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Journal of Virology, February 2006, p. 1700-1709, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1700-1709.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Function of Small Hydrophobic Proteins of Paramyxovirus

Rebecca L. Wilson,1 Sandra M. Fuentes,2 Ping Wang,3 Erica C. Taddeo,1 Alicia Klatt,2 Andrew J. Henderson,1,2,3,4,5 and Biao He1,2,3,4,5*

Department of Veterinary and Biomedical Sciences,1 Graduate Program in Pathobiology,2 Intercollege Graduate Program in Genetics,3 Integrated Biosciences Graduate Program, The Huck Institutes of Life Sciences,4 Center of Molecular Immunology and Infectious Disease, Pennsylvania State University, University Park, Pennsylvania 168025

Received 22 July 2005/ Accepted 15 November 2005


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ABSTRACT
 
Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5{Delta}SH) is viable and has no growth defect in tissue culture cells. However, rSV5{Delta}SH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-{alpha}) and TNF-{alpha} receptor 1 block rSV5{Delta}SH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-{alpha}-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5{Delta}SH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5{Delta}SH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-{kappa}B by TNF-{alpha} in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-{alpha} signaling.


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INTRODUCTION
 
Mumps virus (MuV), a member of the Paramyxoviridae family, causes acute infections in humans. Although infection is often asymptomatic to mildly symptomatic, about 10% of mumps virus infections affect the central nervous system, leading to aseptic meningitis. Mumps virus infection was the most common cause of viral meningitis and encephalitis until the arrival of mass immunization with mumps virus vaccine (6, 12, 24). In unvaccinated populations, mumps virus still poses a threat (27, 34, 41, 50).

Mumps virus is an enveloped, nonsegmented, negative-sense RNA virus that has seven genes, which encode nine known viral proteins (6, 11). The V/I/P gene encodes three proteins, V, I, and P, through a process of RNA editing (43, 46). The V protein plays an important role in inhibiting interferon signaling in infected cells (28, 49, 56). The function of I is not known. The nucleocapsid protein (N), phosphoprotein (P), and large RNA polymerase (L) protein are important for transcription and replication of the viral RNA genome. The fusion (F) protein, a glycoprotein, mediates both cell-to-cell and virus-to-cell fusion in a pH-independent manner that is essential for virus entry into cells (54). Hemagglutinin-neuraminidase (HN), another viral glycoprotein, is also involved in virus entry and release from the host cells (48, 53). The matrix (M) protein plays an important role in virus assembly (39, 40, 47). The small hydrophobic (SH) protein is a 57-amino-acid-residue hydrophobic integral membrane protein and is oriented in membranes with its C terminus in the cytoplasm (10, 44). Due to the variability among different strains of MuV SH, the SH gene has been used as a marker to categorize mumps virus isolates (45). At present, there are 12 different mumps virus groups (A to L) based on their SH gene sequences (20, 21, 55).

The function of the MuV SH protein is not clear. While the SH gene has been identified in all mumps virus isolates, expression of the gene does not appear to be required for virus growth (44). In Enders strain (subtype A) virus-infected tissue culture cells, neither monocistronic mRNA encoding SH nor SH protein has been detected, due to a point mutation at the end of the F gene that causes a failure of transcription initiation at the mumps virus SH gene, suggesting that mumps virus SH is not essential for virus growth in tissue culture cells (44). However, it is possible that SH is expressed below detection levels in vitro and that SH is necessary for growth in vivo.

Simian virus 5 (SV5) is closely related to mumps virus. Both viruses are rubulaviruses in the Paramyxoviridae family and have identical gene orders. Like the MuV SH gene, the SV5 SH gene is located between the F and HN genes. SV5 SH is a type II membrane protein of 44 amino acid residues. SV5 lacking SH (rSV5{Delta}SH) grows as well as the wild type (wt) in tissue culture cells (14), but it induces apoptosis in L929 cells through a tumor necrosis factor alpha (TNF-{alpha})-mediated extrinsic apoptotic pathway (15, 26). Addition of neutralizing antibodies against TNF-{alpha} and TNF-{alpha} receptor 1 (TNF-R1), but not antibody against TNF-R2, blocks rSV5{Delta}SH-induced apoptosis, suggesting that SV5 SH plays an important role in blocking the TNF-{alpha}-mediated apoptotic signaling pathway (26). However, it has not been shown whether SH alone can block TNF-{alpha} signaling.

We postulate that the functions of the mumps virus and SV5 SH proteins are similar and that they both play an important role in inhibiting TNF-{alpha} signaling, even though they have no sequence homology. The ability to prevent infected cells from undergoing apoptosis is beneficial to a virus, since prolonging the lives of cells gives the virus more time to propagate. In this work, we have generated a recombinant SV5 in which the open reading frame (ORF) of SV5 SH has been replaced by the ORF of MuV SH (rSV5{Delta}SH+MuV-SH). We have compared the growth and the apoptotic effects of wt SV5, rSV5{Delta}SH, and rSV5{Delta}SH+MuV-SH. In addition, using a reporter gene assay, we have examined the abilities of MuV SH and SV5 SH to inhibit TNF-{alpha} signaling.


