VIROLOGY

190,

635-644

(1992)

Bacteriophage $6 Envelope Immunodetection, JOHN M. KENNEY,*

Elucidated by Chemical Cross-Linking, and Cryoelectron Microscopy

JARKKO HANTULA,t STEPHEN D. FULLER,* LEONARD P&VI M. OJALA,t AND DENNIS H. BAMFORDtf’

MINDlCH,+

*Biological Structures and Biocomputing Programme, European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Germany; tDepartment of Genetics, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki, Finland; and +Department of Microbiology, Public Health Research Institute, New York, New York 100 16 Received

June 3, 1992;

accepted

June

15, 1992

Bacteriophage 66 is an enveloped dsRNA virus which infects the plant pathogenic Pseudomonas syringae bacterium. Using low dose cryoelectron microscopy we show that the nucleocapsid, spikeless virion, and intact virion have radii of 29, 35, and 43 nm, respectively. Thus, the membrane is 6 nm thick and the surface spikes of the receptor binding protein P3 extend 8 nm from the membrane surface. Cross-linking, immunological, and complementation evidence suggest that the spikes areformed of multimeric P3 molecules and that P3 is associated with membrane-bound protein P6. We observe that the envelope can accommodate up to 400 molecules of P3 but that the average virion contains less than one-fourth of this amount. Assembly of a very small number of P3 or truncated P3 molecules onto inactive virions restores infectivity, showing that only a few spikes are necessary for receptor binding and membrane fusion. 0 1992 Academic

Press,

Inc.

INTRODUCTION

chemical analysis has shown that proteins P3 and P6 are accessible from outside and that P3 is associated with the virus via binding to P6 (Van Etten et al., 1976, Mindich et a/., 1976a; Bamford, 1981; Stitt and Mindich, 1983). The amount of P3 and P6 in the virion fluctuates with the growth temperature (Mindich et al., 1979). In addition, the viral membrane contains three proteins P9, PlO, and P13 (Sinclair et al., 1975; Van Etten et al,, 1976; Gottlieb et a/., 1988). The $6 nucleocapsid (NC) consists of five proteins. The NC surface protein P8 and four proteins (Pl, P2, P4, and P7) which form a particle enclosing the three 46 dsRNA genome segments (Semancik et al., 1973; Sinclair, et al,, 1975; Van Etten et al., 1976). This partcle possesses polymerase activity (Van Etten et a/., 1976; Partridge et al., 1979; Gottlieb et a/., 1990; Olkkonen et al., 1991). Negative staining, thin sectioning, and freeze fracturing electron microscopy have been applied to $6 and its subassemblies (Bamford et al., 1976; Gonzalez et a/., 1977; Bamford and Lounatmaa, 1978). The different techniques have yielded results which differ considerably from each other. For the virus particle freeze fracturing has given the largest (80 nm) and thin sectioning the smallest (65-75 nm) diameters. No morphological differences have been observed between intact viruses and particles lacking P3 or P3 and P6. In addition, the NC is disrupted in tungstate stains commonly used in negative staining (Olkkonen and Bamford, 1987).

The studies of enveloped animal viruses have led to a rather detailed understanding of the receptor-binding and membrane fusion events (White et a/., 1983; Marsh, 1984; Marsh and Helenius, 1989). The receptor-binding and fusion activities can be present on separate parts of a single gene product such as in the case of the hemagglutinin of the influenza viruses (Wrigleyet a/., 1986) or as separate proteins as in the case with the NH and F proteins of the parainfluenza viruses (Scheid, 1987). In the case of the human immunodeficiency virus the receptor-binding protein (gp 120) and the transmembrane protein (gp70) form a single spike complex (Gelderblom, 1987). The discovery of an enveloped bacteriophage (46; Vidaver et a/., 1973) infecting a gram-negative host (Pseudomonas phaseolicola) has brought to light a prokaryotic system with receptor-binding and membrane fusion events similar to those taking place in enveloped animal viruses. Bacteriophage 46 is a dsRNA virus consisting of an icosahedral nucleocapsid surrounded by a membrane. The virus binds to a host pilus through its surface protein P3 (Mindich et a/., 1976a; Bamford et a/., 1987). Upon contact the viral membrane fuses with the host outer membrane. Aviral membrane protein (P6) has been shown to have fusogenie activity (Bamford et al., 1987). Genetic and bio’ To whom

reprint

requests

should

be addressed. 635

0042.6822/92 Copyright All rights

$5.00

0 1992 by Academic Press, Inc. of reproduction in any form reserved

636

KENNEY

scale

(kbp)

