The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex

150

202

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins GN and GC. To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both GN and GC. Using these MAbs, we found that GN predominantly colocalized with a Golgi marker when expressed alone or with GC, while GC was transported to the Golgi apparatus only in the presence of GN. Both proteins remained endo-β-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the GN ectodomain, because a soluble version of GN lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of GN and GC also resulted in localization of soluble GC to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the GN precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of GN when it was expressed alone but were dispensable when GC was coexpressed. In neutralization assays on SW-13 cells, MAbs to GC, but not to GN, prevented CCHFV infection. However, only a subset of GC MAbs protected mice in passive-immunization experiments, while some nonneutralizing GN MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to GC

Section: Resultssupporting

confidence: 78%

Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins

Bertolotti-Ciarlet

Smith

Strecker

et al. 2005

150

202

“…As was found for the animal-infecting bunyaviruses Uukuniemi virus (27,32) and Bunyamwera virus (23), the N-terminal protein G2 of the TSWV glycoprotein precursor could be transported to the Golgi system on its own, though with decreased efficiency, and apparently contains a Golgi retention signal. The C-terminal protein G1 expressed on its own was unable to leave the ER and thus seems to require an interaction with G2 in order to be transported out of the ER.…”

Section: Discussionmentioning

confidence: 97%

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

Kikkert¹,

Verschoor²,

Kormelink³

et al. 2001

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.

“…We recently mapped the signal for Golgi targeting and retention to the transmembrane domain of Gn (46). The requirement for Gn-Gc heterodimerization for efficient trafficking to the Golgi has also been documented for other members of family, such as Uukuniemi, Punta Toro and Rift Valley Fever viruses of the Phlebovirus genus (9,17,31,40), La Crosse virus of the Orthobunyavirus genus (6), Hantaan and Sin Nombre viruses of the Hantavirus genus (42,45,47), and tomato spotted wilt virus of the Tospovirus genus (23).…”

mentioning

confidence: 99%

Role of N-Linked Glycans on Bunyamwera Virus Glycoproteins in Intracellular Trafficking, Protein Folding, and Virus Infectivity

Shi

Brauburger

Elliott

2005

The membrane glycoproteins (Gn and Gc) of Bunyamwera virus (BUN, family Bunyaviridae) contain three potential sites for the attachment of N-linked glycans: one site (N60) on Gn and two (N624 and N1169) on Gc. We determined that all three sites are glycosylated. Digestion of the glycoproteins with endo-␤-N-acetylglucosaminidase H (endo H) or peptide:N-glycosidase F revealed that Gn and Gc differ significantly in their glycan status and that late in infection Gc glycans remain endo H sensitive. The roles of the N-glycans in intracellular trafficking of the glycoproteins to the Golgi, protein folding, and virus replication were investigated by mutational analysis and confocal immunofluorescence. Elimination of the glycan on Gn, by changing N60 to a Q residue, resulted in the protein misfolding and failure of both Gn and Gc proteins to traffic to the Golgi complex. We were unable to rescue a viable virus by reverse genetics from a cDNA containing the N60Q mutation. In contrast, mutant Gc proteins lacking glycans on either N624 or N1169, or both sites, were able to target to the Golgi. Gc proteins containing mutations N624Q and N1169Q acquired endo H resistance. Three viable N glycosylation-site-deficient viruses, lacking glycans on one site or both sites on Gc, were created by reverse genetics. The viability of these recombinant viruses and analysis of growth kinetics indicates that the glycans on Gc are not essential for BUN replication, but they do contribute to the efficiency of virus infection.