The EMBO Journal vol.5 no.12 pp.3103-3109, 1986

Kinetics of endosome acidification detected by mutant and wild-

type Semliki Forest virus

M.C.Kielianl, M.Marsh2 and A.Helenius

Department of Cell Biology, Yale University School of Medicine, NewHaven, CT 06510, USA

'Present address: Department of Cell Biology, Albert Einstein College ofMedicine, Bronx, NY 10461, USA2Present address: Institute of Cancer Research, Chester Beatty Laboratories,Fulham Road, London, UK

Communicated by L.Kaariainen

The fusogenic properties of Semliki Forest virus (SFV) andits mutants were used to follow the kinetics of acidificationduring the endocytic uptake of virus by BHK-21 cells. It haspreviously been shown that the low pH of endocytic vacuolestriggers a conformational change in the SFV spike glycopro-tein, activating membrane fusion and initiating virus infec-tion. This conformational alteration was here shown to occurin endosomes and to follow the same time course as the in-tracellular fusion reaction, demonstrating that fusion occursrapidly after virus exposure to endosome acidity. The kineticsof endosome acidification were monitored using wild type (wt)SFV and fus-1, an SFV mutant with a lower fusion pHthreshold. The results presented here demonstrated that wtand mutant virus were internalized with a tl/2 of 10 min, andthat endosomes were acidified to the wt threshold of pH 6.2with a tl/2 of 15 min. In contrast, endosome pH reached thefus-1 threshold of 5.3 with a much longer tl/2 of 45 min. Thesubsequent degradation of SFV in lysosomes had a t1/2 of 90min. It was found that after the initial uptake of virus fromthe plasma membrane, its transit through the endocyticpathway, exposure to endosome acidity and eventual deliveryto lysosomes were markedly asynchronous.Key words: endocytosis/endosome acidification/Semliki Forestvirus/virus fusion/virus penetration

IntroductionA number of enveloped animal viruses are now known to infectcells by a mechanism that involves endocytosis followed by acid-activated membrane fusion in intracellular vacuoles (reviewedin Helenius et al., 1980a,b; Dimmock, 1982; Lenard and Miller,1982; White et al., 1983; Marsh, 1984; Kielian and Helenius,1986). As best studied for Semliki Forest virus (SFV, an

alphavirus), the endocytic process responsible for internalizingthe virus particles appears identical to the process of receptor-mediated endocytosis described for numerous physiologic ligandssuch as low density lipoproteins and polypeptide hormones(reviewed in Goldstein et al., 1979; Steinman et al., 1983;Helenius et al., 1983). Upon binding to cell surface receptors,SFV is internalized in coated pits and coated vesicles and trans-

ported to the endosome compartment.The endosomes [also called receptosomes (Willingham and

Pastan, 1980) and CURL (Gueze et al., 1983, 1984)] constituterecently recognized prelysosomal organelles that play a central

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role in the endocytic pathway. They serve as intermediates inplasma membrane recycling and in the transport of ligands, fluid-phase components and receptors to lysosomes. They are involv-ed in the dissociation of ligand-receptor complexes and in themolecular sorting, processing, storage and regulation of recep-tor expression (endosome functions are reviewed in Brown etal., 1983; Helenius et al., 1983; Hopkins, 1983; Pastan and Will-ingham, 1983; Bergeron et al., 1985). Many of these functionsdepend on the low endosomal pH (5-6.2), which is generatedand maintained by proton-translocating ATPases present in thelimiting membrane (reviewed in Meilman et al., 1986). Althoughcurrently an active focus of research, the biogenesis, fate, com-partmentalization and biochemical nature of these complexorganelles have not been defined.SFV has proved a useful marker for endosome analysis as it

can be readily followed through the various stations of thepathway both morphologically and biochemically (Marsh et al.,1983; Helenius, 1984; Helenius et al., 1985; Kielian andHelenius, 1985). The exposure to acidic pH in the endosomestriggers a conformational change in the spike glycoproteins ofthe incoming virus particles, which then mediate the fusion ofthe viral membrane with the endosomal membrane. As a resultof the fusion reaction the nucleocapsid is released into the cyto-plasm in infectious form. The virus spikes that are integratedinto the endosomal membrane during fusion and any unfusedvirus particles are subsequently transported to the lysosomes anddegraded. Weak bases and carboxylic ionophores, which elevatethe pH in the endosomal compartment, block virus infection bypreventing the fusion reaction (Helenius et al., 1982; Marsh etal., 1982).Here SFV's fusion activity and the acid-induced changes in

its spike glycoproteins were used to examine the properties ofendosomal acidification in BHK-21 tissue culture cells. Wild type(wt), mutant and revertant viruses were employed. It was foundthat incoming viruses encountered a progressively decreasing pHin the endosomes, that endosomes were heterogeneous in termsof pH and that viruses with different pH thresholds for fusionpenetrated with widely different kinetics.