4061

bp

fragment

2

1

gene

lo

gene

6

3

gene

3

4

gene

13

67

ET At

as previously described (Sinclair et al., 1976) and propagated on LM 169. Plaques were tested on HB and LM169 at 28”. Those which did not grow on HB but grew on the P3complementing strain, LMl69, were plaque purified on LM 169. Eight presumptive mutants were selected from 1500 plaques and were further tested for thermosensitive phenotype (20” permissive and 26“ restrictive temperature). Four mutants were found to be bona fide ts mutants in protein P3. The one used in this study is designated m551.

pLM362

FIG. 1. Restriction map of a cDNA copy of genomic segment M (Gottlieb et al., 1988). Fragment 67 is a cDNA segment that includes gene 3. Fragment 67 was inserted into vector pLM254 to form pLM380. Cells carrying pLM380 can complement mutations in gene 3. pLM382 was formed by deleting the sequences to the left of the SalI site at position 2312. The ORF of gene 3 is then in frame with that of the vector /acZ gene.

The entire 46 genome has been sequenced, the genes have been localized, and a considerable amount of genetic, physiological, and biochemical information is available (for reviews see Mindich and Bamford, 1988; Mindich, 1988). One could better utilize the 46 system if more reliable structural data on the virus and its subviral assemblies were available. In this investigation we apply lowdose ctyoelectron microscopy, cross-linking, and genetic analysis to characterize the virus particle. Special attention is given to the membrane component of the virus. MATERIALS

AND METHODS

Bacterial strains, phages, and plasmids The 46 wt and its host Pseudomonas syringae pv. phaseolicola HBl OY (HB) were originally provided by A. Vidaver (Vidaver et al., 1973). The truncated gene 3 was obtained as follows (Fig. 1): Fragment 67 cDNA of the middle (M) genomic segment of $6 containing gene 3 was ligated to the vector plasmid pLM254 (Mindich et a/., 1985) to obtain plasmid pLM380. The N-terminal SalI fragment was deleted from this construction to obtain plasmid pLM382, which was transformed into HB to obtain strain LM169. Thus, pLM382 encoded a protein containing 11 amino acids from the IacZ’ gene of PUC8 fused to the 368 C-terminal amino acids of P3. This protein product is about 42 kDa in size, while the native P3 is 69.2 kDa (Gottlieb et a/., 1988). 46hIs, a host range mutant with high plating efficiency on both HB and P. pseudoalcaligenes (Mindich et al,, 197613) was mutagenized with nitrosoguanidine

Growth and purification of viruses, P&less particles, and nucleocapsids Bacteriophage 46 (wt) was grown on HB in LB, concentrated, and purified as previously described (Hantula and Bamford, 1988). Briefly, lysed and cleared cultures were concentrated with polyethylene glycol6000 and then purified in 5-200/o linear sucrose gradient in buffer A (10 mM K-phosphate, pH 7.2, 1 mM MgCI,). The virus zone was collected, the viruses sedimented by differential centrifugation and resuspended into bufferA. This virus material was designated as 1X purified. The receptor-binding surface protein P3 was removed from the virus using butylated hydroxytoluene (BHT). The P3-free particles (BHTv) were obtained by sedimentation through a sucrose gradient and resuspension of the pellet to an appropriate volume of buffer A as previously described (Bamford et al., 1987). The NC particles were obtained by Triton X-l 14 phase separation of BHTv particles as previously described (Ojala et a/., 1990). The quality of the particle preparations was determined by titering and by analyzing the protein profiles by SDS-PAGE (not shown), and by immunoprecipitation: anti-P3 monoclonal antibody precipitated 65% of the virions in the virion preparation, whereas no precipitation over background (4%) was detected with nucleocapsid-specific anti-P8 monoclonal antibodies. Seventy-five percent of the particles in the NC preparation could be precipitated with these anti-P8 antibodies. The radioactive labeling was carried out in M9 medium (Maniatis et a/., 1982) supplemented with glucose (0.4%), histidine, serine, phenylalanine, methionine (each at 20 Kg/ml), glutamate (200 pg/ml), thiamine (1 fig/ml), and l/50 vol of the supplement mixture described by Cohen-Bazire et a/. (1957). [‘“Clmethionine (10 &i/ml) was added 40 min postinfection. The virus concentration and purification were done as above and the preparate assayed for infectivity and radioactivity. The specific activities obtained were typically 1 x 1Op5 cpm/PFU. The virus material was stored at -70“.

