A Single Mutation in the Carboxy Terminus of Reovirus ...jvi.asm.org/content/76/19/9832.full.pdf ·...

JOURNAL OF VIROLOGY, Oct. 2002, p. 9832–9843 Vol. 76, No. 190022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.19.9832–9843.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Single Mutation in the Carboxy Terminus of Reovirus Outer-CapsidProtein �3 Confers Enhanced Kinetics of �3 Proteolysis, Resistance

to Inhibitors of Viral Disassembly, and Alterationsin �3 Structure

Gregory J. Wilson,1,2* Emma L. Nason,3 Charles S. Hardy,1,2 Daniel H. Ebert,2,4 J. Denise Wetzel,1,2

B. V. Venkataram Prasad,3 and Terence S. Dermody1,2,4

Departments of Pediatrics1 and Microbiology and Immunology4 and Elizabeth B. Lamb Center for Pediatric Research,2

Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Department of Biochemistry andMolecular Biology, Baylor College of Medicine, Houston, Texas 770303

Received 16 October 2001/Accepted 3 June 2002

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting information of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassemblythat mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassemblypathway is the proteolysis of viral outer-capsid protein �3. Mutant reoviruses selected during persistentinfection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistanceto inhibitors of endocytic acidification and proteolysis. To identify sequences in �3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the �3 proteins of wild-type strain type 3Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant �3 proteins were expressed in insect cellsand used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteasescathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354(Y354H) enhanced �3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in �3 weresubstantially greater than those of rISVPs lacking this mutation after growth in cells treated with eitheracidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions ofelectron micrographs of virus particles containing wild-type or mutant �3 proteins revealed structural alter-ations in �3 that correlate with the Y354H mutation. These results indicate that a single mutation in �3protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanismsof �3 cleavage during reovirus disassembly.

Many viruses use receptor-mediated endocytosis to enterhost cells (reviewed in reference 32). For some viruses, expo-sure to the acidified environment in the endocytic compart-ment or proteolysis by proteases contained in these organellesfacilitates structural alterations in viral capsid components re-quired for delivery of the virus into the cytoplasm. Reovirusesare nonenveloped, double-shelled particles that contain a ge-nome of 10 discontiguous segments of double-stranded RNA.Following attachment to cell-surface receptors, reoviruses areinternalized into cells by receptor-mediated endocytosis (5, 6,43, 46). In the endocytic pathway, the viral outer capsid isremoved by acid-dependent proteases through an ordered se-ries of proteolytic events resulting in the generation of infec-tious subvirion particles (ISVPs) (2, 6, 9, 44, 46). ISVPs areobligate intermediates in reovirus disassembly that mediatepenetration of endosomal membranes, leading to delivery ofthe viral core into the cytoplasm (5, 21, 22, 31, 47). The earliestdetectable proteolytic event in reovirus disassembly is the deg-radation of �3 protein (6, 9, 44, 46), which likely triggers thecascade of disassembly steps that culminate in endosome pen-

etration. Thus, the �3 protein plays a central role in reovirusentry into cells.

Reoviruses are capable of establishing persistent infectionsof many types of cultured cells (reviewed in reference 12).These cultures are maintained by horizontal transmission ofvirus from cell to cell and therefore are more accuratelytermed “carrier cultures.” During maintenance of persistentreovirus infections of murine L929 (L) cells, mutations thataffect viral disassembly are selected in cells and viruses (13).Mutant cells do not support acid-dependent proteolysis of theviral outer capsid (13) and do not express the enzymaticallyactive form of the endocytic cysteine protease, cathepsin L (3).In contrast to wild-type viruses, viruses isolated from persis-tently infected cultures (PI viruses) demonstrate enhanced ki-netics of viral disassembly (53) and can grow in cells treatedwith either ammonium chloride (13, 53), an inhibitor of vacu-olar acidification (34, 41), or E64 (2), an inhibitor of cysteineproteases (4). These findings suggest that PI viruses have al-tered requirements for acidification and proteolysis to com-plete disassembly.

Analysis of wild-type � PI reassortant viruses indicates thatgrowth of PI virus L/C in cells treated with ammonium chloridesegregates with the S1 gene, whereas growth of PI 2A1 and PI3-1 in ammonium chloride-treated cells segregates with the S4gene (53). These results suggest that there are at least two

* Corresponding author. Mailing address: Lamb Center for Pediat-ric Research, D7235 MCN, Vanderbilt University School of Medicine,Nashville, TN 37232. Phone: (615) 343-5731. Fax: (615) 343-9723.E-mail: [email protected].

