key: cord-0004920-tmvebf23
authors: Stephenson, J. R.; ter Meulen, V.
title: A comparative analysis of measles virus RNA by oligonucleotide fingerprinting
date: 1982
journal: Arch Virol
DOI: 10.1007/bf01315058
sha: be351c7034515e0a8411d92518bec270c763d35b
doc_id: 4920
cord_uid: tmvebf23

Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T(1) oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow a differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin.

Measles virus is an ubiquitous and highly contagious agent which infects man and also primates in close contact with man. The disease usually occurs during childhood, and normally causes a relatively mild stereotyped infection. In a few cases, a more serious complication can arise which may include acute encephalitis. In addition to this disorder another CNS disease (subaeute selerosing panencephalitis --SSPE) has been associated with measles virus infection (1--3) . This association has led to many studies on the possible biochemical and antigenic differences betweea measles and SSPE viruses. Several reports demonstrate differences in etectrophoretic mobility between the proteins of measles and SSPE viruses, especially between the P and M proteins (4--6) however the variation found between SSPE and measles isolates was of the same order as that found among the measles isolates themselves (5) . Furthermore, little or no difference has been found in the antigenieity of these viruses when assayed by neutrali-zation, haemolysin inhibition, haemagglutination inhibition (7) . Moreover, all major virus-specific polypeptides from either measles of SSPE viruses can be precipitated equally well by either homologous or heterologous r a b b i t sera (8) . However, b y using short-term i m m u n i z a t i o n schedules with purified protein, antigenic differences were reported between the M protein of one SSPE isolate a n d one measles isolate (9) . I n addition, recent, studies using monoclonal antibodies against measles virus haemagglutinin have demonstrated antigenic differences between several measles and SSPE isolates (10) , b u t no characteristics, specific for SSPE isolates were observed. I n addition a t t e m p t s have been made to analyse the sequence homology between the RNAs of measles and SSPE viruses. YI~H (11) and HALL a n d TE]~ MEULEN (12) reported only 60 and 90 per cent homology respectively between I~NAs from measles and SSPE isolates.

As the methods employed in these previous studies could only detect gross differences in virus-specific products, or examined one protein or ~ small p a r t thereof, we have chosen in this study to analyse the oligonncteotides from a T1 digest of the complete genome. Not only is this technique capable of detecting differences in R N A sequence throughout the genome, b u t is also capable of detecting differences as small as one base change. I n this report, a comparative analysis between isolates from acute measles cases, a case of acute measles encephalitis and several cases of SSPE is described.

The fusion inhibitor SV4814 was obtained from Bachem Inc. Torrance, California, U.S.A., and Actinomycin D (AM])) from SERVA, Heidelberg, F.I£.G. 32p orthophosphate (40 mCi/ml), Adenosine 51-a2p triphosphate (2000 Ci/mmol) and 5 3H Uridine (25--30 Ci/mmol) were obtained from Amersham-Buchler, Braunschwcig, F.R.G. Neutral cellulose powder (Ccllcx N-l) was purchased from Bio-Rad and further purified by the method of SHIlt and MARTIN (13) . Bacterial alkaline phosphatase (BAP) was purchased from Worthington, polynucleotide kinase (from T4 XF-1 infected E. Coli B) from P. L. Biochemicals, and T1 ribonuclease and pancreatic ribonuclease A from Sigma. Xylene-cyanol FF(CF) and Bromophenol blue (BPB) was obtained from Chroma and Trypan red was a gift from Dr. J. J. Skehel (NIMR, Mill Hill, London).

All isolates of measles and SSPE viruses were grown on Veto cell monolayers as described (8) . As the LEC isolate of SSPE virus caused extensive celt fusion which resulted in premature cell death, 10 vg/ml fusion inhibitor was added to the growth medium after the virus had been allowed to adsorb to the cell sheet. The Hall6 isolate of SSPE virus was kindly given by Dr. F. B. Wild, the Mantooth isolate by Dr. Horta-Barbose, and the Braxator isolate was isolated from a case of acute measles encephalitis (14). The "CM" and "Joy" isolates from acute cases of measles were kindly given by Dr. B. Fields, Harvard Medical School, U.S.A.