ResultsEndocytosis and acidification of SFVThe passage of SFV through the endocytic pathway was deter-mined biochemically using metabolically radiolabeled viruses.Four different events involving the virus particles were monitoredusing quantitative assays: (i) endocytosis, (ii) exposure to acidpH, (iii) penetration of RNA into the cytoplasm, (iv) the degrada-tion of viral protein in the lysosomes. In each case trace amountsof virus were allowed to bind to cell monolayers on ice. En-docytosis was initiated by rapidly warming the cells to 37°C andstopped by cooling to 0-4°C.For these analyses, both wt SFV and a mutant SFV calledjfs-1

were used. This mutant has been previously isolated and partial-

3103

M.C.Kielian, M.Marsh and A.Helenius

ly characterized in our laboratory (Kielian et al., 1984). It wasselected to have a lower fusion pH threshold than the parent virus;as determined biochemically or by pH-induced fusion betweeninfected cells, the pH threshold ofJiis-1 is approximately 5.3 com-pared to the wt 6.2 (Kielian et al., 1984). Consistent with thisphenotype, the mutant is more sensitive to lysosomotropic agentsthan wt SFV. However, it enters BHK cells normally andreplicates sufficiently well to allow isolation of radioactively label-ed forms. It has also been shown thatfius-1 penetrates from withinthe endosomal compartment (Kielian et al., 1984), which sug-gests that some endosomes are acid enough to trigger the con-formational change needed for _fus-1 fusion.The rate and efficiency of initial internalization of wt and mu-

tant SFV were determined by allowing radiolabeled virus to bindto cells in the cold. Endocytosis was initiated by rapid warmingof the cultures to 37°C. After varying times at this temperaturethe cells were placed on ice, digested with proteinase K in thecold to remove the viruses that remained on the cell surface andthe remaining radioactivity quantitated. As previously shown(Marsh and Helenius, 1980), > 90% of the cell surface viruseswere removed by the protease, and <10% of the [3H]uridinelabel of endocytosed viruses was released back into the medium.This allowed easy quantitation of virus endocytosis. As shownin Figure lA, both wt and fiis-l SFV were endocytosed with at1/2 of 10 min, and virtually all the bound viruses (>95%) wereinternalized within 30 min (see also Marsh and Helenius, 1980).BHK cells thus appeared to treat both viruses identically duringtheir endocytic uptake.

Taking advantage of the irreversible effect that low pH hason El, one of the transmembrane spike glycopolypeptides ofSFV, the kinetics by which the incoming viruses were exposedto acid pH were determined. Recent studies (Kielian andHelenius, 1985) have shown that in vitro acid treatment causesirreversible conformational alterations in both El (51 kd) andE2 (52 kd). As a result, their sensitivity to trypsin and other pro-teases is altered. While E2 becomes very trypsin-sensitive, Elis converted from a trypsin-sensitive to a trypsin-resistant con-formation. The change in both polypeptides occurs with the samepH threshold and kinetics as the activation of fusion. Further-more, it has been shown that the conversion of El is also observedduring the endocytic uptake of virus into cells (Helenius et al.,1985).

[35S]methionine-labeled wt virus was bound to BHK cells inthe cold, the cells rapidly warmed to 37°C as in Figure 1 andthen lysed in Triton X-100 after various times of uptake. Ali-quots of the lysates were incubated with or without trypsin andanalysed by SDS-PAGE under non-reducing conditions whichallow the resolution of El from E2. The trypsin-resistant Elradioactivity was directly quantitated by excising the gel band.As shown in Figure 2, the conversion of El to trypsin resistanceoccurred with a tj/2 of 15 min. About 45% of the total endocytos-ed El became trypsin-resistant, compared to the conversion of65% when SFV was pretreated at pH 5 in vitro and similarlyassayed in cell lysates. Conversion was blocked by 10 ,tM monen-sin, which dissipates the acid pH of endosomes (Maxfield, 1982;Marsh et al., 1982) or by 4°C incubation to prevent endocyto-sis. Under either of these conditions, trypsin-resistant El com-prised from 7 to 12% of the total E1. The El protein of_fs-ldid not show a clear-cut change in trypsin resistance after lowpH treatment (M.Kielian, unpublished observations). Thus, thisconformational change could not be used to determine the kineticsof endosome acidification to pH 5.3, thefius-I fusion threshold.

