Characterization of the Golgi retention motif of Rift Valley fever virus G(N) glycoprotein.

Rift Valley fever (RVF) virus is a member of the genus Phlebovirus within the family Bunyaviridae. RVF virus is endemic to much of sub-Saharan Africa; however, regions affected by this pathogen are expanding as a result of introductions of the virus from sub-Saharan Africa into neighboring regions, such as Egypt and the Arabian Peninsula (3-5, 22). RVF virus causes severe disease in livestock and humans in a cycle that is linked with periods of unusually high rainfall (17). Heavy rainfall creates pools of standing water, or “dambos,” that provide breeding habitat for the mosquito vectors of RVF virus (18). Although the RVF virus natural infectious cycle has been linked to the Aedes sp. mosquito, many mosquito species can be infected and subsequently transmit RVF virus (9, 10, 16, 21). The fact that many mosquito species can act as vectors increases the likelihood of RVF virus becoming endemic to areas outside of its traditional range. RVF virus, like all viruses of the family Bunyaviridae, has a tripartite negative-stranded RNA genome (28). Most, if not all, members of the family Bunyaviridae mature by budding into the lumen of the Golgi (27). Localization of the envelope glycoproteins is a crucial step in the virus maturation process. The two RVF virus envelope glycoproteins localize to the Golgi apparatus in the absence of other virally encoded proteins (31), and thus one or both of these glycoproteins contains a Golgi localization signal. Following Golgi localization, the remaining structural proteins and the genome are recruited, followed by viral budding. Members of the family Bunyaviridae lack matrix proteins; therefore, the glycoproteins (presumably the cytosolic tail region of one of the glycoproteins) are responsible for the recruitment of the remaining core elements of the virion. Mature virions are then released from the cell through fusion of virion-filled Golgi elements with the plasma membrane (27). The envelope glycoproteins of Uukuniemi and Punta Toro viruses, both belonging to the genus Phlebovirus, have been characterized with respect to their subcellular localization (1, 19, 20, 23, 29). Both the carboxy-terminal glycoprotein (GC) and the amino-terminal glycoprotein (GN) localize to the Golgi apparatus when expressed together as a polyprotein precursor. However, GC does not localize to the Golgi apparatus when expressed in the absence of GN (6, 23); it instead localizes to the endoplasmic reticulum (ER). The GC of all members of the genus Phlebovirus contain lysine-based ER retrieval signals at their extreme carboxy terminus. Therefore, GC is thought to attain Golgi localization through physical interaction with GN. Both viruses have Golgi localization signals in their GN. However, the location of the signal within the GN was not consistent between Uukuniemi and Punta Toro viruses (1, 2, 19). The Golgi localization signal for the Uukuniemi virus GN is contained exclusively within the cytosolic tail and the signal for the Punta Toro virus GN is contained within a region encompassing the transmembrane domain and 10 amino acids of the cytosolic tail. However, there is little amino acid sequence conservation in the carboxy-terminal regions of the GN of members of the genus Phlebovirus (Fig. ​(Fig.1A).1A). It is therefore plausible that within this genus more than one strategy is employed for envelope glycoprotein localization. FIG. 1. (A) ClustalW alignment of the carboxy-terminal region of the GN of viruses belonging to the genus Phlebovirus. The alignment begins 10 amino acids upstream of the transmembrane domain and concludes just prior to the signal sequence for GC. Abbreviations: ... Golgi localization of cellular proteins is thought to involve retrieval and/or retention processes (12, 24, 26). Retrieval of resident Golgi proteins from distal compartments is thought to rely in large part on signals contained within the sequence of the cytosolic tail of the escaped Golgi protein (24, 26). These sequences within the cytosolic tail of resident trans-Golgi network (TGN) proteins, such as those found in furin and TGN-38, recruit adapter proteins that in turn allow for packaging into clathrin-coated vesicles destined for the Golgi apparatus (11). Retention refers to the process by which resident Golgi proteins are prevented from moving into distal compartments. Retention signals are more diverse and can be contained within transmembrane domains, lumenal domains and multimerization domains (24). Unlike the retrieval motifs present in cytosolic tails of TGN proteins, there is no sequence homology among retention signals; these signals may instead be conformational in character. Viruses, such as RVF, which mature in the Golgi apparatus, presumably take advantage of the existing cellular machinery in order to localize their envelope glycoproteins properly. Thus, it is likely that retrieval and/or retention signals present in the RVF virus glycoproteins show similarity, either structural or amino acid sequence, to cellular resident Golgi proteins. Primary to the understanding of RVF virus maturation is a characterization of the mechanism by which the structural elements of the virion are localized to the Golgi apparatus. We have begun a characterization of RVF virus maturation by identifying GN as containing a Golgi localization signal. Furthermore, we have mapped the Golgi localization signal to a 47-amino-acid region that includes the transmembrane domain and 28 amino acids of the cytosolic tail. This region of GN is sufficient to localize a GFP chimera to the Golgi apparatus. Contrary to expectations, deletion of either hydrophobic region within the Golgi localization signal does not lead to secretion to the plasma membrane, but rather to retention in the ER.