Role of the cytoplasmic tail domains of Bunyamwera orthobunyavirus glycoproteins Gn and Gc in virus assembly and morphogenesis

Abstract

The M RNA genome segment of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, encodes a precursor polyprotein that is proteolytically cleaved to yield two structural proteins, Gn and Gc, and a nonstructural protein called NSm. Gn and Gc are type I integral transmembrane glycoproteins. The Gn protein contains a predicted cytoplasmic tail (CT) of 78 residues, and Gc has a shorter CT of 25 residues. Little is known about the role of the Gn and Gc CT domains in the virus replication cycle. We generated a series of mutant glycoprotein precursor constructs containing either deletions or alanine substitutions in the CT domains of Gn and Gc. We examined the effects of these mutations on glycoprotein maturation, cell surface expression, and low pH-induced syncytium formation. In addition, the effects of these mutations were also assessed using a reverse genetics-based virus assembly assay and a virus rescue system. Our results show that the CT domains of both Gn and Gc play crucial roles in BUNV-mediated membrane fusion, virus assembly, and morphogenesis.

FIG. 1.

BUNV M RNA segment and mutagenesis of the glycoprotein precursor. The layout of the BUNV M segment-encoded gene product is shown at the top, with positions of amino acid residues marking protein boundaries (Gn, NSm, and Gc) indicated. ss, signal peptide; TMD, transmembrane domain. Below are shown alignments of the CT domains of Gn and Gc proteins from eight orthobunyaviruses and sequences of the BUNV mutants containing sequential substitutions with strings of five alanine residues. Abbreviations: CEV, California encephalitis virus (GenBank database accession number AAD53039); LACV, La Crosse virus (AAB62804); INKV, Inkoo virus (AAB93841); SRV, South River virus (AAD53044); JCV, Jamestown Canyon virus (AAB93842); MAGV, Maguari virus (AAQ23639); NGAV, Ngari virus (AAT01931); BUNV, Bunyamwera virus (NP_047212).

FIG. 2.

Colocalization of Gc protein with Golgi matrix protein GM130. BSR-T7/5 cells were transfected with BUNV M segment cDNA constructs pTMBUNM (wt BUNM), BUNMΔGnCT (ΔGnCT), and BUNMΔGcCT (ΔGcCT) or were left untransfected (BSRT7 cell), as indicated. The permeabilized samples were stained with a mixture of anti-Gc MAb 742 and anti-GM130 and examined by confocal microscopy. Gc stains red, and the Golgi complex stains green. Colocalization is shown as yellow in the merged images, with an enlarged selected area shown in the upper left corner. The nuclei were stained blue by 4′,6′-diamidino-2-phenylindole (DAPI).

FIG. 3.

Effect of deletion of either Gn or Gc CT on BUNV glycoprotein maturation. Vero cells were infected with vTF7-3 and subsequently transfected with wt or mutant BUNV M segment cDNA constructs. Cells were labeled with [³⁵S]methionine for 20 min and chased in the presence of excess unlabeled methionine for different times, as indicated (in minutes). Labeled proteins were immunoprecipitated (IP) with either MAb 742 (A) or anti-BUNV (B), subjected to endo H digestion (+) or left undigested (−) as indicated, and analyzed by SDS-12.5% PAGE under reducing conditions. BUNV Gn and Gc proteins are indicated to the right of the gels. Quantitative analyses of protein bands by phosphorimaging are shown. Data from the pulse-chase experiment with MAb 742 are shown as the total density at each time point, while data from the pulse-chase experiment with anti-BUNV are shown as percentages of the endo H-resistant form versus the total Gc protein.

FIG. 4.

Detection of BUNV glycoproteins on the cell surface. (A and B) Cell surface staining of nonpermeabilized cells. Cells were fixed with 4% paraformaldehyde and stained with anti-BUNV polyclonal antibody or anti-Gc MAb 742. Surface expression of viral glycoproteins was observed using a Delta Vision restoration microscope, and nuclei were stained blue with DAPI. (A) BSR-T7/5 cells were infected with wt BUNV or transfected with wt BUNV M segment cDNA (wt BUNM), BUNMΔGnCT (ΔGnCT), and BUNMΔGcCT (ΔGcCT) constructs, as indicated. Infected cells were stained with MAb 742, and the transfected cells were stained with anti-BUNV antibody. (B) BSR-T7/5 cells were transfected with BUNV M segment cDNA clones containing alanine substitutions in the Gn CT, as indicated. The cells were stained with anti-BUNV antibody. (C). Cell surface biotinylation of either wt BUNV-infected or alanine substitution mutant-transfected BSR-T7/5 cells. The biotinylated viral glycoproteins expressed on the cell surface were precipitated with anti-BUNV serum and separated in SDS-PAGE gels, and signals were revealed by chemiluminescence. No Gn protein was detected.

FIG. 5.

Low pH-induced syncytium formation. BSR-T7/5 cells were infected with BUNV or transfected with wt or mutant BUNV M segment cDNA constructs. At 24 h postinfection or -transfection, cells were treated with low-pH medium (pH 5.3) for 5 min, and syncytium formation was examined following incubation at 37°C for a further 5 h. Cells were then stained with Giemsa solution. (A) Syncytium formation in cells either infected with BUNV (wtBUN) or transfected with wt BUNV M segment cDNA (wtM), BUNMΔGnCT (ΔGnCT), and BUNMΔGcCT (ΔGcCT) constructs, as indicated. Cells were treated with acidic pH (pH 5.3) or neutral pH (pH 7.2) as shown below. (B) Syncytium formation following low-pH treatment in cells transfected with BUNV M segment mutants containing alanine substitutions in the Gn and Gc CTs. (C) f values. All f values were calculated from cells treated with low-pH (5.3) medium as described in Materials and Methods, except for the wtBUNnf value (second bar), which was obtained from wt BUNV-infected cells under neutral-pH conditions.

FIG. 6.

Effects of mutations in CT domains of Gn and Gc on the formation of infectious VLPs. BSR-T7/5 cells were transfected with minigenome component plasmids together with either wt BUNV M segment cDNA or alanine-substituted mutants. Supernatants from these cells were taken at 24 h posttransfection and used to infect fresh BSR-T7/5 cells. Renilla luciferase activities in extracts of these cells were measured after another 24 h of incubation and are shown in arbitrary light units.

FIG. 7.

Rescue of mutant viruses. The rescue of viruses containing deletions or alanine substitutions in the CT domains was attempted as described in Materials and Methods. Recombinant viruses were obtained from transfections involving BUNMGcCTA1 and BUNMGcA4 DNAs. (A) Yields of transfectant viruses rBUNGcCTA1 and rBUNGcA4. Supernatants from the initial transfection dishes were titrated by plaque assay on Vero cells. The results are the averages for two independent titrations. (B) Comparison of plaque sizes produced on Vero cells. The right column represents typical images of single plaques under a light microscope (magnification, ×100). Cell monolayers were fixed with 4% formaldehyde-PBS and stained with Giemsa solution.