The virus stocks used in these experiments had been passaged as follows. The Braxator and Mantooth isolates had been passaged 20 times in Vero cells, the LEG isolate 15 times in Vero cells and the Hall6 isolate 25 times. The CM and Joy isolates were isolated in HeLa cells, passaged twice in CV~ cells and 10 times in Vero cells.

Cell monolayers were labelled with ~p orthophosphate as follows. Cells (4 X 107) were infected at a m.o.i, of 1 until 20 per cent of the sheet was incorporated into syncytia (15 21 hours post infection depending on the viral isolate). The cells were then in-cuba.ted in phosphate-free M E M c o n t a i n i n g i ixg/ml A M D a n d 50 5im H E P E S pI-I 7.4) for 1 h o u r a n d t h e n in 20 mI of fresh phosphate-free m e d i u m c o n t a i n i n g AMD, H E P E S a n d 1 m C i / m l of a2P-orthophate for a f u r t h e r 3 hours. T h e m e d i u m was t h e n decanted, cells w a s h e d once in saline a n d pelleted b y centrifugation for 5 m i n u t e s at 1000 × g a n d 4 o C.

T h e cell pellet was homogenized with a D o u n c e h e m o g e n i s e r in 20 t i m e s its v o l u m e of R S B (10 m~ NaC1, 10 m~I Tris p H 7.4, 1.5 mM MgCI~), m a d e 0.5 per cent w i t h respect to N P 4 0 a n d elarifed b y two cycles of centrifugation at 1000 × g a n d 4 ° C. The s u p e r n a t e n t fluid was t h e n layered onto a step gradient c o n t a i n i n g 15 a n d 65 per cent sucrose in T K M (150 m~ KC1, l0 m~ MgCI~ 0.5 per cent N P 4 0 a n d 10 m~ Tris: pI-I 7.4) a n d centrifuged for 16 hours a t 1 5 0 , 0 0 0 × g a n d 4 ° C. A discreet b a n d was clearly visible a t t h e j u n c t i o n of t h e 15 a n d 65 p e r cent sucrose layers. This b a n d was n o t visible w h e n similar e x t r a c t s were m a d e from u n i n f e c t e d cells. T h e m a t e r i a l a t t h e interface of t h e two sucrose solution wass diluted. 5 × in R S B plus 0.5 per cent N P 4 0 a n d centrifuged on a similar g r a d i e n t a t 150,000 × g for 16 hours at 4 ° C. The m a t e r i a l a t t h e interface of t h e second gradient was diluted 10 × in R S B plus 0.5 per cent N P 40 a n d pelleted b y c e n t r i f u g a t i o n for 4 h o u r s at 200,000 × g a n d 4 ° C. T h e pellet was vigorously r e s u s p e n d e d in e x t r a c t i o n buffer (100 mM NaC1, 50 m?~ N a a c e t a t e p t { 4.6, 2.5 mM E D T A ) a n d m a d e 1 p e r cent w i t h respect to SDS. 100 ILg of g r a d i e n t purified 4S R N A from u n i n f e c t e d Vero cells was a d d e d as carrier a n d R N A e x t r a c t e d as described (15) . Nucleoeapsid R N A p r e p a r e d b y this m e t h o d was a s s u m e d to be unc o n t a m i n a t e d b y messenger R N A as it dld n o t b i n d to oligo dT a n d was u n a b l e to s t i m u l a t e p r o t e i n synthesis in a cell-free p r o t e i n synthesizing s y s t e m from r a b b i t reticulocytes (data n o t shown).

Messenger IZNA from virus-infected a n d mock-infected cells was e x t r a c t e d b y p h e n o t / S D S a n d oligo dT c h r o m a t o g r a p h y as described (15) .

