Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct;90(Pt 10):2483-2492.
doi: 10.1099/vir.0.013540-0. Epub 2009 Jul 1.

Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein

Affiliations

Functional analysis of the Bunyamwera orthobunyavirus Gc glycoprotein

Xiaohong Shi et al. J Gen Virol. 2009 Oct.

Abstract

The virion glycoproteins Gn and Gc of Bunyamwera orthobunyavirus (family Bunyaviridae) are encoded by the M RNA genome segment and have roles in both viral attachment and membrane fusion. To investigate further the structure and function of the Gc protein in viral replication, we generated 12 mutants that contain truncations from the N terminus. The effects of these deletions were analysed with regard to Golgi targeting, low pH-dependent membrane fusion, infectious virus-like particle (VLP) formation and virus infectivity. Our results show that the N-terminal half (453 residues) of the Gc ectodomain (909 residues in total) is dispensable for Golgi trafficking and cell fusion. However, deletions in this region resulted in a significant reduction in VLP formation. Four mutant viruses that contained N-terminal deletions in their Gc proteins were rescued, and found to be attenuated to different degrees in BHK-21 cells. Taken together, our data indicate that the N-terminal half of the Gc ectodomain is dispensable for replication in cell culture, whereas the C-terminal half is required to mediate cell fusion. A model for the domain structure of the Gc ectodomain is proposed.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
BUNV glycoprotein Gc mutants. 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. Below are shown schematics of the proteins encoded by wt and mutant (MΔ1, etc.) cDNA clones. Deletions start at residue 480 to keep intact the cleavage site between NSm and Gc. The N-terminal position of each mutant Gc protein and the number of residues (aa) deleted are shown. The M segment gene products of MAGV and its two non-ts revertant viruses R1 and R2 (Pollitt et al., 2006) are shown beneath. Predicted transmembrane domains (TMD) and the fusion peptide (FP) in BUNV Gc (residues 1058–1079) are shown as grey and hatched boxes, respectively. ⧫ indicate glycosylation sites. Viruses recovered from mutant M segment cDNAs by reverse genetics are marked with an asterisk (*).
Fig. 2.
Fig. 2.
Proteins expressed from mutant BUNV M segment cDNAs. BSR-T7/5 cells were transfected with cDNAs as indicated or left untransfected (Mock) and labelled with [35S]methionine for 1 h at 24 h post-transfection, and cell lysates were reacted with anti-BUN serum. Immunoprecipitated proteins were analysed on 4–12 % polyacrylamide NuPAGE gels (Invitrogen) under reducing conditions. The positions of glycoproteins Gn and Gc and of protein molecular mass markers (in kDa) are indicated.
Fig. 3.
Fig. 3.
Intracellular localization of BUNV glycoproteins. BSR-T7/5 cells were transfected with wt or mutant BUNV M segment cDNAs as indicated or left untransfected (C), and stained with a mixture of rabbit anti-BUN and mouse anti-GM130 antibodies. BUNV glycoproteins Gn and Gc stain green and GM130 stains red. In merged images, the colocalization of BUNV glycoprotein and GM130 shows as yellow. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI).
Fig. 4.
Fig. 4.
Low pH-induced syncytium formation. BSR-T7/5 cells were transfected with wt or mutant BUNV M segment cDNA constructs or left untransfected as a control (C). At 24 h post-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 transfected with wt (wt M) and mutant BUN M segment cDNAs (MΔ1–MΔ12) as indicated. The small syncytia formed by cells transfected with MΔ6 and MΔ9 cDNAs are marked by white arrows. (b) Fusion indices (f), calculated as described in Methods from cells treated with low-pH medium.
Fig. 5.
Fig. 5.
Effects of deletions in the N terminus of the Gc ectodomain on virus assembly. (a) VLP formation. BSR-T7/5 cells were transfected with minigenome component plasmids together with either wt or mutant M segment cDNA constructs. Supernatants from these cells were used to infect new BSR-T7/5 monolayers previously transfected with BUNV L and N protein-expressing plasmids. Renilla luciferase activities were measured after a further 24 h incubation and are shown as arbitrary light units. (b) Protein profiles of the rescued mutant viruses. BHK-21 cells were infected with either wt BUNV or mutant viruses and cells were labelled with [35S]methionine for 1 h at 24 h post-infection. Cell lysates were analysed on 4–12 % polyacrylamide NuPAGE gels under reducing conditions. The positions of viral proteins are marked.
Fig. 6.
Fig. 6.
Plaque phenotypes, growth kinetics and protein synthesis profiles of wt and mutant viruses. (a) Comparison of plaque size on BHK-21 cells. Monolayers were fixed 6 days post-infection with 4 % formaldehyde and stained with Giemsa solution. (b) Virus growth curves in BHK-21 cells (at 37 and 33 °C). Cells were infected with either wt (⧫) or recombinant (□, rBUNGcΔ4; ▵, rBUNGcΔ7; ○, rBUNGcΔ8; ×, rBUNGcΔ9) viruses at an m.o.i. of 0.01 p.f.u. per cell. Virus was harvested at intervals as indicated and titrated by plaque formation in BHK-21 cells. Results are shown as the mean of two independent titrations. (c) Time-course of protein synthesis in infected BHK-21 cells. Cells were infected at an m.o.i. of 0.01 p.f.u. per cell and were labelled with 80 μCi [35S]methionine for 1 h at the time points indicated. Cell lysates were analysed on 4–12 % polyacrylamide NuPAGE gels. The positions of viral proteins are indicated; * indicates wt Gc.
Fig. 7.
Fig. 7.
Model of the domain structure of the BUNV Gc protein. The lengths of BUNV glycoproteins Gn and Gc are drawn in proportion to their actual sizes. The prediction of disordered residues (a) and consensus secondary structure (b) of Gc, obtained by using the Phyre server (Kelley & Sternberg, 2009), are shown on the left. Disordered residues are indicated as shaded boxes in (a), and α-helices and β-strands are shown as shaded boxes and open arrows, respectively, in (b). The positions that indicate the residues of the N and C termini of Gn and Gc within the precursor protein, and residues defining the deletion mutations (MΔ4, MΔ7–MΔ10 and MΔ12), are shown. The predicted fusion peptide in Gc (residues 1058–1079) is indicated as a hatched box.

Similar articles

Cited by

References

    1. Brandenburg, B. & Zhuang, X. (2007). Virus trafficking – learning from single-virus tracking. Nat Rev Microbiol 5, 197–208. - PMC - PubMed
    1. Bridgen, A. & Elliott, R. M. (1996). Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc Natl Acad Sci U S A 93, 15400–15404. - PMC - PubMed
    1. Buchholz, U. J., Finke, S. & Conzelmann, K. K. (1999). Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73, 251–259. - PMC - PubMed
    1. Cortez, I., Aires, A., Pereira, A. M., Goldbach, R., Peters, D. & Kormelink, R. (2002). Genetic organisation of iris yellow spot virus M RNA: indications for functional homology between the G(C) glycoproteins of tospoviruses and animal-infecting bunyaviruses. Arch Virol 147, 2313–2325. - PubMed
    1. Elliott, R. M. (1990). Molecular biology of the Bunyaviridae. J Gen Virol 71, 501–522. - PubMed

Publication types

Substances

LinkOut - more resources