100 0D 8

0*

2 2

time (hrs)

Fig. 1. Kinetics of endocytosis and penetration for wt (0) and fis-l (0)SFV. (A) Endocytic uptake. A mixture of [3H]uridine-labeled fiis-I and 32p-labeled wt SFV (-1 x 105 c.p.m. each isotope) was prebound to BHKcells for 90 min on ice. The cells were warmed to 370C for the indicatedperiod, extracellular viruses stripped off by proteinase K digestion andinternalized virus assayed by liquid scintillation counting using a double-label program. From 35 to 55% of the added virus bound to the cells and85-98% of the bound virus was internalized. (B) Penetration of viral RNAinto the cytoplasm. [3H]uridine or 32P-labeled virus was prebound andinternalized as in 2A. At the indicated time, the cells were scraped andhomogenized and the accessibility of viral RNA to degradation byexogenous RNase assayed. Data shown are the average of threeexperiments. Efficiency of penetration averaged 37% of internalized wt and32% of internalized fiss-l.

60

4-Jcn4-J,I)CO

cLC-

I-U)

CL

40

20

15 30 45 60

Time of Uptake (min)

Fig. 2. Kinetics of E1 conversion to the acid conformation during endocyticuptake of SFV. [35S]methionine-labeled SFV (-4 x 105 c.p.m./35 mmplate) was prebound to BHK cells on ice for 90 min. The cells werewarmed to 37°C for the indicated time to permit endocytosis, after whichthey were lysed and the conversion of El assayed by incubating duplicatealiquots with or without trypsin, followed by SDS-PAGE. The El bandswere excised and quantitated for each time point and the data expressed asthe % of the El counts which were resistant to trypsinization (0). Controlsincluded virus internalized in the presence of 10 FM monensin (0), andvirus pretreated at pH ( o ) or pH 5 (* ) and bound to cells in the cold.Inset shows the fluorograph of the trypsin-resistant El bands from thisexperiment. The pH-treated, bound controls are shown in the left panel. Theendocytosed samples, plus or minus monensin, are shown on the right.

3104

Kinetics of endosome acidification

E1000

0~~~~~~~~~~E:x ~~~~~0

0~~~~

2 3 4

time (hrs)

Fig. 3. Degradation of internalized wt SFV (0) andfiss-1 (0).[35S]methionine-labeled virus was bound and internalized as in Figures 1 and2. At the indicated time, an aliquot of the medium was removed,precipitated with TCA and soluble counts determined after correction for thetotal volume of medium on the cells.

However, as shown below, the virus penetration assay provedan accurate indicator of acidification and worked well with anyof the SFV strains assayed.Penetration and degradation of SFVPenetration of viral RNA into the cytoplasm was determined bya nuclease assay (Helenius et al., 1982; Marsh et al., 1983). Cellsthat had been incubated with [3H]uridine-labeled SFV for dif-ferent times as above were homogenized in order to allow ac-cess of added RNase to the cytoplasmic compartment. In contrastto RNA of intact viruses (present on the cell surface or in in-tracellular vacuoles), the RNA in nucleocapsids that havepenetrated is exquisitely sensitive to RNase under these condi-tions. The assay confirmed our previous results (Marsh et al.,1983) that wt SFV penetrates with a tI/2 of about 15 min (Figure1B, open circles). The identical kinetics of El conversion (Figure2) and wt penetration (Figure 1B) demonstrated that virus fu-sion occurred immediately upon exposure of virus to acid pH.Thus the penetration assay is an accurate measure of the kineticsof acidification.

