A structurally unresolved head segment of defined length favors proper measles virus

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1 JVI Accepted Manuscript Posted Online 7 October 2015 J. Virol. doi: /jvi Copyright 2015, American Society for Microbiology. All Rights Reserved A structurally unresolved head segment of defined length favors proper measles virus hemagglutinin tetramerization and efficient membrane fusion triggering Chanakha K Navaratnarajah, Quincy Rosemarie and Roberto Cattaneo* Department of Molecular Medicine, Mayo Clinic, and Virology and Gene Therapy Track, Mayo Graduate School, Rochester, Minnesota * To whom correspondence should be addressed: Roberto Cattaneo, Department of Molecular Medicine, Mayo Clinic, and Virology and Gene Therapy Track, Mayo Graduate School, Rochester, Minnesota 55905; E.mail: cattaneo.roberto@mayo.edu, Running title: A leash for the measles virus hemagglutinin heads (51 characters) 1

2 21 ABSTRACT Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin, H) tetramer and a fusion protein (F) trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or co-crystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport, or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion-trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high molecular weight H-oligomers. Some oligomers had reduced fusion-trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H-tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers. (247/250 words)

3 45 IMPORTANCE Understanding the molecular mechanism of membrane fusion triggering may allow developing new antiviral strategies. The fusion apparatus of Paramyxoviruses consists of a receptor-binding tetramer and a fusion protein trimer. Structural analyses of the receptor-binding hemagglutininneuraminidase of certain Paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H-heads on a leash long enough to adopt a triggering-permissive conformation, but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a leash, suggesting a conserved triggering mechanism. (145/150 words) 3

4 63 INTRODUCTION Although targeted for eradication, measles virus (MeV) still caused 120,000 deaths worldwide in 2014 alone (1, 2). Relaxed vaccination discipline has favored measles re-emergence in Europe and North America, now reporting costly epidemics: in 2013 measles cases in the US were triple than in previous years, in 2014 about 10-fold higher (3-6), and in 2015 a Disneylandoriginated outbreak reminded the world of the immediate benefits of high measles vaccination coverage. Moreover, a recent retrospective study of the consequences of the introduction of measles vaccination 50 years ago indicated that elimination of measles-induced immune suppression significantly reduced children death due to opportunistic infections (7). MeV is a negative strand RNA virus of the family Paramyxoviridae (8), which includes deadly emerging viruses like Hendra and Nipah, and prevalent human pathogens like mumps, parainfluenza, respiratory syncytial, and metapneumo viruses. For cell entry, most Paramyxoviridae use a two component fusion apparatus consisting of a receptor binding protein tetramer and a fusion (F) protein trimer. Those attachment proteins that bind sialic acid and have both hemagglutinin and neuraminidase activity are named HN, while those that bind specific proteins are named G or H (8, 9). Paramyxovirus attachment proteins are type II transmembrane glycoprotein tetramers: four globular heads connect to a tetrameric stalk (10). The 617-amino-acid MeV H protein has a N-terminal 33-residue cytoplasmic tail, followed by a transmembrane segment, a 96-residue stalk, and the globular head domain (11). Atomic structures of the H-stalk are not available, but structures of the HN-stalk alone or in the context of the whole ectodomain have revealed a fourhelix bundle structure with a kink in the central region (12, 13) Five atomic structures of the MeV H globular head have been determined, including those of H-dimers covalently linked by Cys-154 (14), H-monomers (14, 15), and co-structures in complex with three receptors: SLAM (16), nectin-4 (17) and CD46 (18). However, no structure is 4

