many use trimeric fusion proteins that share structural characteristics with cellular proteins of similar function 1 .
since refolding of most viral fusion proteins is irreversible , triggering must be strictly regulated .
while many enveloped viruses , including influenza- , rhabdo- , alpha- and flaviviruses , take advantage of low ph in the endosomal compartment to trigger fusion 2 , the filovirus ebola uses proteases 3 .
other enveloped viruses , including measles virus ( mv ) and hiv fuse directly with the plasma membrane at neutral ph 1,4 .
mv , while being targeted for eradication , still causes more than 150,000 deaths yearly 5,6 .
mv is a member of the paramyxoviridae 
 4,7 , a family including deadly emerging viruses like hendra and nipah , and prevalent human viruses like mumps , parainfluenza , and respiratory syncytial viruses .
moreover , the mv entry system has become a paradigm for targeting oncolysis and therapeutic gene delivery 810 because it can be triggered by cell surface proteins differing in size , trans - membrane organization , and quaternary structure 11 . since the mechanism triggering mv cell entry is not understood , we set out to characterize it
the attachment protein mediates receptor binding and triggers a refolding event of the metastable fusion ( f ) protein that results in membrane fusion 4,7 .
the attachment proteins are named hemagglutinin ( h ) for mv and the other members of the genus morbilliviruses , glycoprotein ( g ) for the henipaviruses , and hemagglutinin - neuraminidase ( hn ) for most other genera .
while the h and g proteins bind proteinaceous receptors , hn proteins bind sialic acid 12 .
mv h is a type ii transmembrane glycoprotein comprised of an amino - terminal cytoplasmic tail , a membrane - spanning segment , and an extracellular membrane - proximal stalk region connected to a large cuboidal head with six - blade beta - propeller structure contacting the receptors ( figure 1a - c ) .
mv h proteins form dimers stabilized by two inter - subunit disulfide bonds 13 ( cys139 and cys154 , figure 1a , d ) located at the top of the long helical stalk 14 , below the base of the cuboidal heads .
it was recently shown that h - tetramers or higher oligomeric forms sustain fusion - support function 15 .
specific interactions of the attachment and f proteins of paramyxoviridae are required for membrane fusion because heterotypic glycoprotein pairs can not mediate this process 16,17 .
in particular , the stalk regions of different hn proteins determine the specificity for the cognate f proteins 4,7 .
similarly , the mv h - stalk region interacts directly with the f protein head 18 , an observation suggesting that the h - oligomer is much taller than the f - trimer ( figure 1d ) .
these f - h interactions are relevant to late phases of the fusion triggering process .
novel information revealing structural diversity of the attachment proteins of paramyxoviridae prompted us to analyze early triggering phases .
in particular , the interface of the h - dimer 19 was found to be much smaller than that of hn - dimers 2022 ( 1070  versus 1800  in a direct comparison 23 ) , and the h protein head crystallized as a monomer when cys154 was omitted 24 .
moreover , while the h - heads lean about 40 away from the symmetry axis , and tilt sideways slightly ( schematics in figure 1c ; see also figure 1d , left ) 19 , the hn - heads are more upright 2022 .
the biology of these attachment proteins also differs : while h hetero - oligomerizes with f in the endoplasmic reticulum 25 , hn and f travel separately to the cell surface 26 . as to fusion triggering , the small interface area of the h - dimer suggested to us that the heads might move relative to each other : figure 1c illustrates a re - alignment movement , but adjustment or rotation movements are also possible .
movement hypothesis by adding covalent cross - links that stabilize the dimer in a single conformation .
we also noted that the footprints of the three mv receptors 2735 are located distal to the dimer interface , such that two bound receptors per dimer would have enough leverage to force the heads to move .
twist hypothesis by directing a membrane - anchored receptor to bind tags inserted in strategic locations on the h - head .
indeed , only binding close to the locations used by the natural receptors , and distal to the h - dimer interface , efficiently triggers membrane fusion .
