Formation of Influenza Virus Proteins

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JOURNALOFVIROLOGY, June 1973,p.823-831

Copyright6 1973 AmericanSocietyforMicrobiology

Vol.11, No.6

Printed inU.SA.

Formation of Influenza Virus Proteins

HANS-DIETER KLENK AND RUDOLF ROTI

Institut furVirologie, JustusLiebig-Universitdt, 6300 Giessen, Germany Received forpublication 1 February 1973

Eight virus-specific proteins have been found in chicken embryo fibroblasts

infected with fowlplague virus. Among themaretwoglycoproteins whicharethe

constituentsofthe hemagglutininonthe virus particle.Theyarederived froma

largeprecursor glycoprotein by cleavage ofa covalentlinkage. The reaction can

be blocked by theprotease inhibitordiisopropylfluorophosphate and the amino

acid analoguefluorophenylalanine. This indicates thatapeptide bond is cleaved.

If infected cells are kept at 25 C, a temperature at which virus maturation is

inhibited, theprecursorglycoprotein is cleavedatasignificantlyslower ratethan

at37 C. It appears, however, that areducedsynthesis of the carbohydrate-free

envelope protein is responsible for the block of virus maturation at25 C rather

thanthelower cleavage rate of the precursor.

The protein constituents of influenza virus consist ofcarbohydrate-free polypeptidesand of

glycoproteins. One of the carbohydrate-free polypeptides forms the protein subunit of the nucleocapsid (10, 16), anotherone isa

constit-uent of the viral envelope where it coats the inner side of the lipid layer (2, 20, 21). The glycoproteins are located on the outside of the lipid layerasconstituentsofthe surface projec-tionsorspikes (2, 12, 20).

Inaddition tothe constituent proteins of the virion, nonstructural polypeptides have been described in influenza virus-infected cells (4, 13,

15, 22). Some ofthese nonstructural proteins

are precursors of the viral hemagglutinin, and

theiranalysis throwssomelightonthesequence

ofeventswhichareinvolvedinthebiosynthesis

ofthisenvelopeconstituent. The first identifia-blestepistheglycosylationof apolypeptideto a

largeglycoprotein (13). Itwassuggestedfirstby Lazarowitz et al. (15) that this glycoprotein is cleaved into two smaller glycoproteins. This

concept has been confirmed in the present

communication by fowl plague virus (FPV)

grown in chicken embryo fibroblasts.

Experi-ments will be described which give further

information about the appearance of virus-specific material ininfectedcells, and

particu-larly aboutthenatureof the cleavage reaction.

MATERIALS AND METHODS

Virus and cells. The Rostock strain of FPV was used in all experiments. It was grown in primary cultures of chicken embryo fibroblasts (23). Virus stocksweregrown inembryonated eggs.

Virus assay. Virusyields were assayed by hemag-glutination titrations (3) and plaque assays (23).

Chemicals andisotopes. Reagents for polyacryla-mide gels and diisopropylfluorophosphate were ob-tained fromServa,Heidelberg, Germany; DL-p-fluoro-phenylalanine from Calbiochem, LosAngeles, Calif. Protein [U-_4C]hydrolysate, L-[U-14C]valine (300 mCi/mmol),L-[4, 5-3H]leucine (1.0Ci/mmol),L-

[2,3-3H]valine (1,5Ci/mmol), L-[3,5-3H]tyrosine (1,0Ci/ mmol), and D-[1_-3Hjglucosamine hydrochloride (1,1 Ci/mmol) werepurchased fromAmersham, Bucking-hamshire, England.

Labeling of infected cells. Confluent monolayers

ofinfected cellswereinoculatedat amultiplicityof10 to 50PFU/cell.Aftera30-minadsorption period,the inoculum was removed and replaced by minimal medium (6). When experimentswerecarriedoutat 37 C, the mediumwasremoved andreplaced bymedium containing radioactive isotopes 4 h after infection. When experiments were carried out at 25 C, the medium was replaced 24 h after infection.

Radioac-tive isotopes were used at the following

concentra-tions: [14C

]protein

hydrolysate,5 sCi/ml; [14C]valine,

5 MCi/ml; ['H]amino acids, 50MCi/ml;

[3H]glucosa-minehydrochloride,50,Ci/ml.The medium

contain-ingtheisotopeswasleftonthe cells for10or60min.