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MATERIALS AND METHODS
 
Plasmids, viruses, and cells. All molecular cloning was carried out according to standard procedures (2). Mumps virus SH was cloned from mumps virus strain Enders, obtained from the American Type Culture Collection (ATCC). MuV was grown in Vero cells, total RNAs were purified from infected cells, and reverse transcriptase PCR (RT-PCR) with appropriate primers was carried out to amplify the MuV SH gene. The PCR product was cloned into expression vector pCAGGS (29). The SV5 SH gene was also cloned into expression vector pCAGGS. A sequence (YPYDVPDYA, recognized by monoclonal antibody 12CA5) from the hemagglutinin (HA) protein of influenza virus (23) was placed at the N terminus of SV5 SH, and the HA-tagged SV5 SH was cloned into expression vector pCAGGS. Sequences of pCAGGS-SV5-SH, pCAGGS-MuV-SH, and pCAGGS-SV5-HA-SH are on file and available on request. pNF-{kappa}B-Luc has been described previously (42). Plasmids used for recovery of infectious SV5 pCAGGS-NP, pCAGGS-P, and pCAGGS-L have been described previously (16, 52)

Wild-type SV5 and rSV5{Delta}SH have been described previously (14, 17). Recovery of rSV5{Delta}SH+MuV-SH is described below. It was grown in MDBK cells similarly to SV5 or rSV5{Delta}SH (14, 17). Mumps virus was obtained from the ATCC and grown in Vero cells.

HeLa, L929, L929F, Vero, and MDBK cells were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Mediatech Inc., Holu Hill, FL). BHK and BSR-T7 cells were maintained in DMEM with 10% tryptose phosphate broth, 10% FBS, and 1% penicillin-streptomycin. G418 at 400 µg/ml was added to the medium of BSR-T7 cells. For virus infection, monolayers were washed with phosphate-buffered saline with Mg2+ and Ca2+ (PBS+) and then infected with virus in DMEM plus 1% bovine serum albumin at 5 PFU/cell for 1 to 2 h at 37°C with 5% CO2. For coinfection, 10 PFU/cell of rSV5 or rSV5{Delta}SH+MuV-SH and 5 PFU/cell of rSV5{Delta}SH were used. The monolayers were then washed with PBS+ and incubated with DMEM containing 2% FBS at 37°C with 5% CO2.

Recovery of mutant rSV5 by reverse genetics. Recovery of rSV5{Delta}SH has been described previously (14). The plasmid for SV5{Delta}SH+MuV-SH was generated from an infectious clone of SV5 (pBH276) from which the SH gene was deleted and replaced with MuV strain Enders SH. Cloning of SH from virus strain Enders is described in the preceding section. BSR-T7 cells were transfected with the genomic cDNA clone for rSV5{Delta}SH+MuV-SH along with plasmids encoding NP, P, and L of SV5 by using Lipofectamine and PLUS reagents according to the manufacturer's recommendations (Invitrogen, Carlsbad, CA). Plasmid amounts were as follows: 1 µg pSV5{Delta}SH+MuV-SH, 100 ng pCAGGS-NP, 20 ng pCAGGS-P, and 500 ng pCAGGS-L. Cells were incubated with the transfection mixture overnight at 37°C with 5% CO2. Cells were then washed once with PBS+, and the medium was replaced with DMEM supplemented with 2% FBS. Two to four days posttransfection, the medium was examined for the presence of recombinant SV5 by monitoring syncytium formation on BSR-T7 cells. The supernatants from syncytium-positive wells were then used for plaque assays on BHK cells, and plaques were purified (31). Virus stocks of rSV5{Delta}SH+MuV-SH from a single plaque were grown in MDBK cells and were harvested 5 to 7 days postinfection (dpi) as described previously (32).

Single-step growth rate. Monolayers of MDBK cells in 6-cm plates were washed with PBS+ and then infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH in DMEM-1% bovine serum albumin at a multiplicity of infection (MOI) of 5 PFU/cell for 1 h at 37°C. The cells were then maintained in DMEM-2% fetal calf serum. Medium was collected at 0, 12, 24, 36, 48, and 60 h postinfection (hpi). The titers of viruses were determined by plaque assays on BSR-T7 cells as described previously (14, 17).

RT-PCR and sequencing. Total RNAs from rSV5-, rSV5{Delta}SH-, or rSV5{Delta}SH+MuV-SH-infected MDBK cells were purified using an RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. Total RNAs were dissolved in 50 µl water, and 9 µl was used in an RT reaction with primer BH191 (sequence,5'-TATTGACCATTGTCGTTGCTAATCGAAA-3'), which annealed to the viral RNA (vRNA) minus strand. An aliquot of the cDNA was then amplified in a PCR using primers BH191 and BH194 (sequence, 5'-TCGAAATAATACTCGGCAAGTGGCC-3') (14). The PCR products were electrophoresed on a 1% agarose gel and purified. The RT-PCR product was sequenced by Davis Sequencing (Davis, CA) using ABI 3730.