9832

on August 30, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

acid-dependent disassembly events during conversion of viri-ons to ISVPs: one involving viral attachment protein �1, whichis encoded by the S1 gene (30, 51), and another involvingouter-capsid protein �3, which is encoded by the S4 gene (35,37). Growth of L/C, PI 2A1, and PI 3-1 in cells treated withE64 segregates exclusively with the S4 gene (2), suggesting that�3 alone is the primary determinant of susceptibility of theviral outer capsid to proteolysis during viral disassembly. Thededuced amino acid sequences of �3 proteins of L/C, PI 2A1,and PI 3-1 contain from one to three mutations, including acommon tyrosine-to-histidine mutation at amino acid 354(Y354H) (53). In the case of PI 3-1 �3 protein, Y354H is theonly mutation observed, which provides strong evidence thatY354H influences protease susceptibility of �3. However, ithas not been possible to compare isogenic virus strains with thesingle �3 polymorphism at amino acid 354 for cell entry anddisassembly. Moreover, potential contributions of the othermutations in PI virus �3 proteins in determining susceptibilityto inhibitors of acidification and proteolysis have not beendefined.

In this study, we used ISVPs recoated with mutant �3 pro-teins containing mutations in PI virus �3 proteins alone and incombination to precisely identify sequences in �3 that modu-late acid-dependent and protease-dependent steps in reovirusdisassembly. This technique facilitates generation of particlesdiffering by site-specific mutations for studies of steps in virus-cell interaction during a single cycle of replication (24). PIvirions and recoated ISVPs (rISVPs) were tested for suscepti-bility to growth inhibition mediated by ammonium chlorideand E64 and analyzed by cryo-electron microscopy (cryo-EM)and three-dimensional image analysis. The results demonstratethat Y354H enhances the kinetics of reovirus disassembly,confers resistance to inhibitors of acid-dependent proteolysisin cellular endosomes, and leads to alterations in �3 structure.

MATERIALS AND METHODS

Cells and viruses. L cells were grown in either suspension or monolayercultures in Joklik’s modified Eagle’s minimal essential medium (Irvine Scientific,Santa Ana, Calif.) supplemented to contain 5% fetal bovine serum (Gibco BRL,Grand Island, N.Y.), 2 mM L-glutamine, and 100 U of penicillin per ml, 100 �gof streptomycin per ml, and 0.25 �g of amphotericin per ml (Sigma-Aldrich, St.Louis, Mo.). Spodoptera frugiperda (Sf21) cells (Clontech, Palo Alto, Calif.) weregrown in Grace’s insect cell medium (Gibco BRL) supplemented to contain 10%fetal bovine serum, 2 mM L-glutamine, 50 U of penicillin per ml, and 50 �g ofstreptomycin per ml.

Reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) are laboratorystocks. PI viruses L/C (1), PI 2A1, and PI 3-1 (13) were isolated as previouslydescribed. Purified preparations of reovirus virions were made by using second-passage stocks as previously described (20). Baculovirus vector strains werederived from Autographa californica nuclear polyhedrosis virus (AcMNPV). Re-combinant baculoviruses containing wild-type and mutant S4 gene cDNAs weregenerated by introduction of cDNAs into pBacPAK8 (Clontech), followed bylipofection-mediated cotransfer of plasmid recombinants and linearizedBacPAK6 AcMNPV DNA (Clontech), respectively, into Sf21 cells according tothe manufacturer’s instructions. Recombinant viruses were isolated by plaquepurification on monolayers of Sf21 cells and amplified by three passages in Sf21cells.

Cloning and mutagenesis of S4 gene cDNAs. The �3-encoding S4 gene cDNAsof strains T3D, L/C, PI 2A1, and PI 3-1 were generated by reverse transcription-PCR (27) and introduced into the pCR2.1 vector (Invitrogen, San Diego, Calif.).S4 genes were amplified with primers specific for the noncoding regions of theT3D S4 gene (53).

Site-directed mutations of the T3D S4 gene cDNA in pCR2.1 were generatedby PCR-based oligonucleotide mutagenesis (Stratagene, La Jolla, Calif.) accord-

ing to the manufacturer’s instructions. The following primer sets were used formutagenesis (nucleotides different from those in the T3D S4 sequence areunderlined): mutation A sense (5�-GGTCGTGTATCAATCTGCAGCGCGCAAGAGGG-3�) and mutation A antisense (5�-CCCTCTTGCGCGCTGCAGATTGATACACGACC-3�) and mutation B sense (5