When theexperimentwas stopped atthis point, the

monolayer waswashed three times with cold

phos-phate-buffered saline (PBS) (5), scraped offwith a

rubber policeman in 1 ml ofPBS, and storedat -20 C.When thepulsewasfollowedbyachaseperiod,the radioactive medium was removed and the cellswere

washed three times with mediumcontaininganexcess

of thecoldanalogueoftheradioactive label (10 gmol of aminoacid/ml) andincubated inthismedium. At the endofthechaseperiod the cellswerewashedand scrapedoff asdescribed above.

Polyacrylamide gel electrophoresis. The cells werehomogenized by sonic treatment. Samples of 100

juliterswereused for gelelectrophoresis. Proteins were

dissociatedwithsodiumdodecyl sulfate and mercap-toethanol and separated by electrophoresis on 10% 823

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acrylamide gels as described (1). The slicing and processing of gels for the determination of radioactiv-ity by liquidscintillation has been reported previously (11).

RESULTS

Viral proteinsynthesis in infected cells at 37 C. We reported previously that

polyacryla-mide gel electrophoresis reveals eight fowl plague virus-specific polypeptides in chicken embryofibroblasts (13). Sixofthese are

struc-tural polypeptidesofthe virion.They comprise

two hemagglutinin glycoproteins,

HA,

(mol wt

49,000) and HA2 (mol wt 32,000), glycoprotein

NA (molwt 45,000) which has been attributed

totheneuraminidase,acarbohydrate-free

enve-lope polypeptide M (mol wt 26,500), the

nu-cleocapsid protein NP (mol wt 60,000), and another internal protein P (mol wt 83,500). In

addition to these structural polypeptides,

in-fected cells contain two virus-specific proteins whichare notpresent inthematurefowlplague

virion: protein NS (molwt 25,000) is found in

largeamounts in thenucleus, proteinHA (mol wt76,000)hasbeensuggestedtobeaprecursor of the hemagglutinin glycoproteins

HA,

and HA2 (15). This pattern obtained 4 h after infection in a 1-hpulse with radioactive amino

acids comprises all fowl plague virus-specific

proteins which we were able to detect in

in-fected chickenembryofibroblasts.

The pattern is different ifthe cells obtain a

pulse ofonly 10 min in length (Fig. 1). Ifthe

pulse is not followed by a chase period, the

carbohydrate-free polypeptides P, NP, M, and NSare clearlyrecognizable. Theprotein show-ing the highest incorporation of radioactivity

under these conditions is proteinHA. Proteins

HA1and HA2 cannotbedetected.

There is a shift in the virus-specific protein

pattern ifthe 10-min pulseisfollowed

by

chase

periodsofincreasing length(Fig. 1and2).The radioactivity in proteins P, NP, and M remain

constant and in the same relative proportions

independently ofthelengthofchase. However,

afterachaseof5min,peakHAisdecreased and

peaks HA1 andHA2aredetectable. If the chase period is extended to 60 min

peak

HA has

almost disappeared, whereas the label in

peak

HA1

and that inHA2have increased

concomi-tantly.Theseresults indicatethat

protein

HA is lessstablethantheothervirus-specificproteins

and thatproteins HA1andHA2aretheresult of

the breakdownofprotein HA.

Viral protein synthesis at 25 C. In FPV-infected cells which are kept at 25

C,

virus-specific components such as RNA

polymerase,

ribonucleoprotein antigen, hemagglutinin, and

neuraminidase are synthesized, even though at

aslowerrate than at 37 C. However, infectious

virions are not released. These data suggested

that, at 25 C,virus maturation is inhibited at a

late stage(18).

It was ofinterest which ofthe virus-specific

proteins wereformedat 25C.Cells were kept at

this temperature for 24 h after infection and

then labeled in a 1-h pulse with radioactive amino acids. At the end of the experiment the

cells contained a considerable amount of

he-magglutinin and they showed hemadsorption

and agglutinability by concanavalin A (17).

However, neither hemagglutinin nor infectious

particleswerereleasedintothemedium.Figure 3shows a comparison ofFPV proteins

synthe-sized at 25 C 24 h after infection with those

synthesized at 37 C 4h after infection. In both

cases the proteins have been labeled in a 1-h

pulse with radioactive amino acids. Several

striking differences in the polypeptide profiles

at thesetwotemperatures can beseen. At 25C

protein HA shows the highest incorporation of

radioactivity. Ascan beseen fromdouble-label experiments with radioactive amino acids and

glucosamine,proteinHAisglycosylatedat 25 C

(Fig. 4). The other FPV-specific glycoproteins,

HA1

andHA2, are missing at 25C.