Immunofluorescence. HeLa cells on glass coverslips were either mock infected or infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. At 1 dpi, cells were washed with PBS and then fixed in 0.5% formaldehyde for 15 min at room temperature. The cells were washed with PBS-0.1% saponin solution and were incubated for 30 min in a 1:100 dilution of a rabbit anti-SV5 SH antibody (18) and a 1:500 dilution of a mouse anti-HN antibody (33). Cells were washed three times with PBS-0.1% saponin. Texas Red-labeled anti-rabbit and fluorescein isothiocyanate (FITC)-labeled anti-mouse secondary antibodies were added to the cells. To detect expression of MuV SH in infected cells, an antibody kindly provided by Kaoru Takeuchi (44) was used at a 1:100 dilution. The cells were incubated for 30 min and were then washed three times in PBS-0.1% saponin. Fluorescence was examined and photographed using an Olympus BX-60 digital microscope with ImagePro plus software or a confocal microscope. To examine the localization of SV5 SH, SV5 HA-SH, and MuV SH, L929 cells on glass coverslips were transfected with plasmids encoding the SH proteins and processed similarly.

To detect p65, a subunit of NF-{kappa}B factors, L929 cells on glass coverslips were either mock infected or infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. At 1 dpi, cells were washed with PBS+ and then fixed in 0.5% formaldehyde for 15 min at room temperature. The cells were washed with PBS-0.1% saponin solution and were incubated overnight in a 1:100 dilution of a monoclonal antibody specific for the p65 subunit of NF-{kappa}B (catalog no. sc-8008; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. Cells were washed three times with PBS-0.1% saponin, and an FITC-labeled anti-mouse antibody was added to the cells. The cells were incubated for 30 min and then washed three times in PBS-0.1% saponin. Fluorescence was examined and photographed as described above.

EMSA. To examine DNA binding of NF-{kappa}B factors, nuclear extracts from mock-, rSV5-, rSV5{Delta}SH-, and rSV5{Delta}SH+MuV-SH-infected cells were prepared and assayed using an electrophoretic mobility shift assay (EMSA) as described previously (4). The infected L929 cells were trypsinized and washed in PBS at 1 dpi. The cells were counted, and 106 cells were resuspended in 1 ml PBS without Mg2+ and Ca2+ and pelleted in an Eppendorf tube. The cells were resuspended in 400 µl of cold buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) for 15 min on ice. Then 25 µl of 10% NP-40 was added. The tubes were vortexed and then centrifuged at 10,000 rpm for 30 s. The supernatant was discarded, and 50 µl of cold buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) was added. The tubes were then rocked at 4°C for 15 min. This was followed by centrifugation at 10,000 rpm for 5 min. The supernatant was collected into new Eppendorf tubes and stored at –70°C.

Oligomers containing NF-{kappa}B binding sites (5'-AGCTAAGGGACTTTCCGCTGGGGACTTTCCAGG-3' and 5'-AGCTCCTGGAAAGTCCCCAGCGGAAAGTCCCTT-3') were radiolabeled by using Klenow fragment in an end-filling reaction mixture. NF-{kappa}B oligomers, deoxynucleoside triphosphates (G, A, and T), [{alpha}-32P]dCTP, reaction buffer, Klenow fragment, and water were incubated together at room temperature for 1 h, and then the radioactively labeled oligomers were purified using a Sephadex column. To examine the binding of nuclear extracts from infected cells to the labeled NF-{kappa}B probe, 5 µl of lysate (mock, rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH infected), the NF-{kappa}B probe (about 40,000 cpm), 2 µl of 10x EMSA buffer (25 mM HEPES, pH 7.5, 60 mM NaCl, 9% glycerol, 1 mM EDTA, 7.5 mM DTT, 50 mM MgCl2), 2 µl of poly(dI-dC) (1 µg/µl), and water up to 20 µl were incubated together at room temperature for 30 min. As controls, unlabeled NF-{kappa}B oligomers and nonspecific competitor AP-1 primers (AP1 5', 5'-GGG GAA GCT TTG ACT CA-3') (25) were added to nuclear lysates from mock-, rSV5-, rSV5{Delta}SH-, or rSV5{Delta}SH+MuV-SH-infected cells. Nuclear extracts and labeled oligomer mixtures were resolved on a 6% polyacrylamide gel. The gel was dried, and radioactivity was detected using a Storm System PhosphorImager (Molecular Dynamics).

Immunoprecipitation and immunoblotting. HeLa cells in 6-cm plates were either mock infected or infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. At 18 hpi, cells were starved in DMEM without cysteine and methionine for 30 min and then metabolically labeled with 35S-Promix (Amersham Life Science) (10 µCi/ml) for 3 h. The cells were lysed in 0.3 M NaCl radioimmunoprecipitation assay buffer, containing 0.3 M NaCl, 1% (wt/vol) sodium deoxycholate, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 0.1 M Tris-HCl (pH 7.4), 1 mM PMSF, 210 ng/ml aprotinin, 10 mM iodoacetimide, and 0.24 trypsin-inhibiting unit/ml, and aliquots were immunoprecipitated using either sera against disrupted SV5 virions or sera specific for HN, SH, F2, V, or P. Polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using a 10% or 17.5% polyacrylamide gel (31).