to test the  movement hypothesis we sought to stabilize the h - dimer interface by adding inter - subunit disulfide bonds .
we note that the heads have a solid beta - propeller structure , and may thus move as rigid bodies .
moreover , cd46 binding does not induce substantial conformational changes of the h - head 36 , consistent with a rigid structure suited to conduct torsion .
we identified amino acid pairs across the interface whose c - alpha atoms are less than 7.5  apart ( table 1 ) and mutated these to cysteine .
we sought to introduce two classes of disulfide bonds : single bonds located on the two - fold symmetry axis of the dimer and involving the same residue on both molecules ( table 1 , top four lines ) , and twin bonds located on both sides of the axis and involving two different residues ( table 1 , bottom four lines ) .
twin cysteine bonds , forming a triangle with cys154 , would provide more resistance to a force , increasing the energy barrier for fusion triggering ; larger triangles would provide more resistance than smaller triangles .
figure 2a shows the location of the two key single bonds ( t273c  t273c , blue and w327c  w327c , green ) and the two key twin bonds ( k236c  g264c , brown / red and p330c  l161c , orange / yellow ) in a side view of the dimer interface .
the dimerization capacity of the mutated h - proteins was then assessed ( table 1 and figure 2c ) .
we note that the cysteine substitutions are distal from known receptor binding epitopes , but to control for the ability of each position to tolerate mutations we also made alanine substitutions , and assayed fusion triggering by the alanine mutants .
where twin bonds were involved , we ensured that neither one of the cysteine substitutions in the pair had an effect on fusion function .
as listed in table 1 ( fifth column ) , only one of the alanine mutants , l203a was fusion deficient , and this residue was eliminated from further consideration .
the fact that all the other alanine mutations were fusion - competent implies that the corresponding mutant proteins have intact receptor binding function . since total expression levels of certain mutants were reduced ( figure 2c ) , we sought to measure their cell surface expression .
we measured the mean fluorescence intensity ( mfi ) by facs analysis for each mutant ( supplementary table 1 ) , which was 4768% as that of the standard h - nse protein .
even with this reduction in cell surface protein levels , we note the mv fusion system is very efficient , and that decreased h surface expression may not necessarily translate in decreased fusion 13 .
we also probed the mutants with a conformation - specific monoclonal antibody that recognizes the h - noose epitope 37 .
the mfi measured with this antibody was similar to that recorded with the anti - h cocktail ( supplementary table 1 ) , indicating that the mutants had similar conformations to the unmodified h - protein .
we then assessed whether covalent bonds are formed , taking advantage of an h protein backbone with mutations in both cysteine residues ( cys139 and cys154 ) that normally mediate covalent inter - subunit cross - linking . while this mutant forms minimal amounts of stable dimers , given time it does support low levels of membrane fusion 13 .
c154a mutant loses the ability to form detectable amounts of stable h - dimers , as assayed by non - reducing sds - page .
c154a background the only inter - subunit bridges are those formed at positions with added cysteines .
figure 2d ( lanes 14 ) show that cysteine substitutions at 236 + 264 , 273 , 327 or 330 + 161 restore the dimerization capacity of the h - protein , demonstrating that these substitutions allow inter - subunit disulfide bonds under physiological conditions .
c154a ( lanes 14 ) or the normal h - backbone ( lanes 58 ) , indicated that the cysteine substitution mutants alone are less efficient at forming dimers .
this is not surprising as the introduced cysteines may not promote covalent linkage as efficiently as the two stalk cysteines .
this analysis also revealed that k328c and k236c+l265c did not yield cross - linked dimers , and these residues were eliminated from further consideration ( table 1 , seventh column ) .
moreover , p330c+e162c dimerized but was not pursued further because it is similar to p330c+l161c . thus ,
analysis focused on two single - bond and two twin - bond cysteine mutants . after confirming disulfide bond formation , h - protein mutants were co - expressed with f - protein to test the hypothesis that movement of the subunits is required to allow membrane fusion .
