Tomato Spotted Wilt Virus Glycoproteins Exhibit Trafficking and Localization Signals That Are Functional in Mammalian Cells

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.

Mapping the Golgi Targeting and Retention Signal of Bunyamwera Virus Glycoproteins

“…When BUN Gn is expressed on its own, it localizes to this organelle; in contrast, the glycoprotein located at the carboxy terminus of the precursor, Gc, remains in the endoplasmic reticulum (ER) when it is expressed alone and is only transported to the Golgi after an interaction with Gn (19). Similar findings were reported for the glycoproteins of the plant-infecting Tomato spotted wilt virus (Tospovirus genus) when they were expressed in BHK cells (15). Golgi retention of Uukuniemi virus (Phlebovirus genus) glycoproteins is mediated by a short region in the cytoplasmic tail of the Gn glycoprotein (1,2), but for two other phleboviruses, Punta Tora fever virus and Rift Valley fever virus, the Golgi retention signal has been mapped to a region encompassing the transmembrane domain (TMD) and part of the adjacent cytoplasmic tail (11,23,24).…”

Viral genetic determinants for thrips transmission of Tomato spotted wilt virus

“…The mutation is also outside the RGD motif of G N ͞G C ORF, which has been associated with cellular attachment in some animal pathogens (18,(44)(45)(46). The C1375A mutation may not be involved with glycoprotein processing, even though there is a previous report that amino acid 484 of TSWV G N ͞G C may be a cleavage site within the glycoprotein precursor (47). It may be significant that amino acid 459 resides within a putative transmembrane domain of the glycoprotein, which spans from amino acid 428 to 484 (47).…”

“…The C1375A mutation may not be involved with glycoprotein processing, even though there is a previous report that amino acid 484 of TSWV G N ͞G C may be a cleavage site within the glycoprotein precursor (47). It may be significant that amino acid 459 resides within a putative transmembrane domain of the glycoprotein, which spans from amino acid 428 to 484 (47). The nonsense and frameshift mutations that were also observed in nontransmissible SLIs would prevent G N ͞G C from being translated by terminating translation prematurely or frameshifting of the G N ͞G C ORF, respectively.…”

Expression and Characterization of a Soluble Form of Tomato Spotted Wilt Virus Glycoprotein G N

“…The G N proteins of several virus species within the family Bunyaviridae contain Golgi retention sequences (4,17,28), and the retention signals were mapped to the transmembrane domain and the cytosolic tail for Rift Valley fever virus (17) and the cytosolic tail for Uukuniemi virus (4). The TSWV G N Golgi localization signal has not been mapped, but by analogy with other members of the Bunyaviridae it likely resides in the transmembrane domain and/or cytosolic tail.…”

“…The GPs are designated G N and G C based on their positions relative to the amino and carboxy termini of the polyprotein. For most of the members of the Bunyaviridae studied, the G N protein has a Golgi retention sequence and the G C possesses an endoplasmic reticulum retention sequence (3,28,39,57). When the TSWV glycoproteins are expressed together, they colocalize to the Golgi, the site of virion formation (27,28).…”