugated anti Flag antibodies, which were subsequently subjected to fluorescence microscopy. 2508

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Figure 4. Interaction of SFTSV NSs protein and NP. A) Expression of NSs protein and NP in transfected HEK293 cells. Soluble and insoluble fractions of the cell lysates were prepared for Western blot analysis to examine the distribution of NSs protein and NP. B) Association of NSs protein and NP in cotransfected cells. Cell lysates were coimmunoprecipitated with anti-Flag or anti-HA antibodies, and the immunoprecipitates were subjected to Western blot analyses with anti-HA or anti-Flag antibodies respectively (top 2 panels). Input HA-NSs and Flag-NP in the lysates were shown with anti-HA or anti-Flag antibodies, respectively (bottom 2 panels). C) Detection of NSs protein and NP in the insoluble fraction of lysates. HEK293 cells were transfected with NSs protein or NP alone or cotransfected with NSs and NP cDNA. Insoluble fractions of the lysates were subjected to SDS-PAGE and blotted with anti-flag antibodies on membranes. D) No coimmunoprecipitation between NSs protein and the NP of an influenza virus subtype H9N2. The cells were cotransfected with flag-tagged NSs protein and NP of SFTSV or influenza viral NP. The lysates were immunoprecipitated with anti-NSs antibodies and the membrane was incubated with anti-Flag antibodies (top panel). The inputs of SFTSV NSs protein and NP and influenza NP were shown (bottom panel). E) Colocalization of NSs protein and NP in VLSs. HeLa cells were cotransfected with cDNA of Flag-NSs and HA-NP; the cells were fixed and costained with TRITC-conjugated anti HA and FITCconjugated anti-Flag antibodies, followed by fluorescence microscopy (top panels), or the cells were cotransfected with cDNA expressing EGFP-NSs and DsRed-NP and subjected live to confocal fluorescence microscopy (bottom panels).

associated in the cotransfected cells (Fig. 4B). This may explain why a fraction of NP was found in the insoluble fraction of the cell lysates, even though NP is presumably a soluble protein. There was more NP detected in the insoluble fraction of cells cotransfected with cDNA from NP and NSs protein compared with what was present in cells transfected with NP alone (Fig. 4C). NSs protein is thought to have brought a portion of NP, through an NSs-NP interaction, into the VLS, which is insoluble in the lysates. The specific interaction of NSs protein and NP was SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

confirmed by cotransfection of the cells with NSs protein and the NP of an influenza virus (subtype H9N2); ref. 26). The influenza viral NP did not coimmunoprecipitate with NSs protein (Fig. 4D). We then examined the localization of NSs protein and NP in cotransfected HeLa cells by confocal microscopy and found that in cotransfected cells 20 h posttransfection, NP diffusely dispersed in the cytoplasm mainly disappeared, and a portion of the Flag-NSs and HA-NP was colocalized in the VLS (Fig. 4E, top panels). We cotransfected HeLa cells with plasmids expressing 2509

both EGFP-NSs and DsRed-NP fusion proteins and found that large proportions of the NSs protein and NP were colocalized in VLS 32 h post-transfection in live cells, as well (Fig. 4E, bottom panels). NSs protein associated with viral RNA and promoted viral replication Since NP may be critical to viral RNA replication, we speculated that viral RNA may also be located in VLS. First, we examined whether NSs protein was associated with viral RNA. Vero cells were mock infected or infected with SFTSV. As shown in Fig. 5A, cell lysates were prepared at 24 hpi, using a protocol for preparing mRNP, and immunoprecipitated with anti-NSs antibody. NSs protein was observed in lane 1 from the infected cells but not in lane 2 from the mock infection, as shown in Fig. 5A. NSs protein could not be precipitated by preimmune serum (rabbit IgG) from infected cell lysates (lane 3). The resultant immunoprecipitates were subjected to RT, and cDNA was used for PCR amplification with specific primers for the S segment of SFTSV. The S segment with expected size was visualized on an agarose gel (Fig. 5A, lane 1), while none was detected in the mock infection (lane 2). A very faint S-segment product was detected in the immunoprecipitates with the preimmune serum (lane 3), apparently showing a basal level of nonspecific viral RNA binding on beads. The significant difference in quantities between lanes 1 and 3 as quantified in Fig. 5B suggests

