Notizen 887

Determination o f M easles Virus Protein M olecular W eights on High Percentage,Highly Cross-Linked S D S Polyacrylamide Gels

M. J. C arter and K. Baczko

Institut für Virologie und Im m unologie, Universität W ürzburg, Versbacher Str. 7, D-8700 W ürzburg, BRD

Z. Naturforsch. 38 c, 8 8 7 -8 8 9 (1983); received M ay 6/M ay 30, 1983

Measles, Protein, Electrophoresis, M olecular W eight

A recent exam ination o f m easles virus m RN A molecules has shown that the nucleocapsid and haem agglutin m es­sengers are o f the size expected from a consideration o f their protein products. However, the m R N A for m em ­brane protein is approxim ately 50% larger than the size required. The m olecular weight o f m atrix protein has been determ ined by SD S-polyacrylam ide gel electrophoresis, and this procedure can lead to an underestim ation o f the true size o f hydrophobic molecules which show increased SDS binding. It is therefore appropria te to exam ine the m olecular weight determ ination o f this protein to exclude such an artefactual discrepancy in m R N A and protein sizes. We report here that measles virus m em brane protein does not shown such anom alous behaviour and confirm that the size discrepancy is a true phenom enon.

Six virus-specific structural polypeptides are recognised in m ature measles virus particles. These proteins are designated; the large protein (L); the haem agglutinin (H); the phosphoprotein (P); the nucleocapsid protein (N); the fusion protein which is cleaved into F , and F 2, and the m em brane protein M. There is w idespread agreem ent on the size range o f these proteins [1]. Recently the app li­cation o f recom binant D N A technology has p e r­m itted the cloning o f parts o f several measles virus- specified m R N A molecules [2, 3]. N orthern blotting has then provided an estim ation of the sizes o f the intact virus mRNAs. The nucleocapsid and haem ag­glutinin proteins are specified by mRNAs of sim ilar size, 1735 and 1680 bases respectively, whilst the m em brane protein m RNA is 1515 nucleotides in length [2, 3]. Rozenblatt and co-workers have calculated the coding capacities o f these molecules and it was apparen t that N and H protein m RNAs were approxim ately the size expected from the m olecular weights o f the unm odified proteins p ro ­duced in in vitro translation experim ents. However,

Reprint requests to M. J. Carter.

0341-0382/83/0900-0887 $ 0 1 .3 0 /0

this was not so when the m em brane protein m R N A was considered. Even allowing for a poly-A tail o f 100 residues, these workers derived a predicted molecular weight o f 59 K for the product o f this mRNA. The M protein is produced as a 3 6 -3 7 K molecular weight protein in in vitro translation ex­perim ents [4 -7 ], and is labelled in infected cells by short-pulse labelling procedures. This in form ation excludes any post-translational precursor processing mechanism in the production o f M protein.

Bayreuther et al. [8] have presented da ta concern­ing the electrophoretic m igration o f ano ther m em ­brane-associated protein, the lactose perm ease o f E. coli. This hydrophobic protein shows an ano­malously high binding capacity for SDS resulting in an increased charge-to-m ass ratio o f the complex. Consequently electrophoretic m igration is assisted and m olecular weight determ inations underestim ate the size o f the protein. A sim ilar effect, bu t opposite in direction, has also been reported for som e glyco­proteins which show decreased SDS b inding [9]. Hall and co-workers [10] have isolated m easles virus M protein and found it to be rich in hydrophobic aminoacids. These workers also ob tained some evidence for com plex form ation w ith the non-ionic detergent “triton” , which lead to a m obility shift towards a higher m olecular weight. It is therefore possible that anom alous SDS binding could also occur. Consequently it is appropria te to reexam ine the m olecular weight determ ination o f this peptide.

The electrophoretic m igration o f an SD S-protein complex is affected both by the am ount o f SDS bound (i.e. charge) and by the m olecular sieving effect o f the gel mesh. The latter is governed by the cross-linking w ithin the gel, and Bayreuther et al. [8] have hown that the sieving effect can predom inate in high percentage polyacrylam ide gels w ith a low acrylam ide/bisacrylam ide ratio. In the case o f lac­tose permease, an apparen t m olecular w eight of 30 K determ ined by electrophoresis on a 10% poly­acrylamide gel (acry lam ide:b isacry lam ide= 37.5 :1) was increased by over 50% to 46.5 K if electro­phoresis was perform ed on a 20% gel o f the sam e acrylam ide:bisacrylam ide ratio. Since the true molecular weight o f this protein was known from DNA sequence to be 46.5 K [11], this procedure for molecular weight determ ination effectively over­comes errors due to increased SDS binding. The measles virus M protein m olecular w eight has been determ ined from SDS gels as 3 7 -4 0 K. Exact in-

This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution-NoDerivs 3.0 Germany License.