Unfused viruses and spike proteins inserted into the endosomalmembrane during fusion are transported to the lysosomes andrapidly degraded (Marsh and Helenius, 1980). To follow thekinetics of the degradation reaction, which provides an approx-imation for the kinetics of virus delivery to the lysosomal com-partment, the rate by which [35S]methionine from labeled SFVwas released into the medium in TCA soluble form was deter-mined (Figure 3). It has previously been shown that little of themethionine-labeled digestion product remains associated with thecells (Marsh and Helenius, 1980). Degradation is lysosomal, asjudged by cell fractionation studies and the sensitivity to lyso-somotropic weak bases (Helenius et al., 1982; Marsh et al.,1983). As previously reported, degradation began after a lag ofabout 20 min, and it was complete after about 3 h. The tI/2 fordegradation was approximately 90 min. The degradation kineticsreflect both the time it takes for the viral components to reachlysosomes and the time required for the action of the lysosomalhydrolases.The results obtained for the wt SFV thus showed that 95%

of cell-bound virus was internalized, 45% or more of the incom-ing El was converted to the acid (and presumably fusion-active)form and 30-45% of the RNA was released into the cytoplasm.The kinetics indicated that conversion of El to the acidic con-formation and appearance of non-enveloped nucleocapsids in thecytoplasm both occurred within about 5 min after internaliza-tion. Fusion of the viral membrane, therefore, must occur in anearly endosomal compartment (Helenius et al., 1983; Walkoffet al., 1984; Mellman et al., 1986) and immediately upon ex-posure of the viruses to a pH below the threshold value, whichfor wt SFV is about 6.2. The kinetics suggest, moreover, thatvirus proteins reach the lysosomal compartment at variable ratesranging from 20 min to several hours. Lysosomal delivery andsubsequent degradation thus appears highly unsynchronized.While wt SFV demonstrated how long it takes for incoming

virus to reach a pH of 6.2 or below, the mutant can be used todetermine acidification to pH 5.3 and below. In combination thetwo forms of SFV have the potential for obtaining informationabout the exposure of incoming particles to decreasing pH.When the penetration kinetics of the two viruses were com-

pared using the RNase assay, it was found that the kinetics ofintracellular penetration ofJiis-l were markedly slower than thoseof the wt SFV (Figure 1B). Fus-l penetration began at a com-parable time after shift to 37°C, but the half maximal conver-sion occurred at about 45 min in contrast to 15 min. Maximalpenetration was not reached until about 2 h after the temperatureshift, even though most of the virus was already internalizedwithin 30 min. The efficiency of uncoating of both wt and _fs-lwere comparable in these experiments, with 32-37% of inter-nalized virus penetrating into the cytoplasm.There were several possible explanations for the difference in

penetration kinetics. First, the transit offis-l through the endo-cytic pathway might be slower than that of wt virus. Secondly,fiss-l might be rapidly exposed to a pH of c 5.3, but requirea much longer time to fuse than the wt virus. Finally, the dif-ference in penetration kinetics might directly reflect the time re-quired for endosomes to reach an acidity sufficient to trigger thefusion offis-l. When these possibilities were examined, it wasfound that the difference in kinetics was not due to slowertransport within the endocytic pathway. As shown in Figures 1Aand 3, the rate and efficiency of fus-l uptake and degradationin lysosomes was indistinguishable from wt SFV. Nor could thedifference be explained by a slower rate of fusion after exposureoffis-l to acid. An in vitro fusion assay, as previously describ-ed (White et al., 1980), showed that fusion was rapidly activatedafter acidification. [35S]methionine-labeled viruses (fis-l or wt)were allowed to bind to BHK cells in the cold, the cells werethen exposed to pH 4.8 medium for various times at 37°C, non-fused viruses removed by protease and the remaining radioac-tivity quantitated. As shown in Figure 4, the kinetics and effi-ciency of wt andfius-l fusion with the plasma membrane did notdiffer significantly (see also Kielian et al., 1984). The fusion ofthe mutant appeared slightly slower, but it was maximal within2 min of pH treatment. The difference could not explain the 30min difference in average penetration time.The results therefore suggested that whereas incoming viruses

encountered rather uniformly a pH of 6.2 witiin 5 min after endo-cytosis, a pH c 5.3 was generally encountered in a later en-dosomal compartment. As some fiss-1 viruses penetratedimmediately after endocytosis and others only after several hours,it was apparent that the furtier drop in pH of the virus-containingcompartments was asynchronous.

3105

M.C.Kielian, M.Marsh and A.Helenius

00

a0

4e-co

xco

E

50

2 3 4 5

time of pH treatment (min)

Fig. 4. Kinetics of low pH-induced fusion of SFV with the plasmamembrane of BHK cells. Radiolabeled wt (0) orfus-I (0) was bound toBHK cells for 2 h on ice, the cells washed to remove unbound viruses andtreated at pH 4.8 for the indicated time at 37°C. Non-fused virus was thenremoved by proteinase K treatment and the samples quantitated by liquidscintillation counting.