However,ifthepulseat 25C isfollowed by a

chase periodatthesametemperature, it canbe

seen that proteins

HA1

and HA2 are being formed at the expense of protein HA (Fig. 5).

Similar results were obtained ifthe chase was

carriedout at 37 C (Fig. 6). These experiments indicate that HAisconvertedinto

HA1

andHA2 at 25 C, but at a slower rate than at 37 C.

Therefore, the formation of the hemagglutinin glycoproteins does not seem to be basically altered at 25 C.

A striking difference, however, between the

polypeptide profilesat 25 C andat37Cwasthe

apparent absence of protein M at the lower

temperature. Whereas, at 37C, proteins M and

NS can be identified as a double peak or as a

peak and a shoulder, at 25 C protein M could

notbediscriminatedinthepolypeptide profile.

Thefastest migratingpeakclearly corresponds

to proteins NS (Fig. 4). This was a constant findingin many experiments.

However, it is not clear from the data

pre-sented here whether protein M is completely

missingorwhethersmallamounts arehiddenin a relatively large NS peak. Future studies

employingother means ofdistinguishing these polypeptides, such as peptide mapping or im-munological assays, shouldthrowmorelighton

thisproblem.

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INFLUENZA VIRUS PROTEINS

3H

dm

NP

I

HA1

NS

'I

HA2

I

V0

20

301.0 50W6

70

80 90100

Fraction

number

FIG. 1. Polyacrylamide gel electrophoresis ofFPVproteinssynthesized in chicken embryo fibroblasts. Cells

werelabeled4hpostinfectionbya10-minpulsewith [3H]leucine. Top, Cells were scraped off immediately after thepulse;bottom, the pulse was followed by a chase period with cold leucine for 5 min. Migration in this and all subsequent gels is from the left to the right. Arrows indicate the position of polypeptides. The neuraminidase glycoprotein has not been marked on this and most of the other figures in this paper. There is evidence that it migrates in front of theHA1peak (13).

Inhibition of the cleavage of glycoprotein from glycoprotein HA. Since the sum of the HA. The data described above conform with molecularweightsofHA1 and HA2issimilarto those of Lazarowitz et al. (15). They suggest that of HA, a cleavage reaction might be

that glycoproteins

HA,

and HA2 are derived involved.Thelinkage which is affectedby this

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PHA NPH1

2M

NS

f

30 40 50 6070

Fraction number

FIG. 2. Polyacrylamide gel electrophoresisofFPVproteins. Cellswereexposed toapulse with [3H]leucineas describedinFig.1.Top,Thepulsewasfollowed bya15-minchaseperiod; bottom,thepulsewasfollowed bya 60-minchaseperiod.

cleavagemustbeof covalentnatureandcannot

be a disulfide bond, because protein HA is

stable underthe conditions of the

electrophore-sis employedinthis study, i.e., inthepresence

ofSDS andareducingagent.Thesefindingsare

compatible with the hypothesis that the

cova-lentlinkage which iscleaved is apeptide bond

and that proteolytic enzymes are involved in

this reaction.

To testthis hypothesis, proteaseshave been

blockedby the useof theinhibitor

diisopropyl-fluorophosphate (DFP). This substance had

beenemployed successfully in the elucidation of

large polypeptides as precursors of poliovirus

proteins (9). The effect ofDFPonthesynthesis

of influenza virus proteins is shown in Fig. 6.

Infectedcellswerekeptat25C for 24 h and then

labeled for 1 h with radioactive amino acids.

Theonlyglycoprotein labeled under these

con-ditions is protein HA. If the pulse at 25 C is

followed by a chase period at 37 C, HA

de-creaseswithaconcomitantincrease of

HA,

and

HA2. However, if DFP is addedtothe medium

during the chaseperiod, the shift of the

radioac-tivity from HA to

HA,

and HA2 is clearly

inhibited. Thisprovesthat HA isconverted into

HA,

and HA2by cleavage ofapeptide bond.

