The Cytoplasmic Tails of Uukuniemi Virus (Bunyaviridae) GN and GC Glycoproteins Are Important for Intracellular Targeting and the Budding of Virus-Like Particles

Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Box 240, SE-17177 Stockholm, Sweden.
Journal of Virology (Impact Factor: 4.44). 11/2007; 81(20):11381-91. DOI: 10.1128/JVI.00767-07
Source: PubMed


Functional motifs within the cytoplasmic tails of the two glycoproteins G(N) and G(C) of Uukuniemi virus (UUK) (Bunyaviridae family) were identified with the help of our recently developed virus-like particle (VLP) system for UUK virus (A. K. Overby, V. Popov, E. P. Neve, and R. F. Pettersson, J. Virol. 80:10428-10435, 2006). We previously reported that information necessary for the packaging of ribonucleoproteins into VLPs is located within the G(N) cytoplasmic tail (A. K. Overby, R. F. Pettersson, and E. P. Neve, J. Virol. 81:3198-3205, 2007). The G(N) glycoprotein cytoplasmic tail specifically interacts with the ribonucleoproteins and is critical for genome packaging. In addition, two other regions in the G(N) cytoplasmic tail, encompassing residues 21 to 25 and 46 to 50, were shown to be important for particle generation and release. By the introduction of point mutations within these two regions, we demonstrate that leucines at positions 23 and 24 are crucial for the initiation of VLP budding, while leucine 46, glutamate 47, and leucine 50 are important for efficient exit from the endoplasmic reticulum and subsequent transport to the Golgi complex. We found that budding and particle generation are highly dependent on the intracellular localization of both glycoproteins. The short cytoplasmic tail of UUK G(C) contains a lysine at position -3 from the C terminus that is highly conserved among members of the Phlebovirus, Hantavirus, and Orthobunyavirus genera. Mutating this single amino acid residue in G(C) resulted in the mislocalization of not only G(C) but also G(N) to the plasma membrane, and VLP generation was compromised in cells expressing this mutant. Together, these results demonstrate that the cytoplasmic tails of both G(N) and G(C) contain specific information necessary for efficient virus particle generation.

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    • "Such alterations are expected to greatly affect particle budding. The fact that mutating the K2 or K3 residues of the cytosolic tail of Gc from RVFV led to a mislocalization of Gn at the cell surface corroborates previous observations related to the Gc- K3A mutation in the UUKV-VLP model (Overby et al., 2007b). This supports an additional role of the cytosolic tail of Gc not only for ER retention, but also for the retention of the Gn/Gc heterodimers in the Golgi and/or the process of heterodimerization. "
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    ABSTRACT: The correct folding, heterodimerization and trafficking of Gn/Gc envelope glycoproteins of Rift Valley fever virus, RVFV (Bunyaviridae and Phlebovirus genus) are essential for Golgi assembly and budding of viral particles. The Gn and Gc carboxy-terminus contain a Golgi targeting and an ER-retrieval signal, respectively. We generated RVFV-like particles with mutations in the cytosolic tails of Gn or Gc and identified regions important for release of infectious particles. The role of specific amino-acids in these regions was further investigated by creating recombinant mutant viruses by reverse-genetics. Residues outside the suspected Golgi targeting motif, i.e. the di-lysine K29-K30 motif and the N43, R44 and I46 residues of the Gn cytosolic domain, appeared important for Golgi localization and RNP packaging. Concerning the Gc tail, replacement of K2 or K3 in the di-lysine motif, had a drastic impact on Gn trafficking and induced an important organelle redistribution and cell remodeling, greatly affecting particle formation and release.
    Virology 01/2014; 448C:1-14. DOI:10.1016/j.virol.2013.09.023 · 3.32 Impact Factor
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    • "The receptorbinding surface glycoproteins Gn and Gc, coded by the M segment, play crucial roles in virus assembly and budding through interaction with each other and possibly with host proteins that mediate their passage through the endoplasmic reticulum and the Golgi apparatus, and also by interacting with the nucleocapsid proteinwrapped RNPs. These functions are primarily mediated by the short cytoplasmic tail regions of these proteins (Shi et al., 2007; Överby et al., 2007). Interactions of the also M segment-coded non-structural protein (NSm) are not known and no function is assigned to this product as of yet. "
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    • "Secondly, studies with UUKV led to the identification of a dileucine motif in Gn-CT (Fig. 2) that is responsible for the budding of VLPs. Mutation of this motif to alanines abolished the budding of UUKV VLPs into GC while retaining the proper localization of glycoproteins to the GC membranes (Overby et al., 2007b). Other bunyaviruses are unlikely to share the same budding mechanism, since the expression of UUKV and RVFV glycoproteins in the absence of N or L proteins allow the generation of VLPs (Overby et al., 2006; Piper et al., 2011). "
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