that a portion of viral RNA was bound to NSs protein in infected cells. As controls, inputs of NSs protein (Western blot) and S segment (RT-PCR) were detected, respectively, from infected cells (lane 4) but were negative in the mock infection (lane 5). NSs protein was implicated in viral replication Because viroplasms are formed by some viruses, such as rotavirus, and function as factories for viral replication, we further explored whether VLSs formed by SFTSV NSs protein are also implicated in viral replication. At 24 h before SFTSV infection, HeLa cells were transfected with pRK5-HA-NSs, pRK5-HA-NSs66-205, or pRK5-HA, which expressed full-length NSs protein, truncated mutant NSs66-205, or only HA tags, respectively. The cells were infected with SFTSV at an MOI of 1, and the culture medium was collected at 12, 24, and 36 hpi for infectious virus titration. As shown in Fig. 5C, viral replication was increased in the cells with overexpression of NSs66 –205, as well as NSs in comparison to cells without ectopically expressed NSs. NSs-formed VLSs colocalized with LDs To further understand the nature of NSs-formed VLSs, we transfected HeLa cells with EGFP-NSs, and 12 h later stained the cells with Nile Red, a lipophilic stain (27). LDs were scattered and evenly distributed in the cytoplasm without EGFP-NSs (Fig. 6A). In the presence of

Figure 5. NSs protein was associated with viral RNA and viral replication. A) Association of NSs protein and viral RNA. Cell lysates were prepared from infected (MOI 1; lanes 1, 3, and 4) or control (lanes 2 and 5) Vero cells, and immunoprecipitated with anti-NSs antibodies (lanes 1 and 2) or control serum (rabbit IgG; line 3, top panel), and subjected to Western blot analysis with anti-NSs antibodies. Input NSs in the lysates of the infected cells is shown (lane 4). RNC, immunoprecipitated with anti-NSs antibodies or rabbit IgG, was reverse transcribed, and cDNA was amplified by PCR with the primers specific to the S segment (lanes 1, 2, and 3; bottom panel). The input S segments, which were extracted in total RNA from the infected and uninfected cells and reverse transcribed, were amplified with PCR (lanes 4 and 5, bottom panel). B) Quantitative analysis of the copy numbers of viral S segments, measured with q-RT-PCR from A. C) NSs protein promoted viral replication. HeLa cells were transfected with plasmids expressing full-length or truncated NSs protein (NSs66 –205). After 24 h, the cells were infected with SFTSV. Cultural medium were taken at various time points for infectious virus titration on Vero cells. Controls include the cells nontransfected or transfected with a blank plasmid. 2510

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Figure 6. NSs-formed VLSs colocalized with intracellular lipid droplets. A) Control HeLa cells transfected with empty plasmids. B–D) HeLa cells were transfected with plasmids expressing EGFP-NSs (B, C) or Flag-NSs (D, E) for 8 (B, D) and 16 h (C, E), fixed, and stained with Nile Red and DAPI for observation under confocal fluorescence microscopy (⫻400).

EGFP-NSs, however, NSs protein formed VLSs, and most VLSs colocalized with LDs (Fig. 6B). We noted that, through time, more VLSs became colocalized with LDs, although some large patches of VLSs remained negative by Nile Red staining (Fig. 6C). Uniquely, these large lipid-negative patches possessed the ring-like structures, that are characteristic of LDs morphologically. The colocalization of VLS with Nile Red-stained LDs was repeated in cells transfected with Flag-tagged NSs and was observed, as well, during early and later times (Fig. 6D, E). SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

VLSs were associated with lipid metabolism While we expressed EGFP-NSs in HeLa cells, we also pretreated the cells with triacsin C, which inhibits long fatty acid acyl-CoA synthesis (28). We found that in the presence of triacsin C, the number and size of VLSs, shown as EGFP-NSs, were both decreased compared with those without the treatment of triacsin C (Fig. 7A). Similar effects occurred in SFTSV-infected cells that were pretreated with triacsin C before SFTSV infection. The infected cells were fixed at 16 hpi for immunoflu2511