To examine the expression levels of SV5 SH, SV5 HA-SH, and MuV SH, L929 cells in 6-cm plates were transfected with 3 µg plasmids encoding the SH proteins. At 1 day posttransfection, the cells were lysed in 0.5 ml of protein lysis buffer (2% SDS, 62.5 mM Tris-HCl, pH 6.8, 2% dithiothreitol), and lysates were sonicated briefly to shear DNA. Up to 80 µl of the lysate was subjected to SDS-PAGE using a 17.5% gel. Proteins were transferred to a polyvinylidene difluoride membrane using a wet-gel transfer apparatus. The membrane was incubated first with primary antibodies against SH proteins (anti-SV5 SH at a 1:500 dilution and anti-MuV SH at a 1:500 dilution) and then with anti-rabbit secondary antibodies conjugated to horseradish peroxidase. The proteins on the membrane were detected using the ECL+ kit (Amersham Pharmacia, Piscataway, NJ), and chemiluminescence was detected using a Storm System PhosphorImager (Molecular Dynamics Inc, Sunnyvale, CA).

Luciferase assay. Six wells of L929F cells grown in 12-well plates were transfected using FuGene6 (Roche) according to the manufacturer's recommendations with either an empty pCAGGS vector, pBH462 (pCAGGS-SV5 SH), pACB (pCAGGS-SV5 SH, HA tagged) or pAK1 (pCAGGS-MuV-SH), plus p{kappa}B-TATA-Luc (an NF-{kappa}B-dependent promoter followed by the firefly luciferase reporter gene) and pCAGGS-GFP. Cells were incubated at 37°C with 5% CO2 for 18 to 24 h; then the medium was replaced with either 250 µl of Optima alone (three wells) or 250 µl of Optima containing 10 ng/ml TNF-{alpha} (catalog no. 522-009; Alexis, San Diego, CA) (three wells), and cells were incubated for 4 h at 37°C with 5% CO2. Cells were washed with PBS-, and 0.5 ml trypsin was added to each well, followed by 0.5 ml of DMEM with 10% FBS. Portions (0.5 ml) of the cells were transferred to a flow tube, and 0.5-ml portions were transferred to an Eppendorf tube. The flow tubes were centrifuged for 10 min at 10,000 rpm, the supernatant was decanted, and the cells were resuspended in 0.5 ml of 0.5% formaldehyde. The percentage of cells transfected with green fluorescent protein (GFP) was determined using a flow cytometer (Coulter Epics XL-MXL). The Eppendorf tubes were centrifuged for 1 min at 400 rpm, the supernatant was decanted, and the cells were lysed in 200 µl 1x Reporter Lysis Buffer (Promega, Madison, WI). The tubes were frozen at 70°C for 15 min, thawed, vortexed, and centrifuged at 14,000 rpm for 2 min. Fifty microliters of luciferase substrate (Promega) and 20 µl of lysate were added together and read on a luminometer (TD-20/20; Turner Designs, Inc., Sunnyvale, CA). Statistical significance was calculated using an unpaired t test.


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RESULTS
 
Generation of rSV5{Delta}SH+MuV-SH. Mumps virus and SV5 have the same gene order, and both encode a small hydrophobic protein (Fig. 1A). We speculate that these two SH proteins have similar functions, even though there is no sequence homology between them. Previous studies of SV5 SH indicate that SV5 SH plays a role in inhibiting TNF-{alpha}-induced cell death (26). We reason that if the SV5 and MuV SH proteins have similar functions, they ought to be interchangeable. To study the function of MuV SH protein, a recombinant SV5 was generated in which the open reading frame of SV5 SH was replaced with that of MuV SH (strain Enders, subtype A). Mumps virus obtained from the ATCC was grown in Vero cells, and the SH gene was cloned using RT-PCR with appropriate primers. The cloned mumps virus SH gene was sequenced and confirmed to be identical to the sequence in GenBank (accession number D90231). The ORF of mumps virus SH was used to replace that of the SV5 SH gene in a plasmid containing the SV5 genome cDNA sequence (pSV5{Delta}SH+MuV-SH). To maintain the genome length as a multiple of 6, nucleotides were added immediately downstream of the stop codon of the mumps virus SH gene. To obtain infectious SV5 containing the mumps virus SH gene, BSR-T7 cells, which stably express T7 RNA polymerase (4), were cotransfected with pSV5{Delta}SH+MuV-SH and plasmids encoding the viral proteins NP, P, and L (17, 52). Viable virus was recovered, and a single plaque was used to generate a virus stock for further analysis. To confirm the identity of the virus, total RNAs were obtained from virus-infected cells and subjected to RT-PCR with appropriate primers (Fig. 1B). Cells infected with rSV5{Delta}SH+MuV-SH produced an RT-PCR product (about 706 bp) that was found to be larger than the RT-PCR products from rSV5- and rSV5{Delta}SH-infected cells, as expected (Fig. 1C), indicating recovery of the recombinant virus containing mumps virus SH in place of SV5 SH. The result was further confirmed by sequencing the PCR products: the PCR products have the input DNA sequence (data not shown). The virus was grown in MDBK cells, and stocks of virus with normal titers (about 1 x 108 PFU/ml) were obtained.


Figure 1
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  		FIG. 1. Confirmation of rSV5{Delta}SH+MuV-SH. (A) Schematics of SV5 and MuV SH proteins. Underlined residues are predicted transmembrane domains. (B and C) RT-PCR of vRNA from rSV5-, rSV5{Delta}SH-, and rSV5{Delta}SH+MuV-SH-infected MDBK cells using primers BH191 and BH194. Products from cells infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH were analyzed on a 1% agarose gel. Expected sizes of fragments are given on the right of the gel.

