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. 2001 Jan;75(2):1004-12.
doi: 10.1128/JVI.75.2.1004-1012.2001.

Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

M Kikkert  1 , A Verschoor, R Kormelink, P Rottier, R Goldbach
Affiliations

Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

M Kikkert et al. J Virol. 2001 Jan.

Abstract

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.

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Figures

FIG. 1
FIG. 1
Schematic representation of Semliki Forest virus constructs described in this article. Solid rectangles indicate hydrophobic sequences (signal sequence/transmembrane domain), and open circles stand for potential N-glycosylation sites. Scissor symbols indicate possible (signal peptidase) cleavages in the precursor. Amino acid sequences around the putative signal sequence junction sites are indicated, where an asterisk indicates a cleavage site predicted by the von Heijne (41) algorithm, and a circumflex (∧) indicates a cleavage site predicted by the Jagla algorithm (unpublished data).
FIG. 2
FIG. 2
Western blots using antiserum against TSWV particles, showing the expression (21 h.p.t.) of the TSWV glycoproteins (pSFV-GP), G2 alone (pSFV-G2), G1 alone (pSFV-G1a and pSFV-G1b), and N (pSFV-N) in BHK cells. Expression of the TSWV glycoproteins in the presence of tunicamycin is shown in lane pSFV-GP + G1* and G2* indicate unglycosylated forms of G1 and G2, and prec. is the glycoprotein precursor protein. The positions of size markers are indicated on the left (in kilodaltons). V, purified TSWV particles; C, control (mock-transfected) cells.
FIG. 3
FIG. 3
Immunofluorescence analysis of G1 and G2 expression from transcripts from pSFV-GP (a to l) and of N expression from transcripts from pSFV-N (m). (a) At 6 h.p.t. with anti-TSWV; arrow indicates perinuclear signal. (b) At 21 h.p.t. with anti-TSWV. (c) At 6 h.p.t. with anti-TSWV. (d) Same cell treated with ER marker DiOC6. (e) Merge (double exposure) of c and d. (f) At 21 h.p.t. with anti-TSWV. (g) Same cell treated with Golgi marker WGA-FITC. (h) Merge of f and g. (i) At 21 h.p.t. with anti-TSWV. (j) Same cell treated with Golgi stack marker anti-p58. (k) Merge of i and j. (l) Expression in the presence of the glycosylation inhibitor tunicamycin at 21 h.p.t. with anti-TSWV. (m) At 21 h.p.t. with anti-N. Yellow areas in c, f, and i indicate overexposure of the film to clearly visualize additional (ER) localization elsewhere in the cell.
FIG. 4
FIG. 4
Immunofluorescence analysis of G2 expression from transcripts from pSFV-G2. (a) At 6 h.p.t. with anti-G2. (b) At 21 h.p.t. with anti-G2; arrows indicate perinuclear signal. (c) At 6 h.p.t. with anti-G2. (d) Same cells treated with ER marker DiOC6. (e) Merge (double exposure) of c and d. (f) At 21 h.p.t. with anti-G2. (g) Same cells treated with Golgi marker WGA-FITC. (h) Merge of f and g. Yellow areas in c and f indicate overexposure of the film as explained in the legend to Fig. 3.
FIG. 5
FIG. 5
Immunofluorescence analysis of G1 expression from transcripts from (a) pSFV-G1a, (d) pSFV-G1b, and (g) pSFV-G1ss1. All were taken at 21 h.p.t. with anti-G1. (b, e, and h) Corresponding cells treated with the DiOC6 ER marker. (c, f, and i) Merge (double exposures) of corresponding panels.
FIG. 6
FIG. 6
(a to c) Expression of G1 and G2 from transcripts from pSFV-GP at 6 h.p.t. using antisera (a) 2B6 (monoclonal against Golgi-localized G1) and (b) anti-G2 (polyclonal). (c) Merge of a and b. (d to i) Immunofluorescence analysis of coexpression of pSFV-G2 with pSFV-G1b (d, e, and f) or pSFV-G1ss1 (g, h, and i) at 21 h.p.t. (d and g) 2B6 monoclonal against G1; (e and h) anti-G2; (f and i) merge (double exposures) of corresponding panels.
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References

    1. Adam G, Peters D, Goldbach R W. Serological comparison of tospovirus isolates using polyclonal and monoclonal antibodies: Proceedings of the International Symposium on Tospoviruses and Thrips of Floral and Vegetable Crops. Acta Hortic (Wageningen) 1996;431:135–158.
    1. Andersson A M, Melin L, Bean A, Pettersson R F. A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. J Virol. 1997;71:4717–4727. - PMC - PubMed
    1. Andersson A M, Melin L, Persson R, Rashperger E, Wikström L, Pettersson R F. Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus. J Virol. 1997;71:218–225. - PMC - PubMed
    1. Bar-Peled M, Bassham D C, Raikhel N V. Transport of proteins in eucaryotic cells: more questions ahead. Plant Mol Biol. 1996;32:223–249. - PubMed
    1. De Ávila A C, de Haan P, Smeets M L L, de O. Resende R, Kormelink R, Kitajima E W, Goldbach R W, Peters D. Distinct levels of relationships between tospovirus isolates. Arch Virol. 1993;128:211–227. - PubMed

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