Figure 7. VLSs were affected by the inhibition of lipid metabolism. A) HeLa cells were transfected with pEGFP-NSs, followed by the addition of 10 ␮M triacsin C into the culture medium (bottom) or without treatment (top). At 12 h post-transfection, the cells were fixed for observation of VLS. B) Vero cells were infected with SFTSV at an MOI of 1 in the culture medium containing 10 ␮M triacsin C. The cells were infected with SFTSV in absence (top) and presence (bottom) of triacsin C. Cells were fixed 12 hpi and stained with antiNSs antibodies, followed by incubation with TRITC-conjugated secondary antibodies.

orescence staining with anti-NSs antibodies. The number and size of NSs-formed VLS decreased significantly in treated cells compared with those in nontreated cells (Fig. 7B). These data suggest that the formation of VLS by NSs is dependent on lipid metabolism, and the presence of LDs may be critical to viral replication. It seems that the size and number of VLSs between transfected and infected cells appear to be different, suggesting that VLS formation was more sensitive to triascin C treatment in SFTSV-infected cells than transfected cells (Fig. 7A, B). We consider, however, that the difference is probably due to variable expression levels and accumulating dynamics of NSs between transfected and infected cells. We quantified fold changes of viral M and S gene copy numbers in SFTSV-infected Vero cells treated with triacsin C. No obvious differences in M and S genes were observed by real-time RT-PCR at early times of infection between treated and nontreated cells (Fig. 8A, B). However, we could detect significantly reduced copy numbers of both M and S gene numbers in triacsin C-treated cells at 20 hpi. Furthermore, we treated the cells with 2 additional compounds, C75 and TOFA, a novel inhibitor of fatty acid synthesis and a potent inhibitor of acetyl-CoA carboxylase, respectively. Treat2512

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ment with TOFA alone significantly reduced the S-gene expression, and the combined treatment of TOFA and C75 resulted in the greatest reduction of the S-gene copy numbers in SFTSV-infected cells (Fig. 8C). Collectively, these data demonstrate that SFTSV RNA replication can be lessened through inhibition of lipid metabolism. dsRNA was localized in VLSs To further demonstrate the point that VLSs may be the platform involved in viral replication, we tried to identify a replicative form of viral RNA localized in VLSs. In either untreated or NSs-transfected cells, NSs was either absent or formed VLSs. dsRNA was not detected in either case by anti-dsRNA antibody staining (Fig. 9A, B). dsRNA was not detectable in noninfected cells. We next infected HeLa cells with SFTSV and stained the cells with anti-NSs and anti-dsRNA antibodies at different times postinfection. We observed in an overlay dsRNA and NSs-positive punctuate granular and larger amorphous structures at 24 and 48 hpi (Fig. 9). At 48 hpi, when large VLSs emerged, dsRNA was mostly present in these structures, indicating that an intermediate and replicative form of viral RNA is present in VLSs of the

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Figure 8. Reduced viral M and S gene copy numbers in cells treated with inhibitors of lipid metabolism. A, B) Vero cells were pretreated with or without tracsin C before SFTSV infection at an MOI of 1. Total RNA were extracted from the cells at 5, 10, and 20 hpi followed by RT and real-time PCR with specific primers to viral M (A) and S (B) segments. C) Vero cells were pretreated with C75, TOFA, or a combination of C75 and TOFA before SFTSV infection. Total RNA were prepared for realtime RT-PCR. The assay was performed in duplicates and repeated at least twice. *P ⬍ 0.05.

infected cells and supporting the conclusion that VLSs may serve as a platform for viral RNA replication.