THE

Cryoelectron

46

microscopy

The three 46 specimens: virions, BHTv, and NC were prepared for cryoelectron microscopy (Fuller, 1987). They were transferred to a Gatan cryoholder (Model 626) and inserted into a Philips EM 400 electron microscope. Micrographs of the specimens (at -167”) were recorded under low-dose conditions (Fuller, 1987) at 25,000X magnification on Kodak SO163 film. Pairs of micrographs were recorded (with 4-6 e-/A*) at 0.8 and 2.4 pm underfocus. Negatives which showed, by optical diffraction, no apparent drift, less than 3% astigmatism, and approximately 2.4 pm underfocus (chosen to provide optimal contrast for the image features of interest) were digitized with a 25 pm step size. Image processing The digitized images were processed using a modified SPIDER image processing package (Dr. Joachim Frank, Wadsworth Center for Laboratories and Research, New York State Department Health, Albany, New York). The radial density profiles were generated after centering and removal of background. The background variation in density was removed by subtracting a least squares plane fitted to the density outside the particle. Centering was accomplished by crosscorrelation with a separate reference particle for each class. The reference particles were generated by rotationally averaging a sum of centered particles. Generation of new references and cross-correlation centering were repeated until there was no further improvement in the particle centers. The radial profile average and variance for each particle class were calculated. Additionally, the sectors of the virion with the highest spike density were averaged to make a “Max virion” class. This could be compared to the average over the entire circumference of the virions which was denoted as “virion.” The inverted radial profiles are presented since, in the absence of the microscope contrast transfer function (CTF), the negative of the OD is proportional to the projected potential which at low resolution has the same shape as the electron density. Modeling We used a least squares fitting procedure incorporating the NAG library routine e04 to compare models of the virion and subviral particles with their observed radial OD profiles. The models comprised concentric shells of various densities (di) and radii (ri). The corresponding radial profile was generated by convolving the model projection with the inverse Fourier transform

637

ENVELOPE

of the CTF. The CTF includes both phase and amplitude contrast (Erickson and Klug, 1971; Misell, 1976; Toyoshima and Unwin, 1988) and is a function of the spherical aberration (Cs = 1.6 mm), the electron wavelength (X, = 0.042 A), the defocus (SO, the fraction of the amplitude contrast (F-amp), and an exponential decay (d) which attenuates the high-resolution data. This last factor, not included by Toyoshima and Unwin (1988) incorporates the effects of incoherence of the electron beam, inelastic electron scattering, and imprecision in image alignment. This envelope function has been described in terms of the spatial (Frank, 1973) and temporal (HanBen and Trepte, 1971) coherence of the electron source in the microscope. Under the conditions we used, this envelope function reduces to the expontial form in the CTF. The complete transfer function has the form CTF (v) = ((1 - F-amp)sin[x(v)]

+ F-amp

cos[x(v)]}e-du’,

where x(v) = aX,v’(Gf - CsXEv2/2), and v is the spatial frequency in the inverse units (A-‘). Note, x and v are parameters characterizing the CTF and should not be confused with the standard nomenclature for the reduced ~5 used to describe the quality of the least squares fit. The precision of the least squares fit parameters was evaluated by a Monte Carlo procedure in which 100 simulated data sets with Gaussian distributed errors equal to those of our observations around an ideal (errorless) data set were subjected to the least squares fitting procedure. The reported precision corresponds to an average deviation from the model of the lowest 68.3% of the xf values. All programs are written in VAX Fortran and are available upon request. The CTF and model parameters were determined, in order, for the NC, BHTv, virion, and max virion (see Table 1). The model density (OD/nm3) to mass density (g/cc) conversion was based on the volume of P8 shell, the amount of P8 (16.9 MDa), and the densities of amorphous water at -167” (0.93 g/cc) and protein (1.44 g/cc). The validity of the calculation was confirmed by using the BHTv fit radii and densities to compute a mass comparable with the total mass of protein (31.8 MDa) and lipid (20 MDa) in the BHTv shells as determined by biochemistry (Day and Mindich, 1980). Cross-linking The samples were cross-linked as described in Hantula and Bamford (1988) with DSP (dithiobis[succinimidyl propionate], Pierce) concentrations up to 1.2