figure 3a shows that the most distal twin bonds ( k236c+g264c , 32 from fusion pivot , 533  stabilized area ) abrogated fusion .
on the other hand , the two single bonds ( 22 and 16 from the pivot ) and the pivot proximal twin bond ( 10 from pivot , 68  stabilized area ) had little effect on fusion , in this assay performed 24 hours after transfection .
this analysis was repeated using a luciferase - based quantitative fusion assay that provided additional 12- and 18-hour time points . at 12-hours
the positive control showed high fusion levels , while all the mutants had fusion levels at or near background ( figure 3b ) . at 18-hours the fusion functions of the t273c , w327c and p330c  l161c mutants
the fusion function of the k236c+g264c mutant was reduced by a factor of 13 compared to the wild type protein , while fusion efficiency of the other three mutants was comparable to wild type h , which syncytia had already begun to lyse .
these results indicate that the single bonds at 273 and 327 , and the twin bond at 330 + 161 interfere with fusion temporarily . however , given time fusion is observed , possibly because occasionally disulfide bonds are reduced .
we then assessed whether eliminating the disulfide bonds with a reducing agent restores fusion function .
cells expressing the k236c+g264c mutations were treated with 1,4-dithio - dl - threitol ( dtt ) . in the presence of dtt ,
fusion function was restored in the k236c+g264c h mutant that previously exhibited no fusion ( figure 3c , compare lower left and right panels ) .
similar results were observed for the t273c and w327c mutants when treated with dtt 12-hours post - transfection ( data not shown ) , indicating that this protein is available in sufficient quantities and retains the capacity to interact with receptors .
we next assessed whether the efficiency of fusion triggering depends on interface - distal receptor binding on the h - dimer .
we took a systematic approach and inserted a short hexahistidine tag in different positions at the top half of the h - dimer .
the hexahistidine tag directs binding to vero - his cells which express a membrane - anchored single - chain antibody that recognizes the six - histidine peptide38 .
the insertion points were chosen taking into consideration structural constraints , including n - linked oligosaccharide chains at n168 , n187 , n200 and n215 , and availability of surface accessible loops ( figure 4a , b ) .
the tags were inserted in eight exposed loops ( figure 4a , b , yellow residues ) .
we used mutant 9 with a carboxy - terminal tag as the positive control 38 .
mutants 5 and 6 are located on the top of the h - dimer , between the h - residues in close contact with cd46 , as identified based on the h - cd46 co - crystal 36 ( highlighted in light blue ) and those identified by functional assays to be relevant for slam - dependent fusion 2729 ( highlighted in red ) .
mutants 1 , 3 , 7 and 8 are all located near the top of the h - dimer . while mutant 1 is close to the cd46 footprint , and mutant 7 close to the slam footprint , mutants 3 and 8 are located away from both receptor binding areas .
mutant 2 is also located away from these binding areas and close to the interface .
mutant 4 is the only one in the bottom half of the dimer . to identify functional mutants , dimerization capacity , expression levels , and accessibility of the his - tag
protein extracts from transfected vero cells were separated on sds - page under non - reducing conditions , and immunoblotted . since mutants 2 and 4 dimerized poorly ( figure 4c ) , they were excluded from further analysis .
surface expression levels of the remaining mutants , measured by facs , were at 3999% of standard h - nse ( supplementary table 2 , third column ) , which was considered acceptable for all mutants .
to determine the accessibility of the his - tag we compared the mfi generated by an anti - his antibody to that generated by an anti - h cocktail ( supplementary table 2 , last column ) .
all but one mutant had mfi ratios close to 1 , indicating that their his - tags were equivalently accessible .
since mutant 3 had a lower ( 0.65 ) mfi ratio , it was not considered when drawing conclusions .