DISCUSSION SFTSV appears to be the first tick-borne Phlebovirus found to be pathogenic to humans, while RVFV, an important zoonotic pathogen, is mainly transmitted by a range of mosquito species. There is no significant sequence homology between phleboviruses and the viruses of other genera, and genomic sequence homology is also low even among phleboviruses. Between SFTSV and RVFV, only ⬃15% amino acid identities are found in NSs (1), suggesting that functions or functional mechanisms could be quite different for the NSs proteins of these two phleboviruses. NSs of RVFV is both a nuclear and cytoplasmic protein and forms unique filamentous structures in the nucleus of infected and NSs-transfected cells (8). RVFV NSs protein inhibits cellular RNA polymerase activity through interaction with TFIIH transcription factor, induces degradation of dsRNA-dependent protein kinase (PKR), or functions through other mechanisms, resulting in reduced induction of IFN-stimulated genes (ISGs) or antiviral responses (10, 11, 29 –32). However, SFTSV NSs is a cytoplamic protein, localized in the cytoplasm. In this article we show that NSs of SFTSV is unique among bunyaviruses in forming VLS or IBs in either infected or uninfected NSs-transfected cells. SFTSV NSs protein interacted biochemically with itself, which may be the basis for intracellular VLS formation. The association of NSs protein with viral NP and RNA suggests that NSs may be involved in viral RNA replication. Since NP and the replicative intermediate of viral RNA, in the form of dsRNA, were found in VLS, we hypothesize that NSs may act as a scaffold protein supporting SFTSV RNA replication. Viroplasm or viral replication factories are unique structures generated by viral proteins together with some cellular proteins as a platform where viral proteins, genomes, and host factors are concentrated for SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

efficient viral replication (33). Viral factories have been reported in cells infected with a variety of animal viruses, including nuclear inclusions in large DNA viruses (e.g., herpes viruses and poxviruses) and small DNA viruses (e.g., adenoviruses). Viral factories for RNA viruses have also been reported in several virus families, including togaviruses, reoviruses, flaviviruses, coronaviruses, and bunyaviruses (24, 33). Virus-induced vesicles and double-membrane vesicles are generated in alphaviruses, Dengue flavivirus, and SARS coronavirus, which is followed by the assembly of viral replicases leading to membrane curvature and invagination forming a spherule for viral assembly and morphorgenesis (23). Viroplasm is a structure that has been proposed as a site for viral assembly and genesis in the replication of bunyamwera virus, a member of Orthobunyavirus in Bunayviridae. Studies on bunyamwera virus indicate the presence of a tubular structure harboring the viral replication complexes associated with the Golgi complex. This structure is comprised of Golgi stacks, mitochondria, and rough endoplasmic reticulum (RER) elements and serves as the scaffold for viral replication and morphogenesis (34). The tubular structure, colocalized with viral dsRNA, is composed of nonstructural M segment (NSm) protein (34), a nonstructural protein encoded by the M segment of bunyamwera virus. In contrast to bunyamwera virus, however, no such tubular structures have ever been identified in SFTSV-infected cells under electron microscopic examination (V. Popov, Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA; personal communication). Moreover, NSm has not been identified in the SFTSV genome, suggesting that the tubular viroplasm identified in the replication of bunyamwera virus differs structurally and morphologically from VLSs formed by NSs protein of SFTSV, as reported in this study. Interestingly, SFTSV NSs-formed VLS is similar to the viroplasm in cells infected with rotavirus or other reoviruses (22). Rotavirus factories are composed of electron-dense viroplasm containing nonstructural pro2513

Figure 9. Viral dsRNA colocalized with NSs-formed VLSs in SFTSV-infected cells. Noninfected or transfected HeLa cells (A) or NSs-overexpressing HeLa cells (B) were stained with anti-NSs and anti-dsRNA antibodies as controls. HeLa cells were infected with SFTSV at an MOI of 1 for 24 or 48 h (middle and bottom panels). The cells were fixed and stained with anti-NSs and anti-dsRNA antibodies. Following primary antibody staining, the cells were incubated with FITC-conjugated or TRITCconjugated secondary antibodies. The stained cells were subjected to confocal fluorescence microscopy (⫻400).

teins NSP2 and NSP5, near endothelial reticulumderived membranes (22, 35). In the viroplasm, NSP5 orchestrates recruitment of additional proteins, including viral structural proteins and polymerases (36). Functionally, virus RNA replication and virus assembly all occur in the viroplasm. Viral protein synthesis and virion assembly of SFTSV may occur in the Golgi 2514

