The Erns Carboxyterminus: Much More Than a Membrane Anchor

Abstract

Pestiviruses express the unique essential envelope protein E^rns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of E^rns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of E^rns raises questions with regard to proteolytic processing of the viral polyprotein at the E^rns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of E^rns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the E^rns membrane anchor.

Keywords: ER retention; RNA virus polyprotein processing; amphipathic helix; charge zipper; extracellular vesicles; membrane anchor; pestivirus; secreted T2 RNase; signal peptidase; transport of virus particles; virus assembly; virus budding.

Figure 2

Processing of the pestivirus glycoprotein precursor. Schematic representation of the precursor with the three pestiviral glycoproteins in their precleavage topology (further details in [57]). The SP cleavage sites are indicated by black dots. For the E^rns/E1 site, the flanking sequences are given below the scheme using the colors of the protein representations. The amino acid sequence is derived from CSFV Alfort/Tübingen.

Figure 1

Features of the E^rns membrane anchor. (A) shows the carboxy-terminal sequence of E^rns from BVDV CP7. Amino acids in bold are those that were found to have no water accessibility in peptides in bicelles. Colored in red is the sequence that was used for the molecular dynamics simulation leading to the membrane anchor model shown in (B). The simulation was done for E^rns residues Arg194 to Ala227 at 72 ns in an explicit DMPC membrane. The protein shows a strong helical fold and lies slightly inclined in the hydrophobic region of the membrane just beneath the lipid head groups (see [16] for further information). Location of N and C terminus are indicated. (C) Percentage of E^rns that is membrane associated. Recombinantly expressed E^rns mutants that were successively shorted by a single amino acid from the carboxy-terminus (see stars that indicate the different stop sites in (A) were analyzed for their membrane association). Asterisks denote significant differences from the wt protein as tested with one-way ANOVA and a Dunnett’s post-hoc test with three stars representing p ≤ 0.001. Already the loss of four amino acids from the carboxy-terminus leads to a significant loss of membrane association.

Figure 3

Scheme presenting the E^rns charge zipper hypothesis. The upper part shows the carboxy-terminal amino acid sequence of E^rns (sequence CSFV Alfort/Tübingen). Charged residues are shown in red (negative charge, indicated also by “-“) or blue (positive charge, indicated also by “+”). Cysteine 171, which is responsible for E^rns homodimerization, is highlighted with a turquoise background. Above the amino acid sequence, a scale with 10-residue calibration starting with glutamic acid at Position 170 is shown. Below the amino acid sequence, the net charges of the residues are given. The designations of the amino acids mutated in the studies presented here are given with the short forms used especially in the figures and the full number as used in the text: D7 = Asp177, D4 = Asp184, E1 = Glu191, R4 = Arg194, R9 = Arg199, and R6 = Arg206. The bottom part shows a scheme summarizing the charge zipper theory proposed to be involved in processing the E^rns-E1 precursor. The charge zipper allows the initial formation of a hairpin structure inserted into the membrane, thereby forming a suitable substrate for SP cleavage. After processing, the E^rns carboxy-terminus is proposed to re-orientate and form an amphipathic helix binding in plane to the membrane surface as experimentally determined for the mature protein [16]. The scheme was based on the original presentation of the hypothesis [78] but modified according to recently published data on the role of E1 for the processing step [19].

Figure 4

Effect of mutations of the conserved inner six charged residues in the E^rns carboxy-terminal helix on E^rns-E1 processing. The upper part shows a schematic presentation of the mutants in the putative charge zipper region of E^rns. Amino acids are as described above (D7 = Asp177, D4 = Asp184, E1 = Glu191, R4 = Arg194, R9 = Arg199, and R6 = Arg206). Red color symbolizes negative charge (also indicated by “-“), and blue color positive charge (also indicated by “+”). The tilted helices shown for mutants 10 ErE1, 11 ErE1, 17 ErE1, 21 ErE1 and 32 ErE1 indicate the different degree of repulsion induced by the charge changes. The bar diagram summarizes the results of immunoprecipitation experiments after expression of E^rns-E1 with the mutations shown above. The numbers of the constructs together with the charge distribution patterns are indicated at the X-axis. The bars represent the percentage of uncleaved E^rns-E1 precursor determined in at least three independent experiments. The calculation principle is described in Materials and Methods section. Error bars are indicated and the p-Values for the results determined for the mutants with respect to the wt E^rns-E1 are indicated by asterisks. Significant differences of the E^rns-E1 processing rate were observed for constructs in 10ErE1 and 11ErE1 (p-Value < 0.0001), 15ErE1 (p-Value = 0.0022), 17ErE1 (p-Value < 0.0001), 18ErE1 (p-Value < 0.0001), 0.21 ErE1 (p-Value < 0.0001), 22ErE1 (p-Value < 0.0001), 23ErE1 (p-Value = 0.0093), and 32ErE1 (p-Value < 0.0001), whereas 30ErE1 gave no significant difference (Ns = not significant). p-Value ranges: * ** = <0.01 and **** < 0.0001.

