Function of small hydrophobic proteins of paramyxovirus - PubMed (original) (raw)

Function of small hydrophobic proteins of paramyxovirus

Rebecca L Wilson et al. J Virol. 2006 Feb.

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 (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH 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 rSV5DeltaSH-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 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+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-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.

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Figures

FIG. 1.

Confirmation of rSV5ΔSH+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-, rSV5ΔSH-, and rSV5ΔSH+MuV-SH-infected MDBK cells using primers BH191 and BH194. Products from cells infected with rSV5, rSV5ΔSH, or rSV5ΔSH+MuV-SH were analyzed on a 1% agarose gel. Expected sizes of fragments are given on the right of the gel.

FIG. 2.

Analysis of rSV5ΔSH+MuV-SH. (A) Viral protein expression in rSV5-, rSV5ΔSH-, and rSV5ΔSH+MuV-SH-infected cells. Mock-infected and rSV5-, rSV5ΔSH-, and rSV5ΔSH+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, rSV5ΔSH, or rSV5ΔSH+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.

FIG. 3.

Growth characteristics of rSV5ΔSH+MuV-SH. Single-step growth rates (A) and plaque sizes (B) of rSV5ΔSH+MuV-SH, rSV5, and rSV5ΔSH are shown. Confluent MDBK cells in 35-mm plates were infected with rSV5, rSV5ΔSH, or rSV5ΔSH+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.

FIG. 4.

CPE induced by virus infections. (A) Infection of MDBK, L929, and HeLa cells with rSV5, rSV5ΔSH, or rSV5ΔSH+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, rSV5ΔSH, rSV5ΔSH+MuV-SH, rSV5 plus rSV5ΔSH, or rSV5ΔSH plus rSV5ΔSH+MuV-SH. The cells were photographed at 5 dpi.

FIG. 5.

Inhibition of NF-κB translocation into nuclei by rSV5ΔSH+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, SV5ΔSH, or SV5ΔSH+MuV-SH, and immunofluorescence of p65 was carried out as described in Materials and Methods.

FIG. 6.

Inhibition of TNF-α-induced NF-κB activation by SV5 SH and mumps virus SH. (A) Inhibition of TNF-α-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-κB 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.

FIG. 7.

Model of SH function in the virus life cycle. Virus infection activates NF-κB factors, resulting in low expression levels of cytokines such as TNF-α. 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-α are not interrupted, resulting in high levels of TNF-α expression, triggering cell death.

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Function of small hydrophobic proteins of paramyxovirus - PubMed (original) (raw)