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complex network (GCN) or vesicles. We hypothesize that SFTSV viral RNA replicates in the VLS, where viral nucleocapsids (RNPs) are assembled. RNPs were subsequently transported through an unknown mechanism to the GCN, where virions are assembled. The notion that NSs may act as a scaffold protein forming VLSs, a platform morphologically similar to

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rotavirus viroplasm for supporting viral replication, is unique among bunyaviruses. With the use of minigenome systems, NSs protein of RVFV (37– 40), Bunyamwera (41), and La Crosse (42) viruses have been shown to be either inhibitory or irrelevant for generating recombinant viruses. The function of NSs protein in promoting SFTSV replication as suggested in this study contrasts with that of other viral NSs proteins in the family Bunyaviridae. Drastic sequence differences in NSs between SFTSV and other bunyaviruses, and their distinct subcellular locations, may account for their functional discrepancies. It is interesting to find that structurally, NSs-formed VLSs are associated with LDs, and SFTSV viral replication can be affected by lipid metabolism. On the one hand, VLSs colocalized with intracellular lipid droplets stained with Nile Red, a fluorescent lipophilic stain (27); on the other hand, the number and size of VLSs decreased in infected, as well as transfected, cells pretreated with triacsin C, a compound that interferes with lipid metabolism by inhibiting long fatty acyl CoA synthetase and blocking the synthesis of diglycerides, triglycerides, and cholesterol esters (28). Viral RNA copy numbers of M and S genes were decreased significantly in SFTSV-infected cells treated with tracsin C or a combination of C75 and TOFA (Fig. 8). Morphologically, SFTSV NSs-formed VLSs have ring-like structures, shown in NSs-transfected (Fig. 2C) and SFTSVinfected cells (Fig. 1A) under higher magnification, identical to structurally well-defined LDs. It has been determined that lipid metabolism is critical in the formation of viroplasm in infections with rotavirus and other pathogens. In rotavirus-infected cells, it has been shown that not only triacsin C, but also a combined treatment of isoproterenol and 3-isobutyl1-methylxanthine (IBMX), which leads to the dispersal and lysis of LDs, decreases viral RNA replication and the infectivity of viral progeny (43). In addition, TOFA, which inhibits an early stage of fatty acid biosynthesis, can also inhibit RV replication (44). A detailed analysis of the lipidome of RV-infected vs. uninfected cells has demonstrated an increase of many lipid components in virus-infected cells and confirmed the association of RV viroplasms with LDs (45). LDs are also shown to be of significant importance for the replication of hepatitis C virus (46 – 48), dengue virus (49), and chlamydia (50). Our data suggest that VLSs formed in SFTSV-infected cells are affected by lipid metabolism, as well; of more importance, the NSs-formed VLS, critical in viral evasion of host innate immunity (51), may be similar to the viral factories of other viruses in promoting viral RNA replication. Our study therefore suggests a novel mechanism for phlebovirus replication, in which a nonstructural protein is essential. These findings will help enhance our understanding of bunyavirus replication, which may be implicated in the pathogenicity of SFTSV in human infections. This work was supported by the Minnesota Rapid Agricultural Response Fund (RARF) and a grant-in-aid (to SFTSV NSS FORMS VIROPLASM-LIKE STRUCTURE

Z.X.) from the Vice President Office of Research (VPOR), University of Minnesota at Twin Cities. It was also supported by the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University (grant KF-GN-201407 to Z.X.), and the Jiangsu Province Key Medical Talent Foundation (RC2011084), the Natural Science Foundation of Jiangsu Province (BK20131450), and the 333 Projects of Jiangsu Province (to X.Q.).

REFERENCES 1.

2.

3.

4.

5.

6.

7. 8.

9. 10.

11.

12.

13.