The final ethanolic precipitate from t h e I~NA e x t r a c t i o n procedure was dried, r e s u s p e n d e d in gardient buffer (100 r n~ LiC1, 2.5 m~ E D T A , 0.1 per cent [w/v] SDS, 10 m~ Tris p H 7.4), a n d centrifuged for 5 hours a t 200,000 × g a n d 18 ° C on 15--30 p e r cent linear sucrose gradients. 

This was acheived by allowing the RNA to self-anneal and then separating free single s t r a n d s b y a modification of t h e m e t h o d of FI~A~'KLI~- (16) . A c o l u m n of purified cellulose powder (0.2 ml) was p r e p a r e d in a 2 mt disposable syringe a n d was w a s h e d t h o r o u g l y w i t h a solution c o n t a i n i n g 35 per cent absolute e t h a n o l a n d 65 per c e n t S T E (0. 15 was passed through the cellulose column and washed with 5 ml of a solution containing 35 per cent absolute ethanol/65 per cent STE. The column was then washed with 5 ml of a solution containing 15 per cent absolute ethanol/85 per cent STE. These fractions were pooled, precipitated at --2 0 ° with a further 3 volumes of absolute ethanol and designated "single stranded I~NA". The column was finally washed with 5 ml of STE, precipitated with absolute ethanol as above and designated "double stranded" lgNA.

Single stranded 48S RNA from intracellular nucleocapsids was lyophilysed and dissolved in 50 ~l of buffer (20 mM Tris/HC1 p i t 7.8). Then 2.5 ~1 of T~ RNase (3.3 × 104 units/ml) and 1 ~zl BAP (4.5 units/ml) was added and the solution incubated for 30 rainutes at 37 ° C. After the addition of 150 y.1 of water, the digest was extracted three times with an equal volume of phenol and three times with an equal volume of diethyl ether. The finM aqueous phase was then lyophilysed. The dried RNA was dissolved in 70 ~1 of a 1 m~ solution of spermidine heated, for 3 minutes at 50 ° C and chilled rapidly on ice. The I~NA solution was incubated for 30 minutes at 37 ° C after the addition of 10~tl of 10 × kinase buffer (0.5 M Tris/HC1 p I l 9.5, 100 m~I MgC12 50 m~ Dithiothreitol), 10 al of [-a~p] ATP (ATP was lyophitysed and redissolved in the originM volume of water immediately before use), and 10 ~xl of polynucleotide kinase (1 unit/~l). The reaction was stopped by the addition of 100 al of Ammonium acetate (4 ~) 20 ~1 SDS (10 per cent w/v) and 10 tzl EDTA (0.2 ~). This mixture was extracted three times with an equal volume of phenol, three times with an equal volume of ether and precipitated at--200 with 2.5 volumes of absolute alcohol after the addition of i0 ag of 4S earrier RNA.

The RNA precipitate was dried and analysed by two dimensional gel electrophoresis as described (17) . Gels were dried under vacuum and exposed to X-ray film.

As measles virus grows poorly in tissue culture and the virion has a pleomorphic structure, the isolation of R N A from purified virus is difficult. Subsequently the yield of purified virion R N A is insufficient for adequate analysis. Therefore it was decided to use intraeellular I~NA from eytoplasm:ic nucleoeapsids for this study. As R N A from such a source is k n o w n to consist of a heterogenous p o p u l a t i o n (18, 19) with both positive and negati~e strands of various sizes (S~EPH~SO~-a n d T~R MEULEN, unpublished data), further purification was necessary to ensure t h a t only full-length negative stranded 48S I~NA was used in the final oligonueleotide analysis.