638

KENNEY

mM. The incubation time was 30 min at +24”. The two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Laemmli [1970], modified as described in Olkkonen and Bamford [1989]) analysis of complexes formed by 50 PLM DSP was carried out with radioactively labeled virus (400,000 cpm/gel). The acrylamide concentrations were either 9 or 16% in the first dimension and 16% in the second dimension. Between the first and the second dimension the cross-links were cleaved by 10 min incubation with 2-mercaptoethanol at +37”. Immunological

methods

All of the monoclonal antibodies used in this investigation have been described previously(Olkkonen eta/., 1988). The ability of the antibodies to recognize intact 46 virions and nucleocapsids was determined by radioimmunoprecipitation (RIP) using protein A-Sepharose CL4B (Pharmacia). The RIP buffer used was 50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.25% BSA. The radioactively labeled antigen was first treated with antibody preparation at room temperature for 2 hr. Protein A-Sepharose was then added to the mixture and incubated for 1 hr at room temperature. After incubation the protein A beads were washed twice with 1 ml of the RIP buffer (microcentrifuge 10,000 rpm, 2 min at room temperature) before measuring the protein A-associated radioactivity. The polyclonal rabbit anti-46 serum was used as a positive control and the $6 proteins P8- and PS-specific monoclonal antibodies were used as negative controls. For the virus antibody sedimentation assay a mixture of cold and radioactively labeled virions was incubated with monoclonal antibodies at room temperature for 45 min. The sample was then loaded onto a 5520% sucrose gradient (in buffer A, 150 mM NaCI) and centrifuged in a Beckman SW 50.1 rotor (23,000 rpm, 35 min at 15”). After centrifugation the gradients were fractionated and the pellets collected. The radioactivity in the fractions and in the pellet was measured by liquid scintillation counting. Polyclonal rabbit anti-46 serum and P8- and PS-specific monoclonal antibodies were used as positive and negative controls, respectively. The detection of P3 and its fragment on Western blots (Towbin et al., 1979) of purified viruses was carried out with a mixture of purified anti-P3 monoclonal antibodies (3P1, 3P2, 3N2, 3N3, and 301, final concentrations of 0.51, 1.03, 0.41, 1.07, and 0.71 ,ugl ml, respectively). One-dimensional Western blot analysis of cross-linked complexes was carried out using 9 and 17% gels as previously described (Hantula and Bamford, 1988). The antibodies used were 3Pl (0.26 pg/ml) and 9B2 (4.1 pug/ml) (Olkkonen et a/., 1988).

ET AL.

RESULTS Cryoelectron

microscopy

We obtained low-dose cryoelectron micrographs of the virus, the BHTv, and the NC (Fig. 2A). Six or more particles of each class were analyzed. The BHTv and the NC show smooth spherical particles, but the viruses display spikes on the surface. The radial density profiles of these particles are shown in Fig. 2Ba. To interpret these profiles one must bear in mind that the variations in density at the center of the particle (radius < 24 nm) are not statistically significant and that the phase contrast portion of the transfer function enhances the boundary of the particle by generating a ripple of apparent low density outside the particle. Although the three profiles share a similarly flattened region in the center they differ in the shape and position of their boundaries. The most prominent feature for each particle class is the sharp drop in density representing the particle edge. The zero crossing defines the outer radius for each particle class. The NC and BHTv radii are 29 and 35 nm, respectively. The virus shows a less sharply defined boundary reflecting additional mass outside the 35-nm radius extending to 43 nm, corresponding to the spikes seen in the electron micrographs. Although the positions of boundaries can be determined from a visual inspection of the profiles, the CTF distorts the relative densities of features so that they are not directly interpretable. We were able to characterize the CTF and to account for its effects on the radial profiles, to generate values for the density of the shells in the particles. The CTF parameters C, and X, were fixed and the others allowed to vary so that we were able to fit accurately the observed radial density profiles (Fig. 2B) with different sphere and shell models (see Fig. 6). The best fit to the nucleocapsid radial profile was acceptable (~3 = 0.702, f, > 0.95, see legend to Table 1) and so these parameters were held fixed for all the other fits. The fitted under-focus (2.7 f 1 .O pm) matched the under-focus estimated from the optical diffraction pattern of the original micrograph (2.4 pm). The best fit to the BHTv profile was obtained using two shells around the NC sphere (~5 = 0.621, P, > 0.99). A fit with one shell could be excluded (xf = 0.906, P, < 0.70) as could a fit with three shells (XJ = 0.902, P, < 0.70). For both the virion and the max virion (which included only the spike-rich sectors of the virus particles) radial profiles a satisfactory result was obtained using a single additional shell to the BHTv model. The shell, however, differed in density between these two