Viral protein synthesis in rSV5{Delta}SH+MuV-SH-infected cells. To investigate the effects of replacing SV5 SH with MuV SH on virus growth, growth rate, plaque size, and viral protein production, rSV5{Delta}SH+MuV-SH was examined in comparison to SV5. To examine viral protein expression, HeLa cells were infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH at an MOI of 5 PFU/cell and were metabolically labeled with 35S-Promix. The cells were lysed and proteins immunoprecipitated with specific antibodies against SV5 viral proteins. As shown in Fig. 2A, the use of sera against disrupted SV5 virions or of sera specific for HN, SH, F2, V, or P indicates similar expression levels of these proteins. SV5 SH was detected only in rSV5-infected cells, not in rSV5{Delta}SH- or rSV5{Delta}SH+MuV-SH-infected cells. Furthermore, an immunofluorescence assay of mock-, rSV5-, rSV5{Delta}SH-, or rSV5{Delta}SH+MuV-SH-infected cells using anti-SH and anti-HN antibodies indicates the presence of SV5 SH protein in rSV5-infected cells but not in rSV5{Delta}SH- or rSV5{Delta}SH+MuV-SH-infected cells, whereas HN was present in all infected cells (Fig. 2B). As expected, MuV SH was detected only in rSV5{Delta}SH+MuV-SH-infected cells (Fig. 2C).


Figure 2
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  		FIG. 2. Analysis of rSV5{Delta}SH+MuV-SH. (A) Viral protein expression in rSV5-, rSV5{Delta}SH-, and rSV5{Delta}SH+MuV-SH-infected cells. Mock-infected and rSV5-, rSV5{Delta}SH-, and rSV5{Delta}SH+MuV-SH-infected HeLa cells were metabolically labeled with 35S-Promix for 3 h at 18 hpi. The cell lysates were prepared for immunoprecipitation reactions. Immunoprecipitation was performed using sera against disrupted SV5 virions (right panel) or sera specific for HN, SH, F2, V, or P (left panel). (B) Immunofluorescence using antibodies against SV5 SH and HN. L929 cells on glass coverslips either were mock infected or were infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. At 1 dpi, cells were first incubated with a rabbit anti-SH antibody and a mouse anti-HN antibody and then incubated with Texas Red-labeled anti-rabbit and FITC-labeled anti-mouse secondary antibodies. Fluorescence was examined and photographed using an Olympus BX-60 microscope. (C) Expression of MuV SH. HeLa cells on coverslips were infected, and the cells were processed at 3 days postinfection as described above and in Materials and Methods and were photographed using a confocal microscope.

Growth rate of rSV5{Delta}SH+MuV-SH. It is possible that replacing the ORF of SV5 SH with the ORF of MuV SH could alter the growth rate of the virus. Therefore, the growth rates of rSV5, rSV5{Delta}SH, and rSV5{Delta}SH+MuV-SH were compared in a single-step growth experiment. MDBK cells were infected with the viruses at an MOI of 5, the media were harvested at various times (at 12-h intervals, up to 60 h postinfection), and virus titers were measured by plaque assay. rSV5, rSV5{Delta}SH, and rSV5{Delta}SH+MuV-SH showed similar growth rates, and all viruses reached a plateau around 24 h postinfection, as reported previously (Fig. 3A) (16). It is interesting that rSV5{Delta}SH appeared to grow better than rSV5, and rSV5 better than rSV5{Delta}SH+MUV-SH, at 12 hpi. No difference in plaque size among rSV5, rSV5{Delta}SH, and rSV5{Delta}SH+MuV-SH was observed (Fig. 3B).


Figure 3
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  		FIG. 3. Growth characteristics of rSV5{Delta}SH+MuV-SH. Single-step growth rates (A) and plaque sizes (B) of rSV5{Delta}SH+MuV-SH, rSV5, and rSV5{Delta}SH are shown. Confluent MDBK cells in 35-mm plates were infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH at an MOI of 5 PFU/cell, and the media were collected at 12-h intervals up to 60 hpi. The titers of viruses were determined by plaque assays on BSR-T7 cells.

Growth of rSV5{Delta}SH+MuV-SH in MDBK, L929, and HeLa cells with minimal CPE. SV5 replicates in MDBK cells for many days with minimal cytopathic effect (CPE) (7); however, rSV5{Delta}SH causes CPE in these cells as well as in L929 cells but does not cause CPE in HeLa cells (14, 15). To compare CPEs induced by viruses in MDBK, L929, and HeLa cells, the cells were infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. It was observed that by 4 dpi rSV5{Delta}SH+MuV-SH and rSV5 grew in MDBK and L929 cells with minimal CPE, whereas there was extensive cell loss in rSV5{Delta}SH-infected MDBK and L929 cells. All viruses grew with minimal CPE in infected HeLa cells (Fig. 4A). Previously it has been shown that cell death caused by rSV5{Delta}SH infection can be inhibited by SV5 infection, suggesting that SV5 SH can inhibit cell death induced by virus infection (15). To examine whether MuV SH has a similar function, MDBK cells were infected with rSV5{Delta}SH as well as rSV5{Delta}SH+MuV-SH. Like SV5 infection, rSV5{Delta}SH+MuV-SH infection blocked cell death caused by rSV5{Delta}SH infection (Fig. 4B), suggesting that MuV SH, like SV5 SH, is capable of inhibiting cell death induced by the virus infection.