00

0

0

z

w

z

aI7r

50

5 10

NH4Cl (mM)

Fig. 5. Sensitivity of SFV infection to NH4Cl. BHK cells were preincubatedfor 15 min in medium at pH 7.4 containing the indicated concentration ofNH4Cl. Cells were then infected in these media with 0.5 p.f.u./cell of wt,fuls-I or one of four revertants of Jius-l, for 90 min at 37°C. Infection was

assayed as actinomycin D-insensitive incorporation of [3H]uridine. Fifteenmillimolar NH4Cl was included in all samples during the 5-h uridinelabeling period to prevent further virus infection.

Generation and partial characterization offius-J revertants

To confirm that the difference in penetration rate between wt andfts-l was due to their different pH thresholds, revertants offi*s-1were selected. A revertant with a pH threshold of fusion higherthan fts-l would be expected to penetrate with kinetics more

similar to those of wt virus.To select for revertants offus-1, advantage was taken of the

3106

~~~ 1.ki~~ ~ ~

T~~~~>,! e-%f6 _, W E

fus-1 .. flR>46 oS-t Xv(4i,3j)~~~~~~

pH6.d

Fig. 6. Cell-cell fusion of fits-1 and revertant (R46) infected cells. BHK

cells on coverslips were infected with 1 p.f.u./cell and cultured for 8 h.

Cells were treated for 2 min at 37°C with media of the indicated pH,

returned to neutral pH media for 60 nin at 37°C, fixed and stained with

Giemsa's.

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ _

E 50 ;*Ks-I

0~~~~~~~~~~~~~~~~~~~~~~~CC.-~~~~~~~~~4

1 ~~~~~2

time (hrs)

Fig. 7. Kinetics of virus penetration forfss-1 and revertant (R46) SFV.

[3H]uridine-labeled R46 orifies-1 were bound to BHK cells, internalized and

the penetration of viral RNA assayed as in Figure lB. Data shown are the

average of three experiments. fi*s-l data is that from Figure lB. R46

penetration averaged 513%of the internalized virus.

finding that infection by the mutant is more sensitive to inhibi-

tion by lysosomotropic weak bases than is infection by wt SFV

(Kielian et al., 1984). These agents act by raising endosome pHabove the threshold level required to trigger virus fusion. Fus-l

was grown in BHK cells at low multiplicity in the presence of

10 mM NH4Cl, a concentration chosen because it blockedfi*s-1

infection but still permitted some infection with wt SFV. After

five serial passages in the presence of 10 mM NH4Cl, four rever-

tants were isolated. All were shown to have NH4Cl sensitivity

similar to that of wt virus (Figure 5). One revertant, R46, was

selected for further study. To determine if the NH4Cl resistance

was accompanied by an increase in the fusion pH threshold, BHK

cells were infected with R46 and then treated with low pH buf-

fers at a time when the cells were expressing the virus spike pro-

tein on their surface (Figure 6). R46-infected cells fused to form

REVRTANTS

g

fus- I

Kinetics of endosome acidification

polykaryons at the wt threshold of 6.2 and showed no fusion afterpH 7 treatment. In contrast, fis-l infected cells assayed in parallelshowed little or no fusion at pH 5.6 and substantial fusion afterpH 4.8 treatment (Figure 6). R46 thus appeared to be a revertantas assayed both by fusion activity and NH4Cl sensitivity.R46 was next analysed in the intracellular penetration assay

(Figure 7). In keeping with R46 fusion being comparable to thatof wt virus, its penetration occurred with more rapid kinetics thanthat of fls-1. The t412 for R46 penetration was about 25 min,or 15 min after endocytic uptake. These results confirmed thatthe slow penetration offis-l reflected primarily the time requiredfor the endosome pH to drop to the fis-I threshold of 5.3.

DiscussionKinetics of SFV's interaction with BHK-21 cellsTogether with our previous results, the experiments in this studyallow us to reconstruct a relatively accurate time course for theearliest events that lead to the infection of BHK cells with SFV.They also provide an independent estimate of the pH in earlyand late endosomes. Viruses bind to the cell surface with atl/2of about 30 min (Hahon and Cooke, 1967; Fries and Helenius,1979). Upon warming to 37°C, the bound viruses are internalizedvia coated pits and coated vesicles (t1/2 = 10 min), and almostimmediately thereafter delivered to endosomes (Figure 1,Helenius et al., 1980a; Marsh and Helenius, 1980; Marsh et al.,1983). As early as 5 min after leaving the cell surface, they are

exposed to a pH of < 6.2, judging by the conversion of wt Elspike glycopolypeptides to the 'acid' conformation (Figure 2).wt penetration also occurs at this time, as determined by thetransfer of viral RNA into the cytosol (Figure iB). The encounterof virus with acid, and virus fusion and penetration thus occur