On 01.01.2015 it is planned to change the License Conditions (the removal of the Creative Commons License condition “no derivative works”). This is to allow reuse in the area of future scientific usage.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung-Keine Bearbeitung 3.0 DeutschlandLizenz.

Zum 01.01.2015 ist eine Anpassung der Lizenzbedingungen (Entfall der Creative Commons Lizenzbedingung „Keine Bearbeitung“) beabsichtigt, um eine Nachnutzung auch im Rahmen zukünftiger wissenschaftlicher Nutzungsformen zu ermöglichen.

888 N otizen

1 2 3 4 5 6 7

— ----------14 3

Fig. 1. Measles virus (Edm onston)-infected vero cells were labelled with [35S]m ethionine (A m ersham Buchler, 200 |iC i/m l) for 2 h from 1 8 -2 0 h p. i. Cell lysates were then prepared for im m unoprecip ita tion as described by Stephenson and ter M eulen [20]. Protein-A conjugated to sepharose CL4B (pharm acia) was pre-arm ed w ith rabb it- antim ouse im m unoglobulins (D ako) before use with monoclonal antibodies. Lysates were then im m unopre- cipitated with rabb it hyperim m une anti-m easles serum (track 6) or with m onoclonal antibodies specific for F (track 1); N (track 2); H (track 4) or M (track 5).[ 14]M ethylated protein m olecular w eight m arkers were o b ­tained from A m ersham Buchler and electrophoresed in tracks 3 and 7. These com prised: Myosin, 200 K; Phos- phorylase b, 92.5 K; BSA, 69 K; O valbum in, 46 K; Carbonic Anhydrase, 30 K and Lysozyme 14.3 K. All samples were denaturated and reduced by boiling with dithiothreitol and SDS before loading. Sam ples were analysed on discontinuous gels [21]. Stacking gels con­tained: 3.5% Acrylam ide, 0.15% Bis, 0.14 m tris pH 6.5, 0.1% SDS. Separating gels contained 20% A crylam ide, 0.53% Bis, 0.375 M Tris pH 8.7, 0.1% SDS, electrophoresis was perform ed at 120 V.

formation on gel com position is d ifficult to obtain, but most workers have used highly cross-linked low percentage acrylam ide gels (7 .5% - 10%; acrylam ide: bisacrylamide 3 0 -3 9 :1 ) , [1 2 -1 4 ] or higher percentage gels with lower proportions o f cross­linker (15-25% ; ac ry lam ide: bisacrylam ide = 55 -1 0 0 :1 ) [7, 10, 15, 16]. O ther reports do not give this inform ation [17, 18]. There is one report [19] o f a molecular weight determ ination using a highly crosslinked, interm ediate-strength gel system (15%; acrylam ide:bisacrylam ide = 37.5:1). However, this could still result in a 30% underestim ation of M protein m olecular weight [8].

In order to exclude this possibility we have determined the m olecular weights o f m easles virusH, N, F and M proteins using a highly cross-linked (acrylamide: bisacrylam ide = 37.5:1), 20% poly­acrylamide gel. A typical experim ent is show n in Figure 1. The m olecular weight m arkers show ed a linear relationship between m igration and the logarithm of m olecular w eight over the range

3 0 -9 0 K. The 200 K m olecular weight m arker and virus L protein did not enter this gel. M easles virus polypeptides were identified by im m unoprecip ita­tion using monoclonal antibodies specific for these molecules.

The nucleocapsid protein is very susceptible to proteolytic degradation, the m ajor product o f this process is term ed NC and has a m olecular w eight o f 47 K [20]. The monoclonal antibody used in this work precipitates only the 60 K parental N protein , and although NC is present and runs as the m ajor band immediately above F] (compare tracks 1 and 6, Figure 1), it is not detected by this antibody. This suggests that the site against which this m onoclonal antibody is directed is rem oved during the degreda- tion. A small polypeptide, N C ' (15.8 K) is p rec i­pitated and presum ably corresponds to the p roduct of this cleaveage. The small cleavage product o f the fusion protein (F 2) could not be detected e ither by monoclonal antibody or hyperim m une serum , p re ­sumably because F was present in too small amounts. Long exposures were necessary to dem on­strate the small am ounts o f F and N C '. C onse­quently non-specifically precipitated background proteins were more noticeable in these two tracks but their am ounts were always equivalent to those observed in negative controls. The identification o f H and M was straight forward.

Molecular weights determ ined from analysis on this type of gel and from 10% SDS polyacrylam ide gels were not found to be significantly different, Table I shows the average o f six determ inations. It can thus be concluded that the measles virus m em ­brane protein does not show anom alous SDS bind-

Table I. M olecular weights determ ined in our labora to ry for the m ajor measles-virus (Edm onston) specific p o lypep­tides. The L protein did not enter the high percentage gel, and the N C ' m olecule was electrophoresed out o f the 10% gel. The P protein is present is too low an am oun t for conclusive identification in the absence o f specific m ono­clonal antibodies. NC was not well resolved from F] in the higher percentage gel.