Yu, X. J., Liang, M. F., Zhang, S. Y., Liu, Y., Li, J. D., Sun, Y. L., Zhang, L., Zhang, Q. F., Popov, V. L., Li, C., Qu, J., Li, Q., Zhang, Y. P., Hai, R., Wu, W., Wang, Q., Zhan, F. X., Wang, X. J., Kan, B., Wang, S. W., Wan, K. L., Jing, H. Q., Lu, J. X., Yin, W. W., Zhou, H., Guan, X. H., Liu, J. F., Bi, Z. Q., Liu, G. H., Ren, J., Wang, H., Zhao, Z., Song, J. D., He, J. R., Wan, T., Zhang, J. S., Fu, X. P., Sun, L. N., Dong, X. P., Feng, Z. J., Yang, W. Z., Hong, T., Zhang, Y., Walker, D. H., Wang, Y., and Li, D. X. (2011) Fever with thrombocytopenia associated with a novel bunyavirus in China. N. Engl. J. Med. 364, 1523–1532 Zhang, Y. Z., Zhou, D. J., Qin, X. C., Tian, J. H., Xiong, Y., Wang, J. B., Chen, X. P., Gao, D. Y., He, Y. W., Jin, D., Sun, Q., Guo, W. P., Wang, W., Yu, B., Li, J., Dai, Y. A., Li, W., Peng, J. S., Zhang, G. B., Zhang, S., Chen, X. M., Wang, Y., Li, M. H., Lu, X., Ye, C., de Jong, M. D., and Xu, J. (2012) The ecology, genetic diversity, and phylogeny of huaiyangshan virus in China. J. Virol. 86, 2864 –2868 Sun, Y., Jin, C., Zhan, F., Wang, X., Liang, M., Zhang, Q., Ding, S., Guan, X., Huo, X., Li, C., Qu, J., Wang, Q., Zhang, S., Zhang, Y., Wang, S., Xu, A., Bi, Z., and Li, D. (2012) Host cytokine storm is associated with disease severity of severe fever with thrombocytopenia syndrome. J. Infect. Dis. 206, 1085–1094 Gai, Z. T., Zhang, Y., Liang, M. F., Jin, C., Zhang, S., Zhu, C. B., Li, C., Li, X. Y., Zhang, Q. F., Bian, P. F., Zhang, L. H., Wang, B., Zhou, N., Liu, J. X., Song, X. G., Xu, A., Bi, Z. Q., Chen, S. J., and Li, D. X. (2012) Clinical progress and risk factors for death in severe fever with thrombocytopenia syndrome patients. J. Infect. Dis. 206, 1095–1102 McMullan, L. K., Folk, S. M., Kelly, A. J., MacNeil, A., Goldsmith, C. S., Metcalfe, M. G., Batten, B. C., Albarino, C. G., Zaki, S. R., Rollin, P. E., Nicholson, W. L., and Nichol, S. T. (2012) A new phlebovirus associated with severe febrile illness in Missouri. N. Engl. J. Med. 367, 834 –841 Schmaljohn, C. S., and Nichol, S. T. (2007) Bunyaviridae. In Fields Virology, 5th Ed. (Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., and Martin, M. A., eds) pp. 1741–1789, Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia Bishop, D. H. (1986) Ambisense RNA genomes of arenaviruses and phleboviruses. Adv. Virus Res. 31, 1–51 Yadani, F. Z., Kohl, A., Prehaud, C., Billecocq, A., and Bouloy, M. (1999) The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein. J. Virol. 73, 5018 –5025 Simons, J. F., Persson, R., and Pettersson, R. F. (1992) Association of the nonstructural protein NSs of Uukuniemi virus with the 40S ribosomal subunit. J. Virol. 66, 4233–4241 Le May, N., Dubaele, S., Proietti De Santis, L., Billecocq, A., Bouloy, M., and Egly, J. M. (2004) TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell 116, 541–550 Billecocq, A., Spiegel, M., Vialat, P., Kohl, A., Weber, F., Bouloy, M., and Haller, O. (2004) NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription. J. Virol. 78, 9798 –9806 Streitenfeld, H., Boyd, A., Fazakerley, J. K., Bridgen, A., Elliott, R. M., and Weber, F. (2003) Activation of PKR by Bunyamwera virus is independent of the viral interferon antagonist NSs. J. Virol. 77, 5507–5511 Weber, F., Bridgen, A., Fazakerley, J. K., Streitenfeld, H., Kessler, N., Randall, R. E., and Elliott, R. M. (2002) Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon. J. Virol. 76, 7949 –7955

2515

14.