THE

n-0.06t"""""'

~'J~""~~""'," 0

lo

20 Radius

639

b6 ENVELOPE

1

30

40

50

60

(nm)

FIG. 2. (A) Typical examples of cryoelectron microscopy images of vitrified particles: (a) virus; (b) BHTv; and (c) NC. The bar represents 50 nm. Note the white ring around each particle caused by the underfocus phase contrast. (6) The radial profiles of the particle classes: (a) max virion (solid line); BHTv (large dashed line); and NC (dashed line) showing the average projected density (in OD units) as a function of radius. Note that the projected mass density (modified by the CTF) is proportional to the negative of the optical density (-OD). Least squares fitted radial profiles: (b) NC 6 parameter fit (solid line) and data (points); (c) BHTv 5 parameter fit (solid line) and data (points); (d)virion radial profile 5 parameter fit (solid line) and data (points). The bars are fl SD.

data sets. A final check on the validity of our assumption about the constancy of the imaging conditions was obtained by fitting the outer shell and the imaging parameters to the virion data set (max virion-5 in Table 1) and confirmed the previous determinations within the precision of the fit. Finally, we were able to estimate the maximal amount of P3 in $6 using the max virion data to be 28 MDa + 26%. Given that P3 has a molecular weight of 69.2 kDa, then the maximal number of copies of P3 per virion is about 400.

P3-specific

monoclonal

antibodies

We have previously produced a panel of monoclonal antibodies against 46 structural proteins. When viruses were used as antigens the surface protein P3 turned out to be very antigenic (Olkkonen et al., 1988). The analysis of the anti-P3 monoclonal panel is shown in Table 2. One set of antibodies (303-306) was negative in the Western blot and had low ELISA titers. They, however, recognized native viruses and caused virus

640

KENNEY

ET AL.

TABLE

1

THECTFANDMODELPARAMETERS NC Particles Parameters C, (mm) A, bm) 6f bm) F-amp Decay (nm2) rad, (nm) d, (OD/nm3) 4 (g/cc) rad, (nm) d, (OD/nm3) d, (g/cc) rad, (nm) d, (OD/nm3) d, (g/cc) rad, (nm) d, (OD/nm3) d, (g/cc) 2 X” P&x”, v)

BHTv

6 6 1.60 0.0042 2.7 f 1 .O 0.03 + 0.18 103& 11 29.0 2 0.1 0.042 5 0.008 1.20 f 0.05

7 5

30.9 0.031 1.14 35.0 0.040 1.19

0.702 >0.95

Max

6 3

* * * * * * * * k k ++ * +

Virion-3

2.4 0.003 0.002 0.4 0.003 0.02

0.621 >0.99

* * * * * * * * * * * * * *

43.4 f 0.4 0.0139 + 0.0015 1.02 + 0.01 0.646 >0.98

Virion 6 3

* * * * * * * * * * * * * *

43.0 f 0.3 0.0117 I! 0.0007 1.01 + 0.01 0.316 >0.99

Max

Virion-5 6 5

2.06

* * t 0.19 *

59 f 9 * * * * * * * * * 43.0 + 0.4 0.0172 t 0.0013 1.04 f 0.01 0.640 >0.98

Note. The CTF parameters C, and X, were kept fixed, while 6f, F amp and decay were determined by a least squares fit to the data. The radii (rJ and densities (d,) were determined by the same least squares fitting. The g/cc densities are derived from the OD/nm3 densities and calibrated against the mass of P8 and the density of amorphous water at -170”. An offset parameter (not listed) was evaluated for each data set, but was always insignificant. The asterisk (*) indicates that the parameter is the same as in the column to the left and was fixed during the fit for determining the other parameters in that column. The goodness (~3) and the quality (P,(x’, u)) of the fit are reported as well as the precision of the fitted parameters evaluated by Monte Carlo simulation.