Figure 4
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  		FIG. 4. CPE induced by virus infections. (A) Infection of MDBK, L929, and HeLa cells with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. Cells in 6-well plates were either mock infected (Mk) or infected with one of the viruses. At 4 dpi, the cells were photographed. (B) Coinfection of MDBK cells with viruses. MDBK cells in 6-cm plates were either mock infected or infected with either rSV5, rSV5{Delta}SH, rSV5{Delta}SH+MuV-SH, rSV5 plus rSV5{Delta}SH, or rSV5{Delta}SH plus rSV5{Delta}SH+MuV-SH. The cells were photographed at 5 dpi.

MuV SH blocked NF-{kappa}B p65 translocation. Activation of NF-{kappa}B results in its localization to the nucleus. Previously it has been shown that in rSV5{Delta}SH-infected L929 cells the p65 subunit of NF-{kappa}B is localized to the nucleus, whereas rSV5-infected L929 cells exhibit minimal nuclear localization of p65 (26). To examine whether mumps virus SH is essential for preventing NF-{kappa}B nuclear localization, EMSAs were performed. Nuclear lysates from the same number of mock-, rSV5-, rSV5{Delta}SH-, or rSV5{Delta}SH+MuV-SH-infected cells were collected at 1 dpi, incubated with 32P-labeled NF-{kappa}B probes that included the NF-{kappa}B consensus binding site, and then resolved on a 6% polyacrylamide gel. As previously reported, the NF-{kappa}B probe was shifted by the nuclear lysates from rSV5{Delta}SH-infected cells, whereas a less intense complex was observed when nuclear lysates from rSV5-infected cells were used. As shown in Fig. 5A, the NF-{kappa}B probe was shifted by nuclear lysates from rSV5{Delta}SH+MuV-SH-infected cells to a level similar to that for rSV5 but much lower than that for rSV5{Delta}SH, indicating a lack of activation of NF-{kappa}B by rSV5{Delta}SH+MuV-SH infection. NF-{kappa}B has five subunits, including p65. To examine whether mumps virus SH is essential for preventing the p65 subunit of NF-{kappa}B factors from translocating into nuclei, the localization of the p65 subunit was examined. L929 cells growing on glass coverslips were either mock infected or infected with rSV5, rSV5{Delta}SH, or rSV5{Delta}SH+MuV-SH. The cells were then fixed, permeabilized, incubated with an antibody against p65, and examined under a fluorescence microscope. As previously reported, nuclear localization was observed in rSV5{Delta}SH-infected cells (Fig. 5B). However, nuclear localization of p65 was not observed in rSV5{Delta}SH+MuV-SH-infected cells, suggesting that the presence of MuV SH inhibited NF-{kappa}B p65 nuclear localization.


Figure 5
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  		FIG. 5. Inhibition of NF-{kappa}B translocation into nuclei by rSV5{Delta}SH+MuV-SH. (A) EMSA. L929 cells were infected, and at 1 dpi the nuclear extracts were obtained as described in Materials and Methods. Nuclear extracts and labeled oligomer mixtures were resolved on a 6% polyacrylamide gel. (B) L929 cells were either mock infected or infected with SV5, SV5{Delta}SH, or SV5{Delta}SH+MuV-SH, and immunofluorescence of p65 was carried out as described in Materials and Methods.

Inhibition of TNF-{alpha}-induced NF-{kappa}B activation. While it has been shown that TNF-{alpha} plays an essential role in rSV5{Delta}SH-induced apoptosis, the role of SH is less clear. It is possible that SH inhibits production of TNF-{alpha} and/or signaling of TNF-{alpha}. To examine whether SH inhibits TNF-{alpha} signaling, a reporter gene system was used to examine whether SV5 SH and MuV SH can inhibit TNF-{alpha}-induced NF-{kappa}B activation. Activation of NF-{kappa}B results in its translocation to the nucleus, where it up-regulates the expression of many NF-{kappa}B-dependent genes. A plasmid containing an NF-{kappa}B-dependent promoter followed by a firefly luciferase gene as a reporter was transfected into cells. Cells transfected with this reporter gene produced increased firefly luciferase activity when treated with TNF-{alpha}, as measured using a luminometer (Fig. 6A). In addition to the reporter plasmid, cells were transfected with either an empty vector or a vector containing a gene encoding SV5 SH, HA-tagged SV5 SH, or MuV SH. Cells were also transfected with a GFP-expressing plasmid as a control for transfection efficiency. Figure 6A shows the average increase in luciferase activity (n-fold) in cells transfected with the reporter gene, an empty vector, and GFP and then incubated in a medium with or without TNF-{alpha}. GFP expression was not affected by TNF-{alpha} treatment (data not shown). We have examined the expression levels of SV5 SH, SV5-HA-SH, and MuV SH using immunoblotting and immunofluorescence (Fig. 6B and C). It appeared that the expression levels of SV5 SH and SV5-HA-SH were comparable and that addition of the HA tag did not affect the localization of SV5 SH. In the presence of SV5 SH and MuV SH, the average increases in luciferase activity (n-fold) in cells treated with TNF-{alpha} compared with untreated cells were statistically reduced, indicating that both SV5 SH and MuV SH proteins inhibited TNF-{alpha}-induced NF-{kappa}B activation. Interestingly, the average increase in the luciferase activity (n-fold) of SV5 SH tagged with an HA antigen epitope at its N terminus was similar to that of the positive control even though the expression levels and localization of SV5 HA-SH are similar to those of SV5 SH, suggesting that the addition of a tag at the SV5 SH N terminus affected its function (Fig. 6B and C).