Further evidence for suchaprecursor-product

relationship stems from experiments with an

amino acid analogue. It has been known for

some time that, under appropriate conditions,

L-fluorophenylalanine inhibits the formation of 3H dpm

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PIHA

_4H

_I

NPHAl

_I

3H

dpm

4

30

40

50

HA2

M

NS

14C

dpm

[image:5.493.52.449.49.332.2]

Fraction

number

FIG. 3. Polyacrylamide gel electrophoresis ofFPVproteins synthesized at 25 C (@-*). The cells were

labeled for 1 h with [3H]leucine, ['H]tyrosine, and [3H]valine. For comparison, virus-specific proteins synthesized at 37Candlabeledfor1h with ['4Clproteinhydrolysateweresubjectedtocoelectrophoresisonthe samegel(0--0).

14C

dPm

P

HA

NP

NS

U0

20 30

40Q50

60 70

8090

Fraction number

FIG. 4. Polyacrylamide gel electrophoresis ofFPVproteins synthesizedat25 C. Cellswereexposed for1hto

apulsewith ['4CJaminoacids(@-@)and [3HJglucosamine hydrochloride(0--O).

fowl plague virions and biologically active he-magglutinin although nucleocapsid protein is

synthesized as detected by immunological methods (19, 23). Polyacrylamide gel electro-phoresis reveals that, in the presence of

L-fluorophenylalanine (FPA) only proteins P, HA,

and NPare labeled (Fig. 7).Proteins HA1 and

HA, cannot be detected. On the gel shown in

Fig. 6, proteinsM and NS cannot be detected either. The absence of these latter proteins, however, was not a constant finding. This

experiment indicates that, in the presence of 827

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P

HA NP

NS

I

i

I$I

10

20

30

40

50

60

70

80

90

100 Fraction

number

FIG. 5. Polyacrylamide gel electrophoresis ofFPVproteins synthesized at 25 C. Cells were labeled 24 h postinfection bya1-hpulsewith [3H]leucine. Top,Cellswerescraped offimmediately afterthepulse;bottom, thepulsewasfollowed bya3-hchase at 25 C.

FPA, cleavage of protein HA is inhibited. The

cleavage reaction appears to be completely

blocked, not simply retarded, because even

after chase periods of several hours

HA,

and

HA2cannot be detected. DISCUSSION

Theprofiles of fowl plague virus-specific

pro-teinsreported in this and inaprevious

commu-nication (13) agree well with the findings of

othergroups. Lazarowitzetal. (15)found eight

virus-specific polypeptides in cells infected with

the WSN line of influenza A virus, each of

which can be coordinated to a corresponding

FPV protein. The polypeptide pattern ofFPV

reported bySkehel (22) is, in general, similarto

ourresults, although thereare some

discrepan-cies inthe number aswell asintheproportions

of the various proteins. Skehel described two

additionalpolypeptides (P1 and 9 accordingto

hisnomenclature) whichwehavenotbeen able

to detect. It remains to be seen whether this

discrepancy is duetodifferences inthelabeling

procedures or in the electrophoresis systems.

There are striking differences in the relative

amounts ofradioactivity incorporated into the

various polypeptides between our data and

Skehel's.Theycanbeexplainedbythedifferent

labeling techniques. Whereas Skehel used

35S-methionine as protein label in most of his

experiments, we employed 3H- or "4C-amino

acids. Since methionine isincorporated

prefer-entially into the carbohydrate-free polypeptides

(15), this methodappearstobe lesssuitable for

theanalysis ofthe influenza virusglycoproteins.

Three of the four influenza virus-specific

glycoproteins (HA, HA1, HA2) werefound to be

associatedwith the viralhemagglutinin (8, 14).

There is evidence for the presence of another

glycoprotein which migrates as a shoulder on

3H

dpnm

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14C

dpm

P

HANP

NS

mrn-

I

+ 4

PHIA

4'4

HA2

4

NS

HA2

NS

4 4

20

30

40

50

e

[image:7.493.77.395.55.497.2]

Fraction number

FIG. 6. Polyacrylamide gel electrophoresis ofFPVproteins synthesizedat 25 C. Cells were labeled 24 h

postinfectionbya1-hpulsewith [14C]valine. Top,Cellswerescraped off immediatelyafterthepulse; middle,

thepulsewasfollowed bya1-h chaseat37C; bottom,thepulsewasfollowed bya1-h chase at 37 C, and the

mediaaddedtothecellsduringthe chaseperiodcontaineddiisopropylfluorophosphate (20 Mmol/ml).

the frontslopeofthebroad HA1 peak and which tentatively has been assignedtoasubunit of the viral neuraminidase (13). This assignation

seems to be justified by comparing the gel

patterns of fowl plague virus and other

influ-enzastrains (7). However, because of the poor

resolution and possibly the small amount of

protein NA, little information on the viral neuraminidase can be obtained from the data presented inthis publication.