thus , hexahistidine tagging of exposed protein loops is compatible with efficient protein folding and dimerization , and allows display of accessible tags in a majority of the constructs .
we then measured the fusion efficiency of the dimerization - competent mutants and the control mutant , 9 , in cell lines expressing either the anti - hexahistidine antibody as the designated receptor , or the natural receptor slam , or the vaccine strain receptor , cd46 .
since none of the hexahistidine tags were predicted to interfere with cd46-binding , these fusion assays were considered positive controls .
on the other hand , slam - dependent fusion assays were expected to further define the slam footprint .
finally , hexahistidine receptor - dependent fusion assays were expected to reveal the h - dimer surface area poised for fusion triggering . for consistency in host cell type , we used vero cells , or vero cells expressing anti - hexahistidine ( vero - his ) , or slam ( vero - slam ) . to focus exclusively on entry through one receptor
, we generated a second set of tag mutants in an h - protein backbone exclusively recognizing slam ( h - wt , figure 5a , first and second column ) .
the h - nse vaccine lineage backbone was used only to measure cd46-dependent fusion ( h - nse , figure 5a , third column ) .
this analysis indicated that , with the exception of mutants 7 and 8 , which were reduced in cd46-specific fusion ( fusion scores of 2 and 1 , respectively ) , all the mutants fully retained cd46-dependent fusion function .
analysis of slam - dependent fusion revealed that the h - slam contact area may extend to the region defined by mutants 5 and 6 , because these two mutants are specifically impaired in this fusion function ( figure 5a , first column and figure 5b , red cylinders ; fusion score of 1 and 0 , respectively ) . the slam contact area on the top of the h - dimer may extend also to the position defined by mutant 1 ( fusion score of 1 ) .
we then assessed the efficiency of membrane fusion triggering through the membrane - anchored anti - hexahistidine antibody .
figure 5a , center column and figure 5b , yellow cylinders , show that both mutants ( 5 and 6 ) in which the tags are located between the slam and cd46 footprints , had fusion scores of 2 each .
mutants 1 and 7 , with tags located close to only one of the receptor footprints , and closer to the interface than mutants 5 and 6 , had fusion scores of 1 each .
thus , both hexahistidine tags inserted within the slam - footprint trigger fusion through an artificial receptor , almost as efficiently as the control mutant .
to better compare the fusion efficiency of mutants 5 and 6 , the luciferase - based quantitative fusion assay was used .
figure 5c documents fusion efficiency in vero cells expressing either the membrane - anchored anti - hexahistidine antibody ( yellow bars ) or slam ( red bars ) .
mutant 5 was about 65% as effective as positive control mutant 9 in inducing fusion of vero - his cells , and mutant 6 was about 50% as effective .
thus , an artificial receptor must bind proximal to the locations used by the natural receptors to effectively trigger fusion .
while preferential accessibility of this area of the h - head may influence this striking co - localization of functional binding sites , our findings are also fully consistent with the  twist  hypothesis proposing that receptors have more leverage to force the heads to move while engaging an interface - distal location .
to test the  movement hypothesis we sought to stabilize the h - dimer interface by adding inter - subunit disulfide bonds .
we note that the heads have a solid beta - propeller structure , and may thus move as rigid bodies .
moreover , cd46 binding does not induce substantial conformational changes of the h - head 36 , consistent with a rigid structure suited to conduct torsion .
we identified amino acid pairs across the interface whose c - alpha atoms are less than 7.5  apart ( table 1 ) and mutated these to cysteine .
we sought to introduce two classes of disulfide bonds : single bonds located on the two - fold symmetry axis of the dimer and involving the same residue on both molecules ( table 1 , top four lines ) , and twin bonds located on both sides of the axis and involving two different residues ( table 1 , bottom four lines ) .
twin cysteine bonds , forming a triangle with cys154 , would provide more resistance to a force , increasing the energy barrier for fusion triggering ; larger triangles would provide more resistance than smaller triangles .