5 complete: some include a 17-residue gap (residues ; PDB 2ZB5 and 2ZB6), while others start with residues 184 or higher and thus exclude the entire stalk-proximal region (PDB ID: 2RKC, 3ALW, 3ALX, 3ALZ, 3INB and 4GJT). On the other hand, complete HN ectodomain structures have revealed different arrangements of the heads about the stalk: heads-down, in which the head-dimers align sideways of the stalk; heads-up, with all four heads postulated to be atop of the stalk; and the intermediate 2-heads-up/2-heads-down (13, 19). Functional studies of the Paramyxoviridae receptor binding proteins have tested different models about how they initiate the membrane fusion process (20-22). A conserved Paramyxovirus cell entry mechanism, with interesting genus-specific variations, is emerging (9, 10, 23, 24). In particular, it was shown that the MeV H-tetramer can integrate signals from three different receptors that contact the heads through partially overlapping but substantially different binding surfaces (25-28). All these membrane-bound receptors may pull on head dimers, destabilizing them (11). The central segment of the H-stalk then integrates and relays the triggering signals to the F-trimer, which unfolds to fuse membranes (29-33). Knowing that unstructured linkers connect the globular heads of other Paramyxoviruses with their respective stalks (13), we assessed here whether the primary sequence and length of the 17-residue unstructured MeV H-head segment are critical for three functions: protein oligomerization, transport to the cell surface, and the efficiency of membrane fusion triggering. We conclude that the unstructured segment may keep individual heads on a leash long enough to allow sufficient movement about the stalk for them to adopt a triggering-permissive conformation, but short enough to limit improper contacts with heads of neighboring tetramers. 5

6 109 Materials and Methods Cells. Vero (African green monkey kidney) cells and 293T (human embryonic kidney) cells were grown in Dulbecco s modified Eagle s medium (DMEM ; HyClone, South Logan, UT) supplemented with 10% fetal bovine serum (FBS). Chinese hamster ovary (CHO) cells were maintained in RPMI medium (Corning, Manassas, VA) supplemented with 10% FBS and 0.5 mg/ml of nonessential amino acids. Cell lines were incubated at 37 C with 5% CO 2. Plasmids and mutagenesis. All mutations were made in a vaccine-lineage H-protein backbone (H-NSe) (34, 35). Mutations in the H stalk were introduced into the pcg-h expression plasmid by QuikChange site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) with modifications to the manufacturer s instructions as described previously (36). The clones were verified by sequencing the H-protein gene in the vicinity of the mutation. At least two independent clones were tested for each mutation. Cell-to-cell fusion assays. The semi-quantitative cell-to-cell fusion assay was performed as described previously (33). Briefly, 0.5 µg each of three plasmids, encoding the H protein, the F protein, and green florescent protein (GFP), were transfected into 1.0 x 10 5 Vero cells in 24-well plates using Lipofectamine 2000 (Life Technologies, Grand Island, NY) according to the manufacturer s instructions. Twenty-four hours post-transfection the extent of fusion was recorded in one field of view (about 2,000 cells) using the following criteria: 0, two or fewer syncytia with 4 to 5 nuclei (background); 1, three or more syncytia with 4 to 5 nuclei; 2, one to three syncytia with more than 10 nuclei; 3, four or more syncytia with more than 10 nuclei. The quantitative fusion assay was based on a dual-split reporter assay adopted from (37). CHO cells (2x10 5 in 12-well plates) were transfected with 0.3 µg each of the H and F- expression plasmids and one of the dual split reporter luciferase expression plasmids (DSP 1-7, a kind gift of Z. Matsuda). As control, only the H or the F plasmid was co-transfected with DSP 1-7. Vero cells (4x10 5 in 6-well plates) were transfected with 1.5 µg of the other dual split reporter 6