virtually simultaneously. The previous observation that wt in-fection is not inhibited by chloroquine, a lysosomotropic weakbase which elevates the pH in endosomes, when the inhibitor isadded later than 4-5 min after uptake is consistent with thistime course for penetration (Helenius et al., 1980a). The timecourse of fius-l penetration indicated that an additional 30 minin endosomal vacuoles is needed for the viruses to reach a pHof <5.3.That both wt and fus-I mutant penetrate mainly from the en-

dosomal compartment has been demonstrated morphologicallyby electron microscopy (Helenius, 1984), biochemically usingtemperature blocks (Marsh et al., 1983; Kielian et al., 1984),and by kinetic analysis (Figure 1). It is clear that most fiss-lpenetrates from late endosomes, whereas the wt fuses in earlyendosomes. Both sites thus can lead to productive infection. Thetransfer of unfused viruses and fused viral membrane protein tothe lysosome and their degradation, occurs on the average 90min after endocytosis. Little viral protein is usually recoveredin the lysosomal fraction after Percoll gradient centrifugation(Marsh et al., 1983) suggesting that the viral proteins are quiterapidly degraded after arrival in the lysosome. Whether SFV can

fuse, penetrate and infect once it has reached the lysosomal com-partment remains unclear.

R46, a revertant offis-1 selected for NH4Cl resistance, showedmore rapid penetration kinetics than fis-l. Such revertants pro-ved relatively easy to isolate, without mutagenesis. This may havereflected an inherent instability of the lower fusing phenotype.In this regard, it is interesting to note that growth and infectivityoffiJs-1 are less efficient than wt (Kielian et al., 1984). In con-

trast, R46 resembled wt in both its fusion characteristics and itsgrowth properties (Kielian, unpublished observations). Although

Fig. 8. The asynchrony of transit through the endocytic pathway. Twomodels are proposed to explain the asynchrony of SFV's exposure to moreacidic endosome pH and degradation by lysosomal hydrolases. Both modelsassume that the more peripheral El endosomes are less acidic than theperinuclear E3 endosomes. In Model A, incoming pinocytic vesicles canenter the endocytic pathway at any of the various stages of endosomeprogression, El, E2 or E3. The residence in endosomes is thus determinedby the entry point of the endocytosed ligand. Model B proposes that endo-somes are themselves heterogeneous in both acidification rate and deliveryto lysosomes. Incoming ligands would enter the pathway only through theEl endosomes, the rate of acidification and lysosomal fusion of which isvariable. In either model actual transit from El to E2 etc., might bemediatd by maturation of the endosome itself or by vesicles which shuttlebetween one endosome and the next (see Helenius et al., 1983).

more rapid than fus- 1, the penetration kinetics of R46 were notas rapid as those of wt virus. This difference could reflect otherpleiotropic properties of fiis-1 (Kielian et al., 1984).Gradual acidification and asynchrony of the endosome com-partmentThe results reveal two important characteristics of the endocyticpathway which have not been adequately described in our pre-vious studies. First, they show that while some of the endocytosedvirus particles were rapidly routed to lysosomes, the average timespent by a virus in endosomal compartments was at least 35 min.This seems quite long considering that initial endocytosis andrecycling of physiological receptors from endosomes to theplasma membrane takes only a few minutes (Steinman et al.,1976; Bestenman etal., 1981; Wall and Hubbard, 1981; Schwartzet al., 1982; Anderson et al., 1982; Walkoff et al., 1984).Preliminary results in CHO cells suggest a similar time course(S.Schmidt et al., unpublished results). During their extendedresidence in endosomes the viruses are encountering a graduallydecreasing pH. A possible role for gradual acidification in deter-mining sorting events at different levels of endosomal matura-tion was discussed previously (Helenius et al., 1983). Likeviruses, physiological receptor-ligand complexes have differentpH sensitivities. This could cause dissociation events to occureither at early or late stages of endosomal passage.