Initially, nueleocapsid R N A was analysed by eentrifugation on aqueous sucrose gradients as described in methods. I n Fig. 1 a, a t y p i e a l sedimentation profile is shown for nucleoeapsid R N A labelled with 32p in vivo. The m a j o r i t y of R N A in this sample sediments as a heterogeneously population of molecules below 30S, with only a small proportion sedimenting as an a p p a r e n t l y homogeneous peak at a b o u t 48S. The relative proportions of 48 S a n d heterogeneous IgNA varied from isolate to isolate, b u t the general p a t t e r n shown in Fig. 1 a was always observed. No lgNA species larger t h a n 48S were observed in nucteocapsids from infected cells. The 48S R N A was pooled, as shown in Fig. 1 a precipitated with 50 ~g of 4S carrier I~NA and 3 volumes of absolute ethanol, and analysed on a second sucrose gradient. This sample sedimented as an a p p a r e n t l y homogeneous species at 48S (Fig. l b ) , The sedimentation coefficient of this R N A species was indistinguishable whatever measles isolate was examined. W h e n the 48S R N A was further analysed under denaturing eonditons (Fig. l e ) , heterogeneous low molecular weight I~NA, with a sedimentation profile similar to t h a t of the subgenomie nueleoeapsid R N A seen in Fig. 1 a, is observed. As similar R N A was not a p p a r e n t of the previous aqueous gradient (Fig. l b ) it. was assumed t h a t this I~NA represented subgenomie positive stranded R N A which had hybridized to full length negative s t r a n d e d R N A during the extraetion and purification scheme. This subgenomic R N A was present in all preparations b u t varied in a m o u n t relative to the full-length 48 S RNA. 

As it was apparent t h a t the 48 S I%NA species contained positive sense R N A (see previous section), and t h a t other negative viruses, make full length positive sense strands (20) it was necessary to purify single strands of one sense only, before analysis of their T1 oligonucleotides could be performed. If 32P-labelled 48S R N A from t w o sequential R N A gradients is Mtowed to self-hybridize, and the various types of R N A separated by differential I%NA precipitation, a.pproximately 70 per cent of the radioactivity appears in the "single stranded" fraction and about 30 per cent in the "double stranded" fraction, with less than 2 per cent in the fraction containing small R N A species. This distribution of R N A species did vary from isolate to isolate, and to a lesser extent between experiments, with the highest proportion of double stranded R N A being 40 per cent of the total and the lowest level being 24 per cent. If the single stranded R N A All hybridizations were performed using radioactively labelled virus (LEC) RNA and unlabelled cellular RNA as described in methods. The level of hybridization was calculated as that proportion of molecules which remained TCA precipatable after digestion at 37°C for 30 minutes with 50 ~g/ml of Pancreatic Ribonuclease A and 170 Units/ml of T1 Ribonuelease Fig. 2 a, b from such a. purification scheme is hybridized to unlabelled messenger R N A from infected cells (Table 1) , it can be shown t h a t at, least 85 per cent, is protected from subsequent nuclease digestion and is thus assumed to be negative stranded. 

In order to compare the genomes of various measles isolates: unlabelled, single stranded, negative sense nueleocapsid I~NA was prepared from infected cell cytoplasm and purified in parallel with similar radio-labelled material as described in the previous section. This procedure was adopted as previous a t t e m p t s to aualyse these species of R N A , labelled in vivo, were not successful, due to insufficient incorporation of isotope. The R N A was then digested with T1 endonuclease, labelled with 51-[~ a2p] A T P and the resulting radioactive oligonucleotides analysed b y two dimensional electrophoresis as described in methods. Autoradiograms of such two-dimensional gels with Tt oligonucleotides from two acute measles isolates, one isolate of acute measles encephalitis and three S S P E isolates are shown in Fig. 2 . Translucent tracings of these autoradiograms were m a d e (Fig. 3) and these tracings were then compared.