7 plasmid (DSP 8-11 ). Twenty-four hours post transfection, Versene (0.48 mm Ethylenediaminetetraacetic acid [EDTA] in phosphate buffered saline [PBS], Life Technologies) was used to gently detach the transfected CHO and Vero cells. Equal numbers of CHO (5x10 4 ) and Vero cells were mixed and plated on black-walled 96-well plates. EnduRen (Promega) was added as the substrate to the culture media (DMEM, 10% FBS) according to the manufacturer s instructions. Content mixing between CHO and Vero cells as a result of fusion was monitored at the indicated times using a Topcount NXT luminometer (Packard Instrument Company, Meriden, CT). Three replicates were used for each H construct. Immunoblots. To determine H-protein expression levels Vero cells were transfected with plasmid DNA using Lipofectamine 2000 (Life Technologies) according to the manufacturer s instructions. H expression and oligomerization were documented as described previously (32). Briefly, the standard pcg-h (34) or mutated plasmids (2 µg) were transfected into 1.5 x 10 5 Vero cells in 12-well plates. Thirty-six hours posttransfection, cytoplasmic extracts were made in lysis buffer (25 mm Tris HCl ph 7.4, 150 mm NaCl, 1% NP-40, 1 mm EDTA, 5% glycerol) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland) and 50 mm iodoacetamide (Sigma-Aldrich, St. Louis, MO) and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 4-15% gradient) under reducing conditions. To determine H oligomeric states nonreducing conditions were used. The H proteins were visualized on an immunoblot using an H-cytoplasmic tail-specific polyclonal antibody (38) and an HRP-conjugated secondary antibody. FACS analysis. To determine H-protein cell surface expression levels 293T cells (8 x 10 5 in a 6-well plate) were transfected with H-protein expression plasmids (3 µg) as described above. Thirty-six hours posttransfection, cells were washed with PBS and detached by incubating with Versene (Life Technologies) at 37 C for 10 min. The resuspended cells were washed twice with cold fluorescence-activated cell sorter (FACS) wash buffer (1xPBS, 2% FBS, 0.1% sodium 7

8 azide) and then incubated with a blend of two anti-h monoclonal antibodies (1:100 dilution; MAB8905; Millipore, Billerica, MA) for 1 h at 4 C. Cells were washed three times with cold FACS wash buffer and incubated with a phycoerythrin-conjugated secondary antibody (1:100 dilution; ; Jackson ImmunoResearch, West Grove, PA) for 1 h at 4 C. After three washes with FACS wash buffer, cells were fixed in 4% para-formaldehyde and analyzed by a FACSCalibur (BDBiosciences, San Jose,CA) cytometer and FlowJo software (Tree Star Inc., Ashland, OR). Analysis of H-protein oligomers at the cell surface Vero cells (3x10 6 in a 10 cm dish) were transfected with 8 µg of plasmid DNA encoding MV H constructs as indicated. Twenty-four hours post-transfection cells were washed in cold PBS and then incubated with 0.25 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithio- propionate (Pierce) in PBS for 30 min at 4 C, followed by washing and quenching for 10 min at 4 C. Samples were lysed in lysis buffer (25 mm Tris HCl ph 7.4, 150 mm NaCl, 1% NP-40, 1 mm EDTA, 5% glycerol) supplemented with complete protease inhibitor cocktail (Roche) and 50 mm iodoacetamide (Sigma-Aldrich) for 30 min on ice with vortexing every 5 min. Lysates were cleared by centrifugation for 10 min at 10,000 x g and 4 C. Biotinylated proteins were adsorbed to immobilized streptavidin for 2 hours at 4 C, and then washed three times. Samples were boiled in Laemmli buffer for 5 min at 95 C and then subjected to 4-15% gradient SDS-PAGE and immunobloted using an H-cytoplasmic tail specific antibody (38). 8

9 179 Results The unresolved head-stalk connecting linker (residues ) is located between Cys154 that forms an inter-subunit disulfide bond and defines the top of the stalk, and Cys188 that forms a disulfide bridge with Cys606, defining the globular head (Fig. 1A). Sequence alignment of MeV H with that of five other morbilliviruses reveals weak sequence conservation of the stalkproximal residues, while the adjacent stalk sequence is strongly conserved (Fig. 1B). The primary sequence of the structurally unresolved head segment is not critical for fusion triggering. We used an alanine scan mutagenesis approach to assess whether sequence of the unresolved 17-residue linker is critical for fusion triggering. Five mutants with blocks of 3-9 alanine substitutions were generated (Table 1, Alanine substitutions). The fusiontriggering function of these mutants was tested by a semi-quantitative cell-to-cell fusion assay (32) and found to be similar to that of the standard H-protein (Table 1). To determine if altering the sequence interfered with the kinetics of fusion, we monitored fusion over time using a dual split reporter based quantitative fusion assay (37). In this assay, content mixing between an effector cell expressing the MeV fusion complex and a target cell expressing the MeV vaccine lineage receptor CD46, allows the two halves of Renilla luciferase to functionally interact. Indeed two of the larger alanine substitution mutants, Ala_ and Ala_ , exhibited similar levels and kinetics of fusion as the standard H protein (Fig. 2A). This confirms that the primary sequence of the structurally unresolved head segment is not critical for the fusion trigger function. The length of the structurally unresolved head segment is critical for efficient fusion triggering. Next, we deleted blocks of 3-9 residues from different locations in the unresolved head segment (Table 1, Deletions). In addition, we deleted the entire segment (Table 1, Del_ ). While short deletions of 3 or 5 amino acids on either end of the segment had no impact on function (Table 1, Del_ and Del_ ), an intermediate 9