Previous results from several laboratories using fluorescein-labeled ligands have suggested that short pulse times labelperipheral, less acidic endosome while longer pulse times labelmore acidic, perinuclear endosomes. Due to the nature of thefluorescence experiments, they either provide an average en-dosome pH at a certain time point (Merion et al., 1983; Mur-phy et al., 1984), or give positional and pH information on avery small number of individual endosomes (Tyko and Maxfield,1982; Tanasugarn et al., 1984; Yamashiro et al., 1984). Theseprobes thus cannot be easily used to analyse endosome hetero-geneity. By using radiolabeled virus, information on a largepopulation of individual endosomes can be obtained, each ofwhich should contain 0-1 virus particle (Marsh and Helenius,

3107

M.C.Kielian, M.Marsh and A.Helenius

1980). The most interesting new aspect brought out by thesestudies is the apparent lack of synchrony in the rate of penetra-tion observed for the fiss-l mutant. Incoming individual viruseswhich have been exposed very early to pH values <6.2 pro-ceed to encounter pH 5.3 anytime over a period spanning > 2h.Generation of asynchrony in the endocytic pathwayThe apparent lack of synchrony can be explained by two verydifferent functional models for acidification in the endosomalpathway (Models A and B in Figure 8). Either individual endo-somes acidify at different rates as depicted in Model B, or endo-somes undergo acidification at similar rates but incoming virusesare introduced at different levels of the pathway (Model A). Theseresults alone do not allow distinction between the two pathways.However, numerous studies in other systems have shown thatreceptor recycling and fluid phase reflux involve primarily theearly, peripheral endosomes (Besterman et al., 1981; Walkoffet al., 1984; Wall and Hubbard, 1985), and that incoming ligandsenter the pathway through peripheral endosomes (Geuze et al.,1983, 1984; Mueller and Hubbard, 1986). Thus, since ModelA requires direct delivery of virus at every level of the endosomalpathway, it seems less likely than Model B. While combinationsof the two models are possible, it may be concluded that ModelB is closer to the actual situation in the BHK cell. The relativeasynchrony in fiss-1 penetration would, therefore, be explainedby dramatic differences in the rate by which individual endosomesacidify.The reason for the differing acidification rates revealed by these

results is not clear. It is not known what regulates the pH in anyvacuolar organelle. Conceivably, regulation could depend on thesize of the vacuole, number of vacuolar proton pumps, the con-ductance of the membrane for protons and other ions or thepresence of other electrogenic ion pumps. Recent studies (Fuchsand Meilman, in preparation) have indicated that the higher pHin peripheral, early endosomes could be in part explained by theactivity of Na - K ATPase. The presence of this electrogenicpump would contribute to the generation of an internal positivemembrane potential which would tend to inhibit the electrogenicendosomal proton ATPase. The Na-K ATPase activity appearsto be absent from more mature endosomes (Fuchs, Schmid andMellman, in preparation).

Both models in Figure 8 depict lysosomal fusion occurring onlywith mature endosomes (E3). It is not certain that such fusionis not occurring at every level of the endosomal pathway. Plac-ing this step at the end of the pathway helps, however, to ac-count for the observation that penetration efficiencies for wt andfus-1 are similar (Figure IB, Kielian et al., 1984). If lysosomescould fuse efficiently with early peripheral endosomes, one wouldexpectjfis-I to be considerably less efficient in penetration thanwt SFV. It does seem clear that the pH level of endosomes isnot the controlling factor in their fusion with lysosomes, asdelivery of endosome content to lysosomes and thus the fusionof endosomes and lysosomes are not significantly affected bydissipating endosome acidity (Marsh et al., 1982, 1983).The properties of SFV penetration thus raise new questions

about the nature and regulation of acidification and fusion eventsin the endocytic pathway. They show that after initial uptake andexposure of ligand to mildly acidic pH, the acidification rate andtransit to lysosomes differs for individual endosomes, and theydemonstrate that viruses can be used as pH-sensitive probes tocharacterize this phenomenon. The availability of different virusesand additional mutants with a range of pH thresholds could behelpful in characterizing these important properties of the endo-cytic pathway.3108

Materials and methodsViruses and cellsVirus was propagated, plaque-purified, stored and radiolabeled as previouslydescribed (Kielian et al., 1984), using baby hamster kidney (BHK) cells. Fus-lwas propagated at < 1 p.f.u./cell in all cases, while revertants offjis-l were pro-pagated at c50 p.f.u./cell. Wild type virus for all experiments was a plaque-purified stock derived at Yale from the prototype SFV strain of the virology depart-ment in Helsinki.