figure 2a shows the location of the two key single bonds ( t273c  t273c , blue and w327c  w327c , green ) and the two key twin bonds ( k236c  g264c , brown / red and p330c  l161c , orange / yellow ) in a side view of the dimer interface .
the dimerization capacity of the mutated h - proteins was then assessed ( table 1 and figure 2c ) .
we note that the cysteine substitutions are distal from known receptor binding epitopes , but to control for the ability of each position to tolerate mutations we also made alanine substitutions , and assayed fusion triggering by the alanine mutants .
where twin bonds were involved , we ensured that neither one of the cysteine substitutions in the pair had an effect on fusion function .
as listed in table 1 ( fifth column ) , only one of the alanine mutants , l203a was fusion deficient , and this residue was eliminated from further consideration .
the fact that all the other alanine mutations were fusion - competent implies that the corresponding mutant proteins have intact receptor binding function . since total expression levels of certain mutants were reduced ( figure 2c ) , we sought to measure their cell surface expression .
we measured the mean fluorescence intensity ( mfi ) by facs analysis for each mutant ( supplementary table 1 ) , which was 4768% as that of the standard h - nse protein .
even with this reduction in cell surface protein levels , we note the mv fusion system is very efficient , and that decreased h surface expression may not necessarily translate in decreased fusion 13 .
we also probed the mutants with a conformation - specific monoclonal antibody that recognizes the h - noose epitope 37 .
the mfi measured with this antibody was similar to that recorded with the anti - h cocktail ( supplementary table 1 ) , indicating that the mutants had similar conformations to the unmodified h - protein .
we then assessed whether covalent bonds are formed , taking advantage of an h protein backbone with mutations in both cysteine residues ( cys139 and cys154 ) that normally mediate covalent inter - subunit cross - linking . while this mutant forms minimal amounts of stable dimers , given time it does support low levels of membrane fusion 13 .
c154a mutant loses the ability to form detectable amounts of stable h - dimers , as assayed by non - reducing sds - page .
c154a background the only inter - subunit bridges are those formed at positions with added cysteines .
figure 2d ( lanes 14 ) show that cysteine substitutions at 236 + 264 , 273 , 327 or 330 + 161 restore the dimerization capacity of the h - protein , demonstrating that these substitutions allow inter - subunit disulfide bonds under physiological conditions .
c154a ( lanes 14 ) or the normal h - backbone ( lanes 58 ) , indicated that the cysteine substitution mutants alone are less efficient at forming dimers .
this is not surprising as the introduced cysteines may not promote covalent linkage as efficiently as the two stalk cysteines .
this analysis also revealed that k328c and k236c+l265c did not yield cross - linked dimers , and these residues were eliminated from further consideration ( table 1 , seventh column ) .
moreover , p330c+e162c dimerized but was not pursued further because it is similar to p330c+l161c .
thus , analysis focused on two single - bond and two twin - bond cysteine mutants .
after confirming disulfide bond formation , h - protein mutants were co - expressed with f - protein to test the hypothesis that movement of the subunits is required to allow membrane fusion .
figure 3a shows that the most distal twin bonds ( k236c+g264c , 32 from fusion pivot , 533  stabilized area ) abrogated fusion . on the other hand , the two single bonds ( 22 and 16 from the pivot ) and
the pivot proximal twin bond ( 10 from pivot , 68  stabilized area ) had little effect on fusion , in this assay performed 24 hours after transfection .
this analysis was repeated using a luciferase - based quantitative fusion assay that provided additional 12- and 18-hour time points . at 12-hours
the positive control showed high fusion levels , while all the mutants had fusion levels at or near background ( figure 3b ) . at 18-hours the fusion functions of the t273c ,
w327c and p330c  l161c mutants were reduced by a factor of 6 compared to the positive control . at 24-hours the fusion function of the k236c+g264c mutant
was reduced by a factor of 13 compared to the wild type protein , while fusion efficiency of the other three mutants was comparable to wild type h , which syncytia had already begun to lyse .