10 deletion of 6 amino acids in the middle of the segment decreased fusion (Table 1, Del_ ). Larger deletions of 9 and 17 amino acids completely abolished fusion function in the semiquantitative fusion assay (Table 1, Del_ and Del_ ). In the more sensitive dual split reporter based quantitative fusion assay, low levels of fusion activity for these deletion mutants were detected: after 10 hours the deletion mutants had 5-10% of the fusion activity observed for the standard H protein (Fig. 2B). Thus, shortening the unresolved head segment interferes with efficient fusion function. To assess whether elongating the unresolved head segment interferes with fusion triggering, we inserted 1, 2, or 4 serine/glycine pairs on its sides (Table 1, Insertions). The functional assays indicate that 2, 4 or 8 residue insertions at the stalk-proximal side (before residue 167) reduced fusion function (Table 1). On the other hand, a four amino acid insertion on the stalk-distal side (after residue 183) had no impact on function (Table 1, Ins_183+2xSG). These results are corroborated by the dual split reporter based quantitative fusion assay (Fig. 2C) which indicate slower fusion kinetics and overall lower levels of fusion for Ins_167+2xSG. Finally, we sought to reverse the negative effect on function of the 9- and 17-residue deletion mutants. We compensated for these deletions in mutants Del_ and Del_ by inserting repeating units of serine and glycine (Table 1, Length compensation). Indeed, both the semi-quantitative (Table 1) and the split luciferase fusion assay (Fig. 2D) indicated full or partial functional compensation for the former and latter mutants, respectively. Thus, the length of the structurally unresolved head segment is critical for efficient fusion triggering. All mutants but one are transported efficiently to the cell surface. To characterize the mechanisms causing loss of function we sought to document expression levels and intracellular transport of the mutants. Figure 3A documents by immunoblot that all the key mutants are expressed at comparable levels to the standard H-protein. Next, we measured the amount of H-protein reaching the plasma membrane. 293T cells expressing the different H- 10

11 mutants or standard H-protein were stained with a blend of two monoclonal antibodies that recognize the H ectodomain and analyzed by FACS. Figure 3B plots the mean fluorescence intensity of different MeV H mutants as a percentage of the standard H-protein at the cell surface. Only deletion mutant Del_ reached the cell surface with less than 5% efficiency of standard H. The remaining mutants were present at the cell surface at levels 45-95% of standard H, which is sufficient for full fusion support function (32, 33). Substitution mutant Ala_ reached the cell surface at ~65% efficiency and exhibited full fusion support function. On the other hand 4 and 8-residue insertions of flexible Ser/Gly pairs at the membrane proximal end of the unresolved head segment reached the cell surface at ~90% efficiency (Fig. 3B, Ins_167+2SG and data not shown), but was impaired in fusion function (Table 1, Ins_167+2SG and Ins_167+4SG). Certain mutants express non-standard H-oligomers. We next sought to determine the oligomeric state of the mutants. Cytoplasmic extracts of cells transfected with H expression plasmids were separated on SDS-PAGE. Standard H-protein exists almost exclusively as a stable disulfide-linked dimer under non-reducing conditions (Fig. 4, H lane). As documented in the immunoblot in Figure 4, all the mutants expressed substantial levels of H-dimer (Fig. 4, D bands). The most striking observation was that stable higher-order oligomers were detected for six of the eleven mutants (Fig. 4, O bands). Oligomers were particularly prominent in deletion mutants Del_ and Del_ ; length compensation mutants Sub_ and Sub_ , where repeating serine and glycine residues were used to restore the length of 9 or 17 amino acid deletions; and insertion mutant, Ins_167+2xSG, where 2 repeating serine and glycine pairs were used to extend the length of the unresolved head-segment. Thus, certain mutations resulted in the generation of stable non-standard higher-order H-oligomers under non-reducing SDS-PAGE conditions. 11