Figure 6
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  		FIG. 6. Inhibition of TNF-{alpha}-induced NF-{kappa}B activation by SV5 SH and mumps virus SH. (A) Inhibition of TNF-{alpha}-activated gene expression. L929F cells were transfected as described in Materials and Methods. The percentage of cells transfected with GFP was determined using a flow cytometer. NF-{kappa}B activation was determined using a luminometer. Statistical significance was calculated using the unpaired t test, and all P values were less than 0.05. (B) Expression levels of SH proteins. Immunoblotting was used to compare the amounts of SH proteins expressed in the transfected cells. Ctrl, control. (C) Localization of SH proteins. Localizations of SH proteins in transfected L929 cells were compared. Antibodies used are indicated on the left.


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DISCUSSION
 
Mumps virus encodes an SH protein whose function has not been elucidated. SV5, a closely related member of the paramyxovirus family, encodes an SH protein which has been shown to play an essential role in blocking apoptosis in infected cells through inhibition of the TNF-{alpha} pathway (26). SV5 lacking SH induces more-severe CPE than wild-type SV5 in infected cells, but it is attenuated in vivo. A straightforward approach to understanding the function of SH is to take a genetic approach similar to that used for SV5: deleting MuV SH from MuV to determine whether MuV{Delta}SH might have a phenotype similar to that of rSV5{Delta}SH. However, this approach requires a means to enable the generation of a mumps virus lacking SH. A reverse genetics system does exist for mumps virus (8). However, it is based on a vaccine strain, Jeryl Lynn (JL), that is severely attenuated in vivo and causes more CPE in infected cells than clinical isolates (35-38). Deleting SH from strain JL is unlikely to result in a more attenuated virus in vivo or to cause more CPE in vitro, since the virus already has these characteristics. Thus, using the vaccine strain JL to study the function of MuV SH protein may not be ideal. Using the SV5 genome as a backbone to study the function of MuV SH provides a viable alternative. In this work, we generated a recombinant SV5 in which the SV5 SH gene was replaced with the mumps virus SH gene.

It is known that apoptosis plays an important role in host defense against virus infection. Viruses have often developed strategies to prevent cells from dying from apoptosis in order to prolong the time during which viruses can replicate. Blockage of the TNF-{alpha}-mediated apoptotic pathway by SV5 SH enables SV5 to replicate for extended periods in infected cells, contributing to the ability of SV5 to grow for a long time in many cell types. Because expression of the SH gene does not appear to be required for growth of either MuV or SV5 (14, 44), because both SH genes are localized in the same region of their respective genomes, and because MuV and SV5 are phylogenetically close, we speculate that MuV SH is similar to SV5 SH functionally, even though there is no obvious sequence homology between the SV5 SH and MuV SH proteins and even though MuV SH is a type I membrane protein while SV5 SH is a type II membrane protein (44). In this work we found that by replacing the SV5 SH gene with the MuV SH gene in a SV5 genome background, we restored the phenotype of rSV5{Delta}SH to the wt phenotype in assays carried out in vitro, indicating that mumps virus SH can functionally replace SV5 SH in a SV5 background and that MuV SH has functions similar to those of SV5 SH. Since SV5 SH is not essential for SV5 growth in vitro and is involved in blocking apoptosis in infected cells, it is possible that MuV SH plays a similar role in blocking apoptosis in the mumps virus life cycle. This function of MuV SH is consistent with previous reports indicating that MuV is involved in blocking apoptosis in infected cells and that it may encode an antiapoptotic protein (13). Preventing infected cells from dying or from dying prematurely is beneficial to virus replication. Interestingly, it has been reported that increased CPE induced by mumps virus infection in vitro (tissue culture cells) seems to correlate with decreased pathogenicity of the virus in a rat model system (35-38). Not surprisingly, a mutation in SV5 causing increased CPE in infected cells results in a mutant that is attenuated in vivo (14, 16).

It has been shown previously that rSV5{Delta}SH induces apoptosis in L929 cells through TNF-{alpha}, suggesting that SV5 SH plays an essential role in blocking TNF-{alpha}-mediated apoptosis (26). In the media of rSV5{Delta}SH-infected L929 cells, up to 150 pg/ml of TNF-{alpha} was detected by an enzyme-linked immunosorbent assay. Whereas no TNF-{alpha} was detected in SV5-infected cell media by an enzyme-linked immunosorbent assay, low levels of TNF-{alpha} were detected by using a very sensitive biological assay (26). However, the role of SV5 SH in TNF-{alpha} production is not clear. It is possible that SH can inhibit either production or signaling of TNF-{alpha}, or both. Activation of NF-{kappa}B can lead to expression of TNF-{alpha} (1, 19). TNF-{alpha} is an autocrine cytokine that has opposing effects on cells (3, 9). On the one hand, TNF-{alpha} can activate NF-{kappa}B, leading to activation of antiapoptotic gene expression as well as its own up-regulation. On the other hand, TNF-{alpha}, possibly at higher concentrations, can activate apoptotic pathways through the activation of FADD and caspases (Fig. 7). It is possible that SV5 SH inhibits production of TNF-{alpha} either directly, by preventing activation of NF-{kappa}B; indirectly, by blocking the TNF-{alpha} signaling pathway; or both directly and indirectly. Since wild-type SV5 containing SH can activate NF-{kappa}B moderately and induces expression of TNF-{alpha} at low levels (26), it is proposed that SV5 SH inhibits TNF-{alpha} signaling. Because TNF-{alpha} is an autocrine cytokine, prevention of TNF-{alpha} signaling will also prevent TNF-{alpha} production. The findings that ectopically expressed SV5 SH or MuV SH reduced the average fold increase in reporter gene expression under the control of a TNF-{alpha}-responsive promoter in cells treated with TNF-{alpha} versus cells without TNF-{alpha} treatment indicate that SH proteins are capable of blocking TNF-{alpha} signaling. This is, to our knowledge, the first report to show that paramyxovirus SH proteins are capable of inhibiting host cytokine signaling pathways.