aggregation and adsorption inhibition. All the Westernblot-positive antibodies also had high ELISA titers. Those in this class which caused adsorption inhibition also recognized virus-associated nondenatured P3 (3Pl-3P3). Those that did not cause adsorption inhibition did not recognize virus-associated P3 (3Nl-3N5). Cross-linking The availability of specific monoclonal antibodies against membrane-associated proteins P3 and P9 allowed us to study, using Western blotting, protein complexes crosslinked by DSP, which contained these proteins. P3-specific antibody recognized many highmolecular-weight complexes in addition to monomeric P3 after one-dimensional SDS-PAGE (Fig. 3A). The most abundant are those with molecular weights of about 92, 98, 100-l 18, and 200 kDa. In addition, a weak band was observed at the position of about 130138 kDa and a stronger band above 200 kDa. The 200kDa complex is recognized also with PS-specific antibodies (Fig. 3B). Since the NC proteins form a complex of this size after cross-linking (Hantula and Bamford, 1988) we tested the ability of the P3-specific antibody

to recognize any complexes formed when NCs were cross-linked. As no signal was detected (not shown) we concluded that the complexes detected here are P3specific. The PS-specific antibody did not recognize any complexes from the samples cross-linked with low DSP concentrations. When the concentration of DSP was increased above 0.24 mM, four distinct complexes with approximate molecular weights of 19, 20, 22, and 27 kDa were observed. The 27-kDa complex was located in a position where the antibody also recognized a band in the control lane, and therefore is considered to be artifact. In addition, strong antibody labeling in the upper part of the gel was observed including nonspecific labeling of Pl and P3. Two-dimensional SDS-PAGE analysis of cleavable cross-linked protein complexes allows the identification of participating proteins based on their location in relation to the monomers after the second dimension electrophoresis. In our previous work we observed several NC-specific complexes (Hantula and Bamford, 1988). In the 2D SDS-PAGE analysis of the d6 virion proteins these complexes are also revealed (Fig. 4). In addition to these a P3 + P6 complex can be detected (shown with a large arrowhead in Fig. 4B). This com-

THE TABLE

2

ANALYSIS OF THE PANEL OF MONOCLONAL Data from

Mab” 301 303 304 305 306 3Pl 3P2 3P3 3Nl 3N2 3N3 3N4 3N5

Western blotb

Olkkonen

SDS RIP”

+ + + t t + + + +

Adsorption inhibitiond

-

-

+ + + + -

+ + + + + t + -

+ t + -

ANTIBODIES AGAINST P3

et al., 1988

-

New

ELISA” H L L L L H H H H H H H H

Virus RIP’

data

Virus aggregationg -

+ + + + + + t -

IL + + -

-

ND ND

+ + + -

aThe 3 stands for specificity (anti P3) and the letter for fusion. Purified P3 was used as an antigen for 0 and P fusions and screening was done by measuring virus neutralization. The whole virus was used as an antigen in the N fusion and the screening was carried out by ELISA. b t, detectable; -, nondetectable in Western blot analysis. c Virus preparation disrupted with 0.1% SDS and protein-antibody complexes precipitated with Protein A-Sepharose CL46 beads (see Olkkonen et al., 1988). d The ability of the antibody to inhibit the virus association with its receptor was determined by first treating the virus with antibodies and then measuring the virus-specific radioactivity in the cell and supernatant fractions. e H, high titer (values above 200); L, low titer (values 1 O-50). Purified virus was used as an antigen. ‘Native “C-labeled viruses precipitated with Protein A CL46 beads. Nonspecific background level was below 5% (pellet/input); t indicates values higher than 10%. g See Materials and Methods for the sedimentation analysis. ND, not determined.

plex was also identified in an experiment where the first dimension acrylamide concentration was 9%, where P3 was clearly separated from Pl (not shown). Using this low DSP concentration (50 pn/l) no P9 or PlO complexes were obtained. Complementation

641

46 ENVELOPE

of P3

A plasmid was constructed so that gene 3 would be expressed as a protein missing 42% of its N-terminal sequence. This peptide was able to complement a class of ts mutants in gene 3 represented by mutant ~~557. It could not complement nonsense mutants in gene 3 or most other ts mutants. This suggests that P3 acts as a multimeric complex on the surface of the virion, since the truncated peptide must interact with other molecules of P3 in order to complement.

