A new type of intracellular retention signal identified in a pestivirus structural glycoprotein

Abstract

Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.

The Molecular Biology of Pestiviruses

“…Most of the protein, even when expressed alone, is membrane bound and retained in the cell (Burrack, Aberle, Burck, Ulrich, & Meyers, 2012;Grummer, Beer, Liebler-Tenorio, & Greiser-Wilke, 2001;Tews & Meyers, 2007), but a significant percentage can also be found in the supernatant of infected cells and in the blood of infected animals (Burrack et al, 2012;Magkouras, Mätzener, R€ umenapf, Peterhans, & Schweizer, 2008;R€ umenapf et al, 1993;Tews & Meyers, 2007;Weiland, Weiland, Unger, Saalm€ uller, & Thiel, 1999;Weiland et al, 1992). Intracellular E rns shows a predominantly ER-centered localization.…”

“…Retention depends on the C-terminus of the protein, most notably on three bulky hydrophobic amino acids (L138, I190, L208). These amino acids are not completely conserved among all pestiviruses, but the different E rns proteins also show different levels of retention in the cell (Burrack et al, 2012).…”

“…Intracellular budding has long been proposed for pestiviruses. All three envelope glycoproteins are retained in the cells (Burrack et al, 2012;Grummer et al, 2001;K€ ohl et al, 2004;C. Radtke & B.A.…”

“…Tews, unpublished data), and biochemical data indicate that virus particles are formed intracellularly. Maturation of the carbohydrate chains of the envelope proteins occurs during transport of the particles along the secretory pathway to the cell surface (Burrack et al, 2012;Grummer et al, 2001;Jordan, Nikolaeva, et al, 2002;K€ ohl et al, 2004;Macovei, Zitzmann, Lazar, Dwek, & Branza-Nichita, 2006). Recent ultrastructural analyses on the pestivirus Giraffe-1 revealed the presence of particles containing the core protein or the E2 protein in the ER.…”

Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation

“…The amphipathic helix is important in anchorage of the protein to the viral envelope and was recently shown to possess an intracellular retention signal (Burrack et al, 2012). Change of either of the latter 2 amino acid residues to basic residues introduced a charge change into the polar face of the helix by replacing non-charged amino acids.…”