Generation offus-l revertantsThe startingfus-1 stock was the second liquid passage after two successive pla-que purifications (Kielian et al., 1984). BHK cells were preincubated with 10 mMNH4Cl in MEM containing 0.2% BSA and 10 mM Hepes, pH 7.4 for 15 minat 37°C. The cells were then infected withfus-I at low multiplicity (0.01 p.f.u./cell)in the same medium for 1 h at 37°C, and the medium was aspirated and replac-ed with identical fresh medium without virus. Progeny virus was harvested aftera 24-h growth period. Five such cycles of NH4Cl selection were performed, afterwhich single plaques were picked, eluted and the isolates screened in a cell -cellfusion assay as previously described (Kielian et al., 1984). Putative revertantswere plaque-purified once more and stocks used at the second liquid passage level.

Endocytosis, degradation and penetration assays

Assays for measuring virus uptake, degradation and the penetration of viralnucleocapsid into the cytoplasm have been described. For all assays, radiolabel-ed SFV was prebound to BHK cells on ice with continuous shaking. Unboundviruses were then removed with an ice-cold wash, and the cells were rapidlywarmed to 37°C by adding pre-warmed medium and placing the tissue culturedishes in a 37°C water bath. The endocytosis assay measured internalized virus,which was resistant to removal by proteinase K digestion (Marsh and Helenius,1980). The degradation assay followed the release of trichloroacetic acid (TCA)-soluble [35S]methionine in the medium (Marsh and Helenius, 1980). The penetra-tion assay monitored the accessibility of [3H]uridine-labeled viral RNA to degrada-tion by nucleases added to cell homogenates (Helenius et al., 1982; Marsh etal., 1983).

Intracellular conversion of viral El proteinThe acid-induced conformational change in El (Kielian and Helenius, 1985) wasused to follow the exposure of endocytosed wt virus to the acid pH of endosomes.The conversoin of El to the acid, trypsin-resistant form was assayed as previouslypublished (Helenius et al., 1985). Briefly, [35S]methionine-labeled SFV was boundto cells and internalized as described above. At various times after endocytosis,cells were cooled on ice, washed with ice-cold phosphate buffered saline (PBS)and lysed in 1% Triton X-100 in PBS containing 20 itg of unlabeled SFV pro-tein/ml. After pelleting the nuclei, lysates were warmed to 37°C in the presenceor absence of 100 yg/ml trypsin for 10 min. The digestion was stopped by add-ing soybean trypsin inhibitor, the samples precipitated with TCA and separatedby SDS-PAGE on 10% Laemlli gels under non-reducing conditions. The trypsin-resistant radioactivity in the excised El band was directly quantitated as describ-ed (Walter et al., 1979) using a fluorograph of the dried-down gel as a guide.Fluorography was performed with sodium salicylate (Chamberlain, 1979).

Virus fusion assaysCell -cell fusion of SFV-infected BHK cells was assayed by a 2 min pH treat-ment at 37°C in medium of pH 4.8-7.0, followed by a 60 min incubation at37°C in neutral pH medium. Cells were then fixed, stained with Giemsa's andevaluated by light microscopy (White et al., 1981).

Direct fusion of radiolabeled SFV with the plasma membrane of BHK cellswas assayed by prebinding virus to cells on ice, treating for 0-3 min with lowpH-medium at 37°C and removing the non-fused virus by proteinase K diges-tion on ice (White et al., 1980). Cell pellets were then directly solubilized inscintillation fluid and the radioactivity quantitated.

Uridine incorporation assayInfection of BHK cells by SFV was assayed by the actinomycin D-insensitiveincorporation of [3H]uridine into viral RNA, as previously described (Heleniuset al., 1982). Cells were infected with virus and assayed in RPMI at pH 7.4,without bicarbonate buffer but containing 10 mM Hepes and 0.2% bovine serumalbumin. NH4Cl was included in this medium as indicated.

AcknowledgementsWe thank Barbara Menzel for help with tissue culture, Pam Ossorio forphotography, Margaret Moench for drawing the figures and Barbara Longobar-di for manuscript preparation. Ira Mellman, Renate Fuchs and Susan Froshauerread the manuscript and made many helpful suggestions. This work was sup-ported by grant A118582-5 (to A.H.) from the NIH and by a Sweibilius CancerResearch Award to M.K.

Kinetics of endosome acidification

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Received on 12 September 1986

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