15.

16.

17.

18. 19. 20.

21.

22. 23. 24.

25. 26.

27. 28.

29.

30.

31.

32.

2516

Bridgen, A., Weber, F., Fazakerley, J. K., and Elliott, R. M. (2001) Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis. Proc. Natl. Acad. Sci. U.S.A. 98, 664 –669 Bouloy, M., Janzen, C., Vialat, P., Khun, H., Pavlovic, J., Huerre, M., and Haller, O. (2001) Genetic evidence for an interferonantagonistic function of rift valley fever virus nonstructural protein NSs. J. Virol. 75, 1371–1377 Qu, B., Qi, X., Wu, X., Liang, M., Li, C., Cardona, C. J., Xu, W., Tang, F., Li, Z., Wu, B., Powell, K., Wegner, M., Li, D., and Xing, Z. (2012) Suppression of the interferon and NF-kappaB responses by severe fever with thrombocytopenia syndrome virus. J. Virol. 86, 8388 –8401 Metcalfe, M. J., Huang, Q., and Figueiredo-Pereira, M. E. (2012) Coordination between proteasome impairment and caspase activation leading to TAU pathology: neuroprotection by cAMP. Cell Death Dis. 3, e326 Keene, J. D., and Tenenbaum, S. A. (2002) Eukaryotic mRNPs may represent posttranscriptional operons. Mol. Cell 9, 1161–1167 Reed, L. J., and Muchen, H. (1938) A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497 Pizer, E. S., Thupari, J., Han, W. F., Pinn, M. L., Chrest, F. J., Frehywot, G. L., Townsend, C. A., and Kuhajda, F. P. (2000) Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts. Cancer Res. 60, 213–218 Van de Loosdrecht, A. A., Beelen, R. H., Ossenkoppele, G. J., Broekhoven, M. G., and Langenhuijsen, M. M. (1994) A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J. Immunol. Methods 174, 311–320 Fabbretti, E., Afrikanova, I., Vascotto, F., and Burrone, O. R. (1999) Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo. J. Gen. Virol. 80, 333–339 Netherton, C. L., and Wileman, T. (2011) Virus factories, double membrane vesicles and viroplasm generated in animal cells. Curr. Opin. Virol. 1, 381–387 Novoa, R. R., Calderita, G., Arranz, R., Fontana, J., Granzow, H., and Risco, C. (2005) Virus factories: associations of cell organelles for viral replication and morphogenesis. Biol. Cell 97, 147–172 Eichwald, C., Rodriguez, J. F., and Burrone, O. R. (2004) Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation. J. Gen. Virol. 85, 625–634 Xing, Z., Harper, R., Anunciacion, J., Yang, Z., Gao, W., Qu, B., Guan, Y., and Cardona, C. J. (2011) Host immune and apoptotic responses to avian influenza virus H9N2 in human tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 44, 24 –33 Greenspan, P., Mayer, E. P., and Fowler, S. D. (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100, 965–973 Igal, R. A., Wang, P., and Coleman, R. A. (1997) Triacsin C blocks de novo synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid: evidence for functionally separate pools of acyl-CoA. Biochem. J. 324, 529 –534 Habjan, M., Pichlmair, A., Elliott, R. M., Overby, A. K., Glatter, T., Gstaiger, M., Superti-Furga, G., Unger, H., and Weber, F. (2009) NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase. J. Virol. 83, 4365–4375 Ikegami, T., Narayanan, K., Won, S., Kamitani, W., Peters, C. J., and Makino, S. (2009) Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and posttranscriptional downregulation of protein kinase PKR. Ann. N.Y. Acad. Sci. 1171(Suppl. 1), E75–E85 Kalveram, B., Lihoradova, O., Indran, S. V., Lokugamage, N., Head, J. A., and Ikegami, T. (2013) Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR. Virology 435, 415–424 Ikegami, T., Narayanan, K., Won, S., Kamitani, W., Peters, C. J., and Makino, S. (2009) Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation. PLoS Pathog. 5, e1000287

Vol. 28

June 2014

33.