Figure 5

E^rns is statically retained in the ER. BHK-21 cells were transfected with plasmids coding for different GFP tagged variants of E^rns, namely E^rns of BVDV CP7 (GFP-E^rns CP7), E^rns of BVDV KE9 (GFP-E^rns KE9) and E^rns of BVDV CP7, in which the membrane anchor had been replaced by a hydrophobic sequence (GFP-E^rns-TM CP7). The transfected cells were used in FRAP experiments. Shown are images directly before the bleaching and at the indicated time intervals after bleaching. The bleached areas are magnified in the insets in the corners.

Figure 6

Schematic drawing of the envelope proteins and p7 before (A) and after (B) processing. Before cleavage the membrane regions of all three proteins start and end in the ER-lumen, with the E^rns membrane anchor in the more likely in plane configuration, but slightly tilted, and the other two most probably in a banana-like or hairpin conformation. After cleavage the E^rns membrane anchor stays in plane in the membrane while E2 and E1 adopt single span transmembrane conformations [14,57,103].

Figure 7

Charge exchange mutations in the membrane anchor can increase E^rns secretion. Results of immunoprecipitation experiments from extracts or supernatants of BHK-21 cells transiently expressing E^rns (from construct SE^rns [19]) or mutants thereof with exchanges affecting the charged residues in the E^rns carboxy-terminus. The radioactively labelled proteins were precipitated from the supernatant and cell extract with E^rns specific mab 24/16, treated with PNGase F and separated by SDS-PAGE. The diagram summarizes the results of at least three independent experiments quantified via phosphorimager analysis. The number of the individual construct together with the charge distribution in the E^rns carboxy-terminus are given at the X-axis. The bars represent the amount of secreted protein as percent of total recovered E^rns. For calculation, the counts determined for extra- and intracellular E^rns were set to 100% expression product as basis for calculation of the secretion value. Error bars are indicated as well as the p-Value of E^rns mutants compared to E^rns wt with **** representing p < 0.0001, ** p= 0.002 for 10Er and ns = not significant for 23Er. “-“ indicates a negative, “+” a positive charge.

Figure 8

Effect of a KDEL retrieval signal on the secretion of E^rns and a mutant thereof. (A) Representation of the basic features of the constructs used in the experiments. The upper panel shows the constructs based on the wt sequence of E^rns whereas the lower panel lists the constructs based on mutant 17Er (see also legend to Figure 3 and [80] for further information on the mutant). On the left the names of the constructs are given whereas on the right the charge distribution in the amphipathic helix and the carboxy-terminal sequences of the encoded E^rns proteins specifying the type of the KDEL modification are shown. Changes with regard to the wt sequence are in red. (B) Result of an immunoprecipitation experiment of transiently expressed proteins using E^rns-specific monoclonal antibody 24/16 and separation of precipitates by SDS-PAGE. Proteins were precipitated either from supernatant (S) or from equivalent amounts of lysate of the transfected cells (CL). On top of the gels the expressed constructs are specified. Left part: constructs based on the wt construct SE^rns [19]; right part: constructs based on mutant 17Er. Please note that the secreted proteins exhibit a significantly higher molecular weight due to maturation of carbohydrates in the Golgi apparatus. (C) Diagram summarizing the results of three independent experiments quantified via phosphorimager analysis. The names of the constructs are given at the X-axis. The bars represent the amount of secreted protein as percent of total recovered E^rns. The counts determined for extra- and intracellular E^rns were set to 100% expression product as basis for calculation of the secretion value. Error bars are indicated as well as the p-Value of E^rns mutants compared to E^rns wt without KDEL (black lines and asterisks) and of the mutants containing KDEL compared to mutant 17Er without KDEL (red lines and asterisks) with **** representing p < 0.0001, * p = 0.0109, *** p = 0.0002 and ns = not significant.

Figure 9

Pulse/chase analysis of E^rns secretion. The diagram summarizes the results of pulse chase studies comparing the secretion of wt E^rns (SE^rns) and the super-secretion mutant SE^rnsE1:10 over time. The bars represent the amount of secreted protein as percent of total recovered E^rns. The counts determined for extra- and intracellular E^rns quantified via phosphorimager analysis were set to 100% expression product as basis for calculation of the secretion value. At the X-axis the information on the samples is given with SS standing for steady state (26 h labelling) and for the other samples the time of chase following a 2 h pulse labelling time.