Protein 10% PAGE 20% PA G E

L 190 _

H 78 76

N 58 61NC 47 —

F, 45 44

M 39 39N C ' — 15.8

Notizen 889

ing and consequently the disparity in m R N A and protein size m ust represent a real phenom enon. In general, with the notable exception o f the corona- viruses [22], m ost RN A virus m RNAs are related to the size o f their product. Exceptions being spliced mRNAs which m ight contain inform ation function- less in the processed product, or proteins form ed by the processing o f precursors. Measles is a cyto­plasmic virus and splicing m echanism s are therefore unlikely. Post-translational processing m echanism s can also be excluded although a co-translational cleavage could still occur. Selection of m em brane protein m R N A and its translation in vitro leads to the production o f only one protein [3]. However, this work has utilised im m une precipitation. A small peptide, produced as above, m ight not be

[1] B. K. Rim a, J. Gen. Virol, in press.[2] M. Gorezki and S. R ozenblatt, Proc. Natl. Acad. Sei.

USA 7 7 ,3 6 8 6 -3 6 9 0 (1980).[3] S. R ozenblatt, S. Gesang, V. Lavie, and F. S. N eu ­

m ann, J. Virol. 4 2 ,7 9 0 -7 9 7 (1982).[4] A N iveleau and T. F. W ild, Virology 96, 2 9 5 -2 9 8

(1979).[5] S. R ozenblatt, M. G orecki, H. Shure, and C. L. Prives,

J. Virol. 2 9 ,1 0 9 9 -1 1 0 6 (1979).[6] J. Sprague, L. J. Eron, A. S. Siefried, and J. B. Mil-

stein, Virology 3 2 ,6 8 8 -6 9 1 (1979).[7] J. R. Stephenson, S. G. Siddell, and V. ter M eulen,

J. Gen. Virol. 57,191 - 197 (1981).[8] K. Bayreuther, B. Bieseler, R. Ehring, H. W. Griesser,

M. M ieschendahl, B. M üller-H ill, and I. Triesch, Biochem. Soc. Trans. 8 ,6 7 5 -6 7 6 (1980).

[9] P. Lam bin, P. Anal. Biochem. 8 5 ,114-125 (1978).[10] W. W. Hall, K. N agashim a, W. Kiessling, and V. ter

M eulen, in: negative Strand Viruses and the Host Cell. (B. W. J. M ahy and R. D. Barry, eds.), pp. 765 -7 7 0 , Academ ic Press, London 1978.

[11] D. E. Büchel, B. G ronenbom , and B. M üller-H ill, N ature (Lond.) 283, 5 4 1 -5 4 5 (1980).

incorporated into virus particles, and not therefore recognised by antiserum . A lternatively the excess inform ation in the m R N A could be non-coding and perhaps serve some regulatory or structural func­tion. However, the reason for the disparity in protein and m R N A size, confirm ed in this report, is at present unknown and m ust aw ait sequence deter­mination.

Acknowledgements

This work is supported by the D eutsche F or­schungsgemeinschaft, H ertie-S tiftung and Volks­wagenstiftung. We thank Dr. Bayreuther for helpful discussion, and Helga Kriesinger for typing the manuscript.

[12] J. M. Hardwick and R. H. Bussell, J. Virol. 25, 6 8 7 -692 (1978).

[13] M. C. Graves, S. M. Silver, and P. W. C hoppin, Virology 8 8 ,2 5 4 -2 6 3 (1978).

[14] B. K, R im a and S. J. M artin , J. Gen. Virol. 44, 135-144(1979).

[15] D. L. J. Tyrrell and E. J. N orrby, Gen. Virol. 39, 2 1 9 -2 2 9 (1978).

[16] K. C. Stallcup, S. L. W echsler, and B. N. Fields, J. Virol. 3 0 ,1 66-176 (1979).

[17] R. S. Fujinam i, J. G. P. Sissons, and M. B. A. Old- stone, J. Im m unol. 1 2 7 ,9 3 5 -9 4 0 (1981).

[18] S. L. W echsler and B. N. Fields, N ature 2 7 2 ,4 5 8 -4 6 0(1978).

[19] W. E. M ountcästle and P. W. C hoppin , Virology 78, 4 63-474(1977).

[20] J. R. Stephenson and V. ter M eulen, Proc. N atl. Acad. Sei. USA 76,6601 -6 6 0 5 (1979).

[21] U. K. Laem m li, N atu re 2 2 7 ,6 8 0 - 685 (19 71).[22] S. Siddell, H. W ege, and V. ter M eulen, J. Gen. Virol.

6 4 ,7 6 1 -7 7 6 (1983).