The temperature-sensitive gene product was expressed in normal amounts in phage-infected cells and was assembled onto the phage surface (Fig. 5, lanes c and d). The plasmid-encoded truncated P3 was also assembled onto phage particles, but the amount of incorporation was low. This low level of P3 integration from plasmid-directed synthesis was seen with wild type P3 as well. On the basis of detection of P3 by Western blotting, the plasmid-coded synthesis was at least five times greater than that found during normal infection (data not shown). The low level of integration appears to be due to the inhibition of host protein synthesis during infection. The low level of incorporation into virus is, however, sufficient to produce infectious particles. DISCUSSION The result of the model fittings to the radial density profiles of the cryo EM images resulted in a “one sphere-three shells” model of 46 (Fig. 6). The assignment of the phage proteins and the dsRNA to these layers is based on previous biochemical and genetic experiments (Mindich and Bamford 1988, Mindich 1988). Previous EM methods gave virus diameters far from the one we obtained with cryo EM (86.0 f 0.8

A

abed

E

B

abed 85.0 69.2 34.9

116 85.0 P3

23.2 17.3 16.0 P9

FIG. 3. Western blot analysis of the DSP-cross-linked complexes of 66 virion. (A) Complexes recognized by P3-specific antibody 3Pl (final concentration 0.26 pglml) in 9% gel. DSP concentrations are: (b) 0.12 mM; (c) 0.24 mM; and (d) 0.48 mM Sample (a) is the noncross-linked control. (B) Complexes recognized by PS-specific antibody 982 (4.1 *g/ml) in 17% gel. DSP concentrations are: (a) 1.2 mM; (b) 0.72 mM; and (c) 0.24 mM. Sample (d) is the non-crosslinked control. Molecular weight markers are myosine (205 kDa), p-subunit off. co/i RNA-polymerase (155 kDa). /3-galactosidase (1 16 kDa), and 46 proteins Pl (85, 0 kDa), P3 (69, 2 kDa), P4 (34, 9 kDa), P5 (23, 2 kDa), P7 (17, 3 kDa), and P8 (16, 0 kDa). The monomeric P3 and P9 (9.5 kDa) are indicated in the figure. The complexes described in text are shown with arrows,

642

KENNEY

Pl P2 P3

z

P4

-

P5

-

P9

-

PlO

-

ET AL.

FIG. 4. 2D SDS-PAGE analysis of crosslinked $6 virion. (A) Non-cross-linked control. (B) 96 cross-linked with 47.5 gM DSP. The P3 + PfZi and P5 + P8 complexes are shown by larger and smaller arrowheads, respectively. The acrylamide concentration of both directior is in 2D gels was 16%. At the left are noncrosslinked ~$6 proteins run only in the second dimension.

nm). However, the value obtained is close to that given by turbidity measurements (82 f 6 nm; Day and Mindich, 1980). We conclude that these two values accurately reflect the diameter of the virion in solution. Two new morphological observations derive from the cryo EM data: (i) This is the first time that it has been possible to assign protein P3 to a morphological location in the virion (to the spike). (ii) The best fit for the membrane region included two shells of different mass densities. This suggests an uneven distribution of protein mass between the outer and inner membrane leaflet. The higher mass density in the outer leaflet could be due, at least in part, to the asymmetry of P6 in the membrane; more mass being in the outer surface so as to form an anchor for the P3 spike. Radial profile analy-

a

b

c

d

e

f

9

P3 -,

AP3-+ FIG. 5. Western blotting analysis of m551, a $6 mutant with thermosensitive P3. Proteins recognized by P3-specific monoclonal antibodies. The acrylamide concentration was 16%. Samples from left to right are: (a) his propagated on HB at 20”; (b) his, HB, 26”; (c) m557, HB, 20”; (d) m551, LM169, 20”; (e) m557, LM169, 26”; (f) whole cell preparation of noninfected HB; (g) whole cell preparation of noninfected LM169.