Figure 10

E^rns copurifies with extracellular vesicles. (A) Electron microscopic picture of a typical preparation of extracellular vesicles from the supernatant of cells expressing SE^rnsE1:10. Differences from this picture with regard to number or morphology were not observed when preparations from cells expressing wt E^rns or naive cells or pestivirus infected cells were analyzed. (B) Western Blot with samples produced during EV enrichment. Cultures of non-transfected BHK-21 cells infected with Vaccina virus MVA-T7 (mock), and cells expressing E^rns wt (SE^rns) or the super-secretion variant mutant SE^rnsE1:10 served as starting material. Supernatant of the cultures was subjected to vesicle enrichment via the ExoQuick-TC kit resulting in a vesicle containing pellet fraction (P-EV) and a vesicle-free supernatant (S-EV). For control purposes, lysates of the corresponding cells were analyzed (CL). Antibodies against calnexin (upper panel, marker for cell resident proteins), TSG101 (marker for EVs/exosomes, middle panel) and E^rns (lower panel) were used for Western blot. (C) same as in B but with SK6 cells infected with CSFV Alfort/Tübingen or SK6 mock control.

Figure 11

SK6TO_E^rns cells stably expressing E^rns under control of an inducible promoter secrete E^rns in a vesicle bound form. (A) Electron microscopic pictures of vesicles enriched from the supernatant of SK6TO_E^rns cells with or without doxycycline induction (lower or upper panel, respectively). Bar represents 100 nm. (B) Western Blot with samples produced during EV enrichment. Cultures of SK6TO_E^rns cells minus or plus doxycycline induction served as starting material. Supernatant of the cultures was either loaded without fractionation (total S) or subjected to vesicle enrichment via the ExoQuick-TC kit resulting in vesicle containing pellet fraction (P-EV) and vesicle-free supernatant (S-EV). For control purposes lysates of the corresponding cells were analyzed (CL). Antibodies against E^rns were used for Western blot with samples from the different fractions separated via SDS-PAGE. (C) Similar as in (B) but including a TSG101 control showing that treatment with doxycycline for induction of E^rns expression has no dramatic effect on EV secretion.

Figure 12

Electron microscopic pictures of immunogold-labelled pestivirus particles and enriched vesicles. CSFV particles enriched from the supernatant of infected SK6 cells by centrifugation (A) or vesicles enriched from supernatant of SK6TO_E^rns induced with doxycycline were analyzed by immunogold-labelling using E^rns specific mab 24/16 and gold-labelled secondary antibody. In (B) vesicles with gold grains are highlighted with red arrows. Bars represent 50 nm.

Figure 13

Exchange of charged residues in the E^rns membrane anchor can block production of infectious virus. Immunofluorescence analyses conducted after transfection of CSFV genome-like wt RNA or RNA with the given selected exchanges of one triplet coding for a charged amino acid in the carboxy-terminal region of E^rns. The upper row shows results of analyses conducted ca. 24 h post electroporation and proves that all tested RNAs represent functional replicons. The lower row shows the results obtained after three passages of cell culture supernatant used for infection of fresh cells. Immunofluorescence was done with mab a18 directed against the E2 protein [33]. “-“ represents a negative, “+” a positive charge. Magnification 100×.

Figure 14

Determination of viral RNA levels in cells and cell culture supernatant. (A) Schematic presentation of the method used for propagation of mutant viruses. Step 1: SK6_TO cells were transfected via electroporation and E^rns production (by the cells) was induced with doxycycline. Viral particles were recovered from wt or defective mutant RNAs due to the trans complementation with wt E^rns. Step 2: The recovered infectious viruses were used in single cycle experiments for infection. Viral RNA was harvested from cells and supernatant and then used for quantification of viral RNA displaying the wt sequence or an E^rns deletion mutant or mutants with deleterious exchanges. Viral RNA in the supernatant should result from secretion of viral particles shown here as gray dots. (B) Results of viral RNA quantification via qRT-PCR in lysates (left) or equivalent amounts of supernatant of SK6 cells infected with wt CSFV or stocks of mutants propagated in complementing SK6TO_E^rns cells. Infection was done with an MOI of one. Harvest for total RNA isolation was done separately for cells and supernatant at 4h or 48 h p.i. and the viral genome copy number was determined via real time RT-PCR. The bar diagrams summarize the results of three independent experiments showing the mean log10 values of the viral genome copy numbers with standard deviation indicated.