34.

35. 36. 37.

38.

39.

40. 41. 42.

43.

44. 45.

46.

47.

48.

49.

50. 51.

Netherton, C., Moffat, K., Brooks, E., and Wileman, T. (2007) A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv. Virus Res. 70, 101–182 Fontana, J., Lopez-Montero, N., Elliott, R. M., Fernandez, J. J., and Risco, C. (2008) The unique architecture of Bunyamwera virus factories around the Golgi complex. Cell. Microbiol. 10, 2012–2028 Altenburg, B. C., Graham, D. Y., and Estes, M. K. (1980) Ultrastructural study of rotavirus replication in cultured cells. J. Gen. Virol. 46, 75–85 Contin, R., Arnoldi, F., Campagna, M., and Burrone, O. R. (2010) Rotavirus NSP5 orchestrates recruitment of viroplasmic proteins. J. Gen. Virol. 91, 1782–1793 Billecocq, A., Gauliard, N., Le May, N., Elliott, R. M., Flick, R., and Bouloy, M. (2008) RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses. Virology 378, 377–384 Habjan, M., Penski, N., Spiegel, M., and Weber, F. (2008) T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus. J. Gen. Virol. 89, 2157–2166 Ikegami, T., Won, S., Peters, C. J., and Makino, S. (2006) Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J. Virol. 80, 2933–2940 Ikegami, T., Peters, C. J., and Makino, S. (2005) Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system. J. Virol. 79, 5606 –5615 Weber, F., Dunn, E. F., Bridgen, A., and Elliott, R. M. (2001) The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system. Virology 281, 67–74 Blakqori, G., Kochs, G., Haller, O., and Weber, F. (2003) Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids. J. Gen. Virol. 84, 1207–1214 Cheung, W., Gill, M., Esposito, A., Kaminski, C. F., Courousse, N., Chwetzoff, S., Trugnan, G., Keshavan, N., Lever, A., and Desselberger, U. (2010) Rotaviruses associate with cellular lipid droplet components to replicate in viroplasms, and compounds disrupting or blocking lipid droplets inhibit viroplasm formation and viral replication. J. Virol. 84, 6782–6798 Gaunt, E. R., Cheung, W., Richards, J. E., Lever, A., and Desselberger, U. (2013) Inhibition of rotavirus replication by downregulation of fatty acid synthesis. J. Gen. Virol. 94, 1310 –1317 Gaunt, E. R., Zhang, Q., Cheung, W., Wakelam, M. J., Lever, A. M., and Desselberger, U. (2013) Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms. J. Gen. Virol. 94, 1576 –1586 Miyanari, Y., Atsuzawa, K., Usuda, N., Watashi, K., Hishiki, T., Zayas, M., Bartenschlager, R., Wakita, T., Hijikata, M., and Shimotohno, K. (2007) The lipid droplet is an important organelle for hepatitis C virus production. Nat. Cell Biol. 9, 1089 –1097 Shavinskaya, A., Boulant, S., Penin, F., McLauchlan, J., and Bartenschlager, R. (2007) The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly. J. Biol. Chem. 282, 37158 –37169 Boulant, S., Targett-Adams, P., and McLauchlan, J. (2007) Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus. J. Gen. Virol. 88, 2204 –2213 Samsa, M. M., Mondotte, J. A., Iglesias, N. G., AssuncaoMiranda, I., Barbosa-Lima, G., Da Poian, A. T., Bozza, P. T., and Gamarnik, A. V. (2009) Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 5, e1000632 Kumar, Y., Cocchiaro, J., and Valdivia, R. H. (2006) The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets. Curr. Biol. 16, 1646 –1651 Wu, X., Qi, X., Qu, B., Zhang, Z., Liang, M., Li, C., Cardona, C. J., Li, D., and Xing, Z. (2014) Evasion of antiviral immunity through sequestering TBK1/IKKe/IRF3 into viral inclusion bodies. J. Virol. 88, 3067–3076

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Received for publication October 14, 2013. Accepted for publication February 18, 2014.

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