these results indicate that the single bonds at 273 and 327 , and the twin bond at 330 + 161 interfere with fusion temporarily . however , given time fusion is observed , possibly because occasionally disulfide bonds are reduced .
we then assessed whether eliminating the disulfide bonds with a reducing agent restores fusion function .
cells expressing the k236c+g264c mutations were treated with 1,4-dithio - dl - threitol ( dtt ) . in the presence of dtt ,
fusion function was restored in the k236c+g264c h mutant that previously exhibited no fusion ( figure 3c , compare lower left and right panels ) .
similar results were observed for the t273c and w327c mutants when treated with dtt 12-hours post - transfection ( data not shown ) , indicating that this protein is available in sufficient quantities and retains the capacity to interact with receptors .
we next assessed whether the efficiency of fusion triggering depends on interface - distal receptor binding on the h - dimer .
we took a systematic approach and inserted a short hexahistidine tag in different positions at the top half of the h - dimer .
the hexahistidine tag directs binding to vero - his cells which express a membrane - anchored single - chain antibody that recognizes the six - histidine peptide38 .
the insertion points were chosen taking into consideration structural constraints , including n - linked oligosaccharide chains at n168 , n187 , n200 and n215 , and availability of surface accessible loops ( figure 4a , b )
. the tags were inserted in eight exposed loops ( figure 4a , b , yellow residues ) .
we used mutant 9 with a carboxy - terminal tag as the positive control 38 .
mutants 5 and 6 are located on the top of the h - dimer , between the h - residues in close contact with cd46 , as identified based on the h - cd46 co - crystal 36 ( highlighted in light blue ) and those identified by functional assays to be relevant for slam - dependent fusion 2729 ( highlighted in red ) .
mutants 1 , 3 , 7 and 8 are all located near the top of the h - dimer . while mutant 1 is close to the cd46 footprint , and mutant 7 close to the slam footprint , mutants 3 and 8 are located away from both receptor binding areas .
mutant 2 is also located away from these binding areas and close to the interface .
mutant 4 is the only one in the bottom half of the dimer . to identify functional mutants , dimerization capacity , expression levels , and accessibility of the his - tag
protein extracts from transfected vero cells were separated on sds - page under non - reducing conditions , and immunoblotted . since mutants 2 and 4 dimerized poorly ( figure 4c ) , they were excluded from further analysis .
surface expression levels of the remaining mutants , measured by facs , were at 3999% of standard h - nse ( supplementary table 2 , third column ) , which was considered acceptable for all mutants . to determine the accessibility of the his - tag we compared the mfi generated by an anti - his antibody to that generated by an anti - h cocktail ( supplementary table 2 , last column ) .
all but one mutant had mfi ratios close to 1 , indicating that their his - tags were equivalently accessible .
since mutant 3 had a lower ( 0.65 ) mfi ratio , it was not considered when drawing conclusions .
thus , hexahistidine tagging of exposed protein loops is compatible with efficient protein folding and dimerization , and allows display of accessible tags in a majority of the constructs .
we then measured the fusion efficiency of the dimerization - competent mutants and the control mutant , 9 , in cell lines expressing either the anti - hexahistidine antibody as the designated receptor , or the natural receptor slam , or the vaccine strain receptor , cd46 .
since none of the hexahistidine tags were predicted to interfere with cd46-binding , these fusion assays were considered positive controls . on the other hand ,
finally , hexahistidine receptor - dependent fusion assays were expected to reveal the h - dimer surface area poised for fusion triggering . for consistency in host cell type , we used vero cells , or vero cells expressing anti - hexahistidine ( vero - his ) , or slam ( vero - slam ) . to focus exclusively on entry through one receptor
, we generated a second set of tag mutants in an h - protein backbone exclusively recognizing slam ( h - wt , figure 5a , first and second column ) .