sis of cryo EM images (accounting for the effects of the CTF) shows that the isolated NC and the BHTv particles have the same radial density profile as the corresponding structures in the virion. Based on these results we conclude that cryo EM is superior in preserving sensitive enveloped virus structures in comparison to the conventional methods by dehydration and negative staining. Although this novel analysis involving the paramerization of a spherical virus model and the CTF has given us valuable insight into the average radial structure of 46, we are in the process of reconstructing in three dimensions the virus and its subparticles in order to elucidate its structure in more detail. In Table 3 we present the summary of the cross-linking data together with suggested composition of the complexes detected. The overall conclusion of this data is that all the membrane-associated 46 proteins are in very close proximity to each other. The P3 + P6 complex was obtained both in 2D gel analysis and with PS-specific antibody in a 1D gel. Biochemical stoichiometry determinations (Day and Mindich, 1980) have given a ratio of 1:2 for P3 and P6 in the virion. A P3-specific complex of the size P3+(P6), could also be detected here. This suggests that the spike complex has this stoichiometry. Finding P3-specific complexes corresponding in size to P3 homotrimers suggests that the spike is multimeric in nature. Western-blot-negative monoclonal antibodies, recognizing native virusassociated P3, do not recognize their epitopes effectively in an ELISA assay, in which the conditions are rather severe for the virus structure. This, in addition to the capability of truncated P3 to complement nonfunc-

THE

46

tional P3, could be taken as further support for the multimeric structure of the spike complex. Analysis of the radial density profiles indicates that P3 extends from the membrane surface by 8 nm. Hence P3 is associated with the distal portion of P6. This rules out models in which P3 contacts the membrane directly or interacts laterallywith P6. The molecular weight of P3 (69.2 kDa) and its 8-nm extent suggest that it must have an axial ratio of at least 1.5. The heterogeneity of the densities of P3 on the virion surface which is observed by cryo EM indicates that the virus does not contain a full complement of the P3-P6 spike complexes. The number of copies of P3 can be estimated from our density data and the comparison with known components of the virion. The value for the most densely packed regions of the virion

643

ENVELOPE TABLE THE CROSS-LINKED

3

COMPLEXES RECOGNIZED MONOCLONALANTIBODIES

BY P3- AND PS-SPECIFIC

Mab specificity/ measured size P3 200 130-138 loo-118 98 92 -

P9

ZD”

200 -

-

22 20 19

+ -

Theoretical size

Suggested composition (P3),, (P3), P3 + P3 + P3 + (P9),

? (P6), P6 -t (PlO or P13) P6 +(PlO or P13)

(W, W),

a Detected in 2-D SDS-PAGE. b In this position there is probably

more

than

207.6 or?b 138.4 103,6 90.7 or 94.2 86, 4 23, 3 or 26, 7 19 19

a single

complex.

surface (max virion) corresponds to about 400 copies of P3 per virion or a surface density of 27,000/pm3. This is close to the density of spikes in the alphaviruses (Fuller, 1987). The number of copies of P3 in the virion determined by biochemical methods (Day and Mindich, 1980) is only 70-80. In addition, the assembly of only a very small number of P3 or truncated P3 molecules onto inactive virion restores infectivity to a normal level. These observations show that far less than the full complement of proteins P3 and P6 are needed for effective adsorption and membrane fusion of 46. ACKNOWLEDGMENTS Ms. Sisko Litmanen and Mr. Marek Cyrklaff are acknowledged for skillful technical assistance and Dr. Vesa Olkkonen for help in preparing fresh virus. This investigation was supported in part by a grant from the Academy of Finland (D.H.B.). Additional support was obtained from Alfred Kordelin, Leo and Regina Weinstain (J.H.), and Emil Aaltonen (P.O.) Foundations. We thank W. KClhlbrandt for his critical reading of the manuscript.

REFERENCES 0

IO

radius

20

30

40

50

(nm)

FIG. 6. Best fit model of 46 particle. The internal solid ball represents the virus NC [r, = 29.0 nm, p, = 0.042 OD/nm3 (1.20 g/cc)]. The membrane is presented by two concentric shells [r2 = 30.9 nm, pe = 0.031 OD/nm3 (1.14 g/cc), and r, = 35.0 nm, p3 = 0.040 OD/ nm3 (1.19 g/cc)], representing approximately the membrane leaflets. Note the higher density of the outer shell indicating a higher protein concentration in the outer part of the membrane. The outermost low density shell [r4 = 43.0 nm, p4 = 0.017 OD/nm3 (1.04 g/cc)] represents the space occupied by the spike protein P3. The density (p, = 0.93 g/cc) of amorphous water at -170” is indicated in the lower schematic by the arrow.

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Bacteriophage phi 6 envelope elucidated by chemical cross-linking, immunodetection, and cryoelectron microscopy.

Bacteriophage phi 6 is an enveloped dsRNA virus which infects the plant pathogenic Pseudomonas syringae bacterium. Using low dose cryoelectron microsc...
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