The observations of Lazarowitz et al. (15)

suggested that glycoproteins HA1 and HA2 are

formed by cleavage of the large glycoprotein HA.Theirfindingsareconfirmedby the results

ofourpulse-chase experimentsat25 C as well as at37 C which also substantiate that a precur-sor-product relationship exists between HA and

HA,

and HA2. The block ofthisreaction by a

protease inhibitor proves that it is the cleavage

ofapeptide bond by which HA is converted into

HA1 andHA2.

Further evidence for proteolytic cleavage of

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PFHA

NtPLNA

I,

II

I II I

I

HA2

M NS

lMNS

14C

dpm

10

30

40

50

60

70

80

90

[image:8.493.57.449.61.302.2]

Fraction number

FIG. 7. Polyacrylamide gelelectrophoresisofFPVproteins synthesizedin thepresenceof p-fluorophenylala-nine. Two hourspostinfection theamino acid analogue (300 ug/ml) was added tothe medium. Four hours

postinfection the cells were exposed to a pulse with [3H]valine for 1 h (a-_). During the pulse

p-fluorophenylaianine waspresent in the medium. Inacontrolexperiment, infected cells weregrown in the

absence ofp-fluorophenylalanine, labeled4hpostinfection for1hwith [14C]valine,andsubjectedto

coelectro-phoresison thesamegel(0- -0).

HAstemsfrom theexperimentswith FPA. Itis

known that this amino acid analogue is incor-porated into

poliovirus-specific

precursor

pro-teins, thereby

blocking

their

cleavage

(9).

Ac-cordingly, HA is still

being

formed in the presence ofFPA, but cleavage asindicated by

the appearanceof

HA,

andHA2isinhibited.It

appearsthat, bytheincorporationofthe amino acid analogue, the configuration of the HA

polypeptide chain is altered insuchawaythat

it can no longer be properly cleaved. This

concept is also supported

by

the

finding

that HA synthesized in thepresence of FPAcan no longer exert

hemagglutination

(20). Our

find-ings agree with those of Lazarowitz et al.

(Virology, in press), who demonstrated the proteolytic nature ofthe cleavage with trypsin invitro. These authors also havegoodevidence that the enzymes involved are host cell

speci-fied.

At 25 C cleavage ofglycoprotein HA takes

placeat asignificantlyslowerratethanat37C.

This, however, does not

explain

the block of virusmaturation at25C, because thereisgood

evidence that thecleavagereaction isnot

essen-tial forvirusassembly.Thisissuggested

by

the

finding that large amountsof

glycoprotein

HA

are present in mature virions of several

influ-enzastrains (2, 8, 12, 20). Furthermore,

experi-ments ofLazarowitz et al. (Virology, in press)

show that cleavage of glycoprotein HA is not required for the expression of the biological

properties ofthe virion, including hemaggluti-nation, butthat it appearstobe a nonessential

result of eventsoccurring in infected cellswhich

are undergoing cytopathic effects. At 25 C the

hemagglutinin glycoproteins are not only

prop-erly formed,buttheyarealsoincorporated into

the plasma membrane ofthe cell. This can be shown by a positive hemadsorption reaction (unpublished results) and byagglutinability of infected cells withconcanavalin A (17).

There-fore, the block of virus maturation at 25 C appears to be neither at the stage of the biosynthesisofthehemagglutinin glycoproteins

nor at the stageoftheirincorporation into the

cellsurface.

Our data suggest that at 25 C the

carbohy-drate-free envelope protein M is made insuch

small amounts that it cannot be detected on

polyacrylamide gels as a distinct peak in the polypeptide profile. Evenat 37Cthis proteinis

found in the infected cell in relatively small

amounts compared to those in thevirion, and

therefore it hasbeen suggested that the

synthe-sis ofproteinM mightbetightlycontrolled and

3H dpm

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arate-limiting stepin virusreplication (13, 15). These findingsarecompatible with the

hypoth-esis that at 25 C the synthesis of protein M is reduced to a rate which no longer permits proper envelope assembly and, therefore, virus maturation.

ACKNOWLEDMENT

We thankElfriede Otto and Michaela Orlich for excellent technical assistence. This workwassupported by

Sonderfor-schungsbereich47(Virologie).

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