the h - nse vaccine lineage backbone was used only to measure cd46-dependent fusion ( h - nse , figure 5a , third column ) .
this analysis indicated that , with the exception of mutants 7 and 8 , which were reduced in cd46-specific fusion ( fusion scores of 2 and 1 , respectively ) , all the mutants fully retained cd46-dependent fusion function .
analysis of slam - dependent fusion revealed that the h - slam contact area may extend to the region defined by mutants 5 and 6 , because these two mutants are specifically impaired in this fusion function ( figure 5a , first column and figure 5b , red cylinders ; fusion score of 1 and 0 , respectively ) .
the slam contact area on the top of the h - dimer may extend also to the position defined by mutant 1 ( fusion score of 1 ) .
we then assessed the efficiency of membrane fusion triggering through the membrane - anchored anti - hexahistidine antibody .
figure 5a , center column and figure 5b , yellow cylinders , show that both mutants ( 5 and 6 ) in which the tags are located between the slam and cd46 footprints , had fusion scores of 2 each .
mutants 1 and 7 , with tags located close to only one of the receptor footprints , and closer to the interface than mutants 5 and 6 , had fusion scores of 1 each .
thus , both hexahistidine tags inserted within the slam - footprint trigger fusion through an artificial receptor , almost as efficiently as the control mutant . to better compare the fusion efficiency of mutants 5 and 6 , the luciferase - based quantitative fusion assay was used .
figure 5c documents fusion efficiency in vero cells expressing either the membrane - anchored anti - hexahistidine antibody ( yellow bars ) or slam ( red bars ) .
mutant 5 was about 65% as effective as positive control mutant 9 in inducing fusion of vero - his cells , and mutant 6 was about 50% as effective .
thus , an artificial receptor must bind proximal to the locations used by the natural receptors to effectively trigger fusion .
while preferential accessibility of this area of the h - head may influence this striking co - localization of functional binding sites , our findings are also fully consistent with the  twist  hypothesis proposing that receptors have more leverage to force the heads to move while engaging an interface - distal location .
we have used information from the static crystal structures of the attachment proteins of four paramyxoviruses to formulate and test two hypotheses aiming at inferring the mechanism of a necessarily dynamic process , such as the triggering of membrane fusion .
our findings are consistent with the following model of h - based fusion triggering : first , two membrane - anchored receptor molecules bind the h - dimer heads opposite to the interface .
this movement transmits a signal to the f protein , triggering irreversible f - trimer refolding and membrane fusion .
our model provides a framework for interpreting recently published structural information documenting one type of interaction between two external cd46 short consensus repeats ( scr ) and the h - heads 36 ( figure 1d ; scr12 are shown in pale green ) .
surprisingly , cd46 binds h in an  upside - down  orientation : scr2 points towards the viral membrane . very recently
, almost the entire cd46 ectodomain ( scr 14 ) was co - crystallized with the adenovirus knob . in this complex cd46
when this structure is modelled into the h - dimer , scr 4 is parallel to the membranes ( figure 1d ) .
remarkably , the slam footprint suggests that its h - binding domain is positioned tangentially to the h - dimer interface ( figure 5b ) and thus also parallel to the membrane .
we propose that tangential engagement sustains efficient transfer of forces arising from the receptor s lateral movement in the plasma membrane to h - heads .
these observations also suggest that cd46 and the h - oligomer initially interact at another angle , or in a different conformation , than those shown in figure 1d . indeed , there are indications that these molecules have structural plasticity and assume different conformations during the binding and fusion processes .
in particular , it was observed that cd46 domains 3 and 4 enhance binding of domains 1 and 2 to mv particles , but impair binding to soluble h 40 .
since the domain 12 interface possesses some flexibility 41 , and flexibility may also be a feature of the similarly - sized domain 23 interface 39 , it is conceivable that before h - binding cd46 is shaped as a  hook 
( figure 1d ; scr 12 shown in pale green , linked to yellow ovals representing scr 34 ) . the  hockey stick  could be an alternative conformation occurring after binding and facilitating membrane fusion .