Only the oligonueleotides beneath the line drawn in Figs. 2 and 3 were included in the analysis. I t was not possible to co-electrophorese the digests of two or more isolates for this comparison, as the large n u m b e r of oligonueleotides in one gel would preclude an accurate analysis of the p a t t e r n thus obtained. As seen in Fig. 2 , the a p p a r e n t m o l a r i t y of the larger oligonueleotides is not uniform. This is assumed to be either the result of the co-migration of 2 or more oligonucleotides or an effect of the seconda~" structure of the oligonucleotides affecting the relative efficiency of the kinase reaction. Similar observations have been noted when the genomes of several R N A viruses were analysed b y this method (2I). Also the mobility and relative concentration of the smaller oligonueleotides differs in some aspects from t h a t obtained from in vivo labelled I~NA molecules. I t is presumed t h a t this loss of small oligonucleotides from the digest occured during the ethanol precipitation step, and is also a result of the decrease in the concentration of bisaerylamide in the second dimension of P A G E , which was introduced in order to facilitate the drying of the gels. However, these observations were judged not to affect the v a l i d i t y of the conclusions as identical results were obtained from duplicate or triplicate preparations from each isolate; and identical p a t t e r n s were also obtained when R N A s from different passage numbers of the same isolate were compared. Table 2 summarizes the main differences and similarities between the various isolates examined. Thus it can be demonstrated that all isolates examined show clear differences, even though they appear virtually identical when examined by classical serology. Although 8 oligonucleotides were common to all isolates examined, none were found to be specific for SSPE isolates or for measles isolates. Similarly none were found to be characteristic for all isolates from cases of encephalitis. If the total number of characteristic changes (i.e. the sum of the oligonueleotides shown to be specific for an isolate and the oligonucleotides specifically absent in an isolate) observed in individual isolates are compared, no clear distinctions can be made between isolates from acute cases of measles acute encephalitis or SSPE. In addition, when the LEC isolate of SSPE was compared to either the Edmonston strain of measles vaccine (J. 1~. SWEPHENSO~ and V. TER MEULEN, unpublished observations) or to another isolate from acute measles, WOODFOLK (22) , again no clear differences were found in the oligonucleotide maps to distinguish SSPE isolates from measles isolates.

Although many attempts have been made by morphological, antigenic and biochemical techniques to distinguish SSPE viruses from isolates derived from acute infections, no-one has been able to establish criteria specific for SSPE viruses (2, 7, 23) . As previous studies have relied either on detecting gross genetic, structural or antigenic changes, we have chosen to analyse the complete genome of various viral isolates by comparing oligonucleotides generated by digestion with T1 endonuetease. By using this technique, differences as small as one base change, occurring throughout, the genome of the virus can be detected.

As it has proved difficult to obtain sufficient quantities of RNA from purified virus, intracellular nueleocapsids have been used as a source of viral I~NA. However, as such material can contain significant amounts of cellular ribosomal RNA, messenger RNA and DNA, a purification scheme was devised to minimise the possibility of contamination from these cellular components. The nueteoeapsids were therefore purified by eentrifugation under conditions which ensured that all ribosomes, ribosomal subunits, messenger RNP, and nuclei were pelleted, while the viral nucleocapsids remained suspended at the sucrose interface. Nueleocapsid RNA prepared from such material contained no detectable amounts of ribosomal or transfer RNA when centrifugated on a sucrose gradient and scanned at 0.D260. In addition, when in vitro-labelled oligonucleotides from nucleoeapsid RNA were compared to those oligonucleotides from 28 S ribosomal RNA, no similarities were observed (data not shown). Thus indicating that no contaminating 28S ribosomal I~NA could be detected in RNA isolated from viral nueleocapsids. Furthermore, 48S nucleocapsid RNA did not bind to oligo dT cellulose nor was it capable of stimulating protein synthesis when added to a nuclease-trcated cell-free system from rabbit reticulocytes. A further complication of using infected cell material is that a significant proportion of the nucleocapsid RNA consists of subgenomic species (18, 19) . These species appear to have been successfully removed by sedimentation on sucrose gradients. Also in infected cells, both positive and negative strands are necessary for the replication of paramyxoviruses (20) , and can contaminate preparations of 48S I%NA from infected cells. These positive strands have been removed b y allowing the R N A to self hybridize and then separating the excess single-strands b y differential ethanol precipitation. R N A p r e p a r e d b y this technique was shown to be at least. 85 per cent measles-specific negative sense R N A b y hybridization to infected cell messenger I~NA.