12 Higher order H-oligomers of deletion mutants efficiently reach the cell surface, but fail to trigger fusion. Finally, we focused on two mutants with moderate or strong defects in membrane fusion function, which are the two deletion mutants Del_ and Del_ , respectively. We analyzed their cell surface expression levels by protein biotinylation, and as control we also assessed the cell surface expression levels of standard H and of the fully functional Del_ mutant. Figure 5 (left half) shows that dimeric and oligomeric species of the two functionallyimpaired mutants are transported to the cell surface about as efficiently as the corresponding species of the two functional H proteins. As expected, β-actin was detected only intracellularly (bottom panels, right five lanes). Thus, while higher-order H-oligomers of mutants with larger deletions efficiently reach the cell surface, they fail to trigger fusion. Downloaded from on September 26, 2018 by guest 12

13 266 Discussion We show here that a structurally unresolved 17-residue segment of the MeV H protein head must have a defined length to favor proper H-tetramerization and promote efficient membrane fusion triggering. We propose that during H-tetramer maturation this segment would act as a leash long enough to allow for the H-heads to adopt a triggering-permissive conformation, but short enough to limit their roaming and improper contacts with heads of neighboring tetramers. Genus-specific variations of the conserved Paramyxovirus membrane fusion mechanism are currently being characterized (9, 10, 23, 24). The Paramyxovirus head re-positioning model was suggested based on structural analyses of entire HN-protein ectodomains, which crystallized in distinct conformations about the stalk: 4-heads-down, 4-heads-up, and 2-headsup/2-heads-down (12, 13, 19). While in all these conformations the globular heads behave like rigid bodies maintaining similar structures, they might take advantage of flexible linkers to relocate from a metastable down to the up position, a movement that may trigger membrane fusion. As to the H-proteins of MeV and the other morbilliviruses, atomic structures of neither their entire ectodomain, nor their isolated stalks are available. However, others and we have obtained data suggesting that morbilliviruses developed a variation of the head re-positioning model accounting for intracellular H-stalk to F-trimer complex formation without premature membrane fusion (24, 39). Namely, it was proposed (24) that in the down position the H-heads contact an additional tetrameric spacer segment (32) located above the signal-transmitting central stalk segment, rather than contacting the central stalk segment directly as the HN-heads (13). This arrangement provides room for morbillivirus F-trimers to interact laterally with H- tetramers already during intracellular transport, as it is known to happen (40). A third variation on this theme may be played by the G-proteins of henipaviruses, which stalks also interact laterally with the respective F-protein (9, 23). 13