a precedent for this is the piv5 hn - tetrameric ectodomain : electron microscopy revealed a staggered arrangement of the heads , and it was proposed that flexibility of the stalk is important in membrane fusion 42 .
h - tetramers may bind cd46 molecules in the  hook  conformation , and subsequently these complexes may adjust to form a scaffold of optimal height to support fusion .
such a scaffold was previously proposed based on the analysis of the fusion - support efficiency of receptor molecules of variable length 43 .
based on the analysis of the h - oligomer s intracellular transport , it was proposed that paramyxoviruses binding either proteinaceous receptors ( h - class ) , or sialic acid ( hn - class ) , regulate fusion by different mechanisms 25 .
this suggestion was strengthened by the documentation of additional structural and functional differences between h- and hn - class proteins 12 .
we now propose that h - heads switch partners to transmit the fusion - triggering signal , while hn - heads must use another mechanism . we know
1d ) 19 , and we propose that they make them rotate 90 about their stalk .
h - heads will then switch partners while the stalk remains covalently linked with the stalk of the original partner , cross - linking two h - dimers into an irregular h - tetramer . since the stalks of these h - tetramers would have anomalous contacts with f - trimers 18 , their accumulation would progressively de - stabilize the viral envelope , eventually triggering membrane fusion .
our model considers that fusion is supported by tetrameric or higher order h - oligomers 15 , and implies that alteration of residues critical for formation of the interface between dimers would interfere with transmission of the fusion - triggering signal .
however , we do not know whether this interface is formed between only one subunit in each dimer , analogous to the staggered arrangements of hn pairs - of - dimers revealed by electron microscopy 42 , or between both subunits in each dimer , analogous to the tightly packed , quadratic arrangements of hn - head crystals 2022 . towards identifying residues in this interface and testing this aspect of the model we generated 10,000 docked poses of h - head pairs - of - dimers using the program zdock 44 ( n.o . and w.b . ,
we expressed mutated h - proteins , focusing initially on mutants representing tightly packed poses , but all mutated proteins maintained fusion function , and formed tetramers as documented by blue native gel electrophoresis ( c.k.n .
since we have identified many potential interface residues , we plan iterative cycles of mutagenesis and functional tests .
hn - based triggering can not be accounted by head partner switch because intermolecular disulfide bonds in the hn - dimer interface do not block fusion and thus both heads of a hn - dimer must move as a unit 45 . since receptors engage these units from the top 2022 , hn - head dimeric units may slide relative to another rather than rotate .
sliding would not cross - link subunits within hn - tetramers , but may catenate the tetramers , which would liberate individual f - trimer spikes and trigger fusion 46 .
 the measles virus entry system , constituted of attachment ( hemagglutinin , h ) and fusion proteins , operates based on a variety of natural and targeted receptors . 
 however , the mechanism triggering fusion of the viral envelope with the plasma membrane is not understood . 
 here we tested a model considering that the two heads of an h - dimer , which are covalently linked at their base , after binding two receptor molecules , move relative to each other to transmit the fusion - triggering signal . indeed , stabilizing the h - dimer interface by additional inter - molecular disulfide bonds prevented membrane fusion , an effect reversed by a reducing agent . 
 moreover , a membrane - anchored designated receptor efficiently triggered fusion , provided it engaged the h - dimer at locations proximal to where the natural receptors bind , and distal to the h - dimer interface . 
 we discuss how receptors may force h - heads to switch partners and transmit the fusion - triggering signal . 
INTRODUCTION
RESULTS
Stabilization of the H-dimer interface
Twin pivot-distal disulfide bonds reversibly inhibit fusion
Directing receptor binding with hexahistidine tags
Only interface-distal binding efficiently triggers fusion
DISCUSSION
Supplementary Material