Figure 7
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  		FIG. 7. Model of SH function in the virus life cycle. Virus infection activates NF-{kappa}B factors, resulting in low expression levels of cytokines such as TNF-{alpha}. The small amount of cytokines directly induced by viral replication or viral proteins is not sufficient to induce cell death. However, due to autocrine effects of many cytokines, the cytokines induced by virus infection have the potential to amplify the expression of cytokines. In cells infected with an SH-encoding virus, the amplification of cytokine expression is blocked by SH, thus preventing the production of higher levels of cytokines that would be sufficient to induce cell death. In the absence of SH, signals mediated by cytokines such as TNF-{alpha} are not interrupted, resulting in high levels of TNF-{alpha} expression, triggering cell death.

Sequences of mumps virus SH genes are relatively diverse compared with those of other viral genes. It is possible that other mumps virus SH proteins have functions different from those of strain Enders (subtype A) SH. However, considering the difference between SV5 SH and mumps virus SH, together with the finding that MuV SH can replace SV5 SH, it is likely that SH proteins of other subtypes of mumps virus may have functions similar to that of strain Enders SH. Other paramyxoviruses that encode SH proteins are respiratory syncytial virus and human and avian metapneumoviruses (30, 51, 57). The functions of those SH proteins are not known. However, it has been reported that deletion of SH from respiratory syncytial virus results in a recombinant virus that has a normal growth rate in tissue culture cells but is attenuated in vivo, much like the phenotype of rSV5{Delta}SH (5, 15). Although speculative, we propose that the SH proteins of paramyxoviruses play an important role in blocking host cell signaling, such as the TNF-{alpha} signaling pathway.

Because SV5 infection activates NF-{kappa}B and increases TNF-{alpha} expression, albeit at a low level, and because SH inhibits TNF-{alpha} signaling, we propose a model of SH function in the virus life cycle (Fig. 7). Virus infection activates NF-{kappa}B at a low level, resulting in increased expression of TNF-{alpha} in infected cells. Because TNF-{alpha} is an autocrine cytokine, it has the potential to amplify its own expression in virus-infected cells. SH, possibly through its interaction with TNF-R1, blocks TNF-{alpha} signaling and prevents more TNF-{alpha} from being produced. This low level of TNF-{alpha} in infected cells is not sufficient to induce apoptosis. This model is consistent with our observations that low levels of TNF-{alpha} are produced in SV5-infected cell media (26) and with the detection of low levels of activation of NF-{kappa}B factors only when the sensitive EMSA method was used, not when the less sensitive immunofluorescence assay was used (26) (Fig. 5).

The mechanism of the inhibition of TNF-{alpha} signaling by SV5 and MuV SH is not clear. Because SV5 SH is a membrane protein and is localized primarily to the endoplasmic reticulum and Golgi (with a small portion on the plasma membrane), a pattern very similar to that of TNF-R1 localization (22), we speculate that SV5 SH may interact with TNF-R1 directly or affect TNF-R1 functionalities indirectly. Identification of an SH-interacting protein, which we are pursuing, will be very informative for our understanding of the mechanism. While SV5 SH and MuV SH are different integral membrane proteins with regard to their orientation, both have cytoplasmic tails that are longer than their ectodomains. Interestingly, addition of an HA antigen epitope tag at the cytoplasmic tail of SV5 SH abolished the ability of SV5 SH to inhibit TNF-{alpha} signaling (Fig. 6), suggesting that the cytoplasmic tail plays an important role for SH function.


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ACKNOWLEDGMENTS
 
We thank Elaine Kunze and Susan Magargee for technical help with the flow cytometer and fluorescence microscope. We thank R. A. Lamb for providing numerous reagents, Kaoru Takeuchi for the antibody against mumps virus SH protein, and Shao-Cong Sun for pNF-{kappa}B-TATA-Luc. We thank members of our lab for support and lively discussions and Michael Teng for carefully reading the manuscript prior to publication.

This work was supported by a grant from the NIH (AI 051372) and a grant from the American Heart Association to B.H.


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FOOTNOTES
 
* Corresponding author. Mailing address: Center of Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 115 Henning Bldg., University Park, PA 16802. Phone: (814) 863-8533. Fax: (814) 863-6140. E-mail: bxh40{at}psu.edu. Back


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Journal of Virology, February 2006, p. 1700-1709, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1700-1709.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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