14 Here, we observed that excessive shortening of the unresolved H-head segment impacts on proper H oligomerization and efficient fusion triggering. Specifically, deletion of 6 residues or more resulted in the accumulation of stable higher-order H-oligomers under non-reducing SDS-PAGE conditions. In contrast, the standard H-protein presents exclusively as a dimer under these conditions. Similarly, smaller deletions of 3 or 5 residues ran exclusively as dimers and maintained fusion function. Length compensation with Ser/Gly repeats fully restored the fusion-triggering function of a 9-residue deletion, and partially restored the function of the entire 17-residue deletion. Thus, in combination with the dimeric top segments of the H-stalk (32, 33), the unresolved H-head segment may act as a leash, which must be long enough for the heads to move about the stalk and adopt the metastable heads-down conformation. Interestingly, mutants with added Ser/Gly repeats at the N-terminal side of the 17- residue segment, as well as Ser/Gly compensation mutants, also accumulated stable higherorder H-oligomers. In contrast, none of the alanine substitution mutants generated higher-order oligomers. The absence of a β-carbon in Gly makes Gly-rich sequences more flexible than sequences of similar length without Gly (41). It was previously observed that very short, or very long linkers promote cross-folding between subunits of oligomeric proteins, which can interfere with function (41, 42). Analogously, we propose here that H-heads with longer, or more flexible linkers have high propensity to roam, contact the heads of other tetramers, and cross-link. We note that, lacking a mutant with alanine substitution of the entire segment, we cannot discount some sequence dependence on proper H-oligomerization. However, blocks of 3 amino acid deletions across the entire length of the segment did no affect function (data not shown), further suggesting that sequence is not critical Finally, the presence of higher-order H-oligomers is not diagnostic of fusion function. Two large deletions and a 4 amino acid flexible insert at the beginning of the 17-residue 14

15 segment resulted in an accumulation of stable higher-order H-oligomers and impacted fusion function. On the other hand, restoring the length of the segment with a flexible Ser/Gly insert also resulted in the accumulation of stable higher-order oligomers, while maintaining function. This may be a reflection of the relative ratio of standard to non-standard H-oligomers generated by the different mutations. Mutations that allow the formation of sufficient amounts of the standard H-tetramer may retain function. It is also likely that different types of higher-order H- oligomers are generated. However, at this point we cannot discriminate between these H- oligomer types. How the structurally unresolved H-head segment favors proper tetramerization and membrane fusion triggering is not fully understood. However, residues are part of a longer segment beginning with Cys154 and ending with Cys188. Cys154 forms a disulfide bridge with Cys154 of another H-monomer within an H-tetramer subunit, defining the top of the stalk dimeric linker. Cys188 forms a long-range disulfide bridge with Cys606 of the same subunit, stabilizing the six-blade propeller globular head (14). Thus, it is possible that the entire H-head segment rearranges during membrane fusion triggering. Indeed, we previously showed that crosslinking of H-dimers through the introduction of Cys at position 161 reversibly blocks H-heads movement and fusion triggering (11). Since sequence alignment of morbilliviruses indicates that residues are poorly conserved (Fig. 1B, conservation level), we suggest that these residues, in combination with the dimeric top segments of the stalk, may act as leashes for the heads of all morbilliviruses. Acknowledgments We thank Z. Matsuda for the kind gift of the dual split reporter plasmids. We thank Patricia Devaux, Mathieu Mateo and Christian Pfaller for helpful discussions; Marie Frenzke for 15

16 technical support and Surendra Negi for help with structure alignment. Q.R. was supported in part by the Mayo Graduate School Summer Undergraduate Research Fellows program. 16

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20 Table 1: Sequence and fusion function of unresolved head-segment mutants Class Name Fusion score a H VNSTLLETRTTNQFLAV +++ Alanine Ala_ AAA substitutions Ala_ AAAAAA Ala_ AAAAAAAAA Ala_ AAAAAA Ala_ AAAAAAAA +++ Deletions Del_ ΔΔΔ Del_ ΔΔΔΔΔ +++ Del_ ΔΔΔΔΔΔ Del_ ΔΔΔΔΔΔΔΔΔ Del_ ΔΔΔΔΔΔΔΔΔΔΔΔΔΔΔΔΔ 0 Insertions Ins_167+SG SG Ins_167+2xSG SGSG Ins_167+4xSG SGSGSGSG Ins_183+2xSG SGSG +++ Length Sub_ SGSGSGSGS compensation Sub_ SGSGSGSGSGSGSGSGS ++ a Fusion levels were determined and scored by a cell-to-cell fusion assay as described in the Materials and Methods. +++ indicates full fusion function; ++ and + indicate lower levels of fusion; and 0 indicates no fusion. 20