W h e n single-stranded, full length, negative sense I%NA from several measles isolates were compared b y analysis of the oligonucleotides generated from ~ T1 digest, a discreet family of oligonucleotides was generated from each isolate. This family of oligonucleotides was similar in number and distribution to those generated from negative-strand viruses with similar size genomes, such as VSV (24) or Spring Viraemia of Carp Virus (25) . No evidence of homopolymeres, such as the poly A or poly C tracts found in positive strand viruses like poliovirns, FMDV, Coronavirus or R N A t u m o u r viruses was found. Although all isolates a p p e a r to be virtually identical antigenically, when examined b y classical serology they have only about 15 per cent or less of their specific T1 oligonueleotides in common. Moreover, no oligonueleotides, specific for SSPE isolates or measles isolates, were observed, and concomitantly, no oligonucleotides were characteristie of encephalitic isolates. W h e n the n u m b e r of detectable specific differences in all viral isolates was compared, all isolates whether from eases of acute measles, acute measles encephalitis or S S P E show similar differences from each other.

Therefore, if we assume t h a t all measles isolates have a common p a r e n t a l ancestor, viruses isolated from a chronic infection, i.e. S S P E do not a p p e a r to have m u t a t e d a t a faster rate t h a n those isolated from eases of acute measles.

However, the techniques employed in these studies cannot analyse all the potential differences in the genomes of these viruses, and such studies m u s t await the future application of molecular cloning and eDNA sequencing to this question.

Subaeute selerosing paneneephalitis, A review

Subacute sclerosilIg panencephMitis

Slow virus infections of the nervous system: virological, immunological and pathogenetic eonsiderations

Differences between the intra-eellular polypeptides of measles and subacut.e selerosing panencephalitis virus

A comparison of polypeptides in measles and SSPE virus strains

Intranuelear polypeptides of measles and subacute selerosing paneneephMitis virus-infected cultures

Measles Virus and Its Biology

Antigenic relationship between measles and canine distemper virus. Comparison of immune response in animals and humans to individual virus-specific polypeptides

Membrane protein of subaeute sclerosing panencephalitis and measles viruses

Antigenic characterization of measles and SSPE virus haemagglutinin by monoelonat antibodies

Characterisation of virus-specific RNAs from subacute sclerosing panencephalitis virus-infected CV-1 cells

R N A homology between subaeute sclerosing paneneephalitis and measles virus

ChemieM linkage of nucleic acids to neutral and phosphorylated cellulose powders and isolation of specific sequences by affinity chromatography

Isolation of infectious measles virus in measles encephalitis

Characterisation of virus-specific messenger RNAs from avian fibroblasts infected with fowl plaque virus

Purification and properties of the replicative intermediate of the R N A bacteriophage R 17

Genetic variation of neurotropic and non-neurotropie murine eoronaviruses

Persistent infection of cells in culture by measles virus. III. Comparison of virus-specific R N A synthesized in primary and persistent infection in HeLa cells

Biological and biochemical characterization of a latent subaeute sclerosing paneneephatitis virus infection in tissue cultures

Pius and minus strand leader RNAs in negative strand virus-infected cells

01igonueleotide mapping of non-radioactive virus and messenger RNA

The Virological State in Subacute Selerosing Paneneephalitis

Subaeute Sclerosing Panencephalitis: Characterization of the Etiological Agent and its Relationship to the Morbilliviruses. Aspects of Slow and Persistent Virus Infection

Spring Viremia of Carp Virus R N A and Virion-associated Transeriptase Activity

Oligonueteotide fingerprints of R N A species obtained from Rhabdoviruses belonging to the Vesicular Stomatitis Virus subgroup

We would like to thank Magdalene Pfohler for excellent technical assistance and Jane Brctt and Helga Kriesinger for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinsehaft.

Authors' address: Prof. Dr. V. :rEg MEULEN, Institut f/ir Virologie, Universit/~t Wfirzburg, Versbacher Strasse 7, D-8700 W/irzburg, Federal Republic of Germany.Received October 21, 1981