21 Figure 1: Schematic of MeV H protein, sequence alignment of morbillivirus H stalks and stalkproximal head segment and structure of a tetrameric stalk with a head dimer. (A) H linear structure. From left to right: C, cytoplasmic tail; T, transmembrane region; S, stalk; and β1 to 6, beta sheets 1 through 6. The two Cys residues that cross-link the H dimer (Cys139 and Cys154) and the two Cys residues that stabilize the H-head (Cys188 and Cys606) are shown as gray lines. The 17-residue structurally unresolved head-segment is shown in red. (B) Sequence alignment of the MeV H stalk and unresolved head-segment to five other morbilliviruses. The alignment was made using Clustal Omega (43). Canine, canine distemper virus; Phocine, phocine distemper virus; Porpoise, porpoise morbillivirus; PPRV, peste-des-petits-ruminants virus; Rinderpest, rinderpest virus; Measles, measles virus. (Top) Shades of blue represent the degree to which the identity of an amino acid is conserved at a given position. (Bottom) The conservation level of each residue is indicated by the height of the bars below each residue and the associated score below the bar (0 to 9). (C) Model of the tetrameric MeV stalk with a head dimer in the down position. The 2-heads-up/2-heads-down HN crystal structure was used as template to align the H dimer crystal structure (PDB ID: 2ZB5) in the down position relative to the stalk. Residues of the two H-monomers are shaded differently (green and cyan) for clarity. Residues are shaded grey in both monomers. The structurally unresolved segment (residues ), which constitutes a 17-residue gap in the H-head dimer crystal structure, is visualized as a red ribbon on the green subunit. These residues are not visible in the cyan subunit as they are on the opposite face of the monomer. The two residues that flank the gap are shaded yellow. Cys 154 at the N-terminal end of the H-monomers are shaded magenta for the cyan subunit and orange for the green subunit. The stalk is illustrated with the aid of a structural model and schematics for regions which lack a structural template. Red: central fusion activation segment (residues ). Green: spacer segment (residues ). Blue: dimeric linker (residues ). 21

22 Figure 2: Fusion kinetics of unresolved head-segment mutants. Cell-to-cell fusion was monitored over a 20-hour period for: (A) two alanine scan mutants, (B) two deletion mutants, (C) two insertion mutants and (D) two length compensation mutants. The names of the mutants and symbols used to represent them are indicated in the insets. As negative controls, cells transfected with only one or the other standard glycoprotein was monitored. Only one control (Fonly) is shown in the figure for clarity. Similar results were obtained for the H-only control. Figure 3: Protein expression levels of unresolved head-segment mutants. (A) Total protein expression. Cytoplasmic extracts of Vero cells transfected with the H expression plasmids indicated below the gel were separated on a 4-15% SDS-PAGE under reducing conditions and immunoblotted with an anti-h (cytoplasmic tail) antibody. M: H-monomer band. A; β-actin was used as a loading control. (B) Cell surface protein expression. 293T cells transfected with the indicated H construct were stained with a blend of two anti-h ectodomain antibodies and phycoerythrin-conjugated secondary antibody. The mean fluorescence intensity of each mutant is presented as a percentage of the standard H-protein levels. Error bars indicate standard deviations of three experiments. Figure 4: Oligomeric state of the mutants. (A) Cytoplasmic extracts of Vero cells transfected with the indicated H expression plasmid were separated on a 4-15% SDS-PAGE under nonreducing conditions and immunoblotted with an anti-h (cytoplasmic tail) antibody. The different H forms are indicated. O, stable higher-order H-oligomers; D, dimers; and M, monomers. A; β- actin was used as a loading control. 22

23 Figure 5: Higher order H-oligomers of two deletion mutants are efficiently transported to the cell surface. Vero cells transfected with the indicated expression plasmids were lysed 24-hours post-transfection. Biotin-labeled surface proteins (left) or total cell protein extracts (right) were separated on non-reducing SDS-PAGE and subjected to anti-h-protein immunblot (top gel). The same extracts were then blotted for β-actin (bottom gel). Molecular weight markers (kda) are indicated on the left side. O, stable higher-order H-oligomers; D, dimers; M, monomers; A, β- actin. Downloaded from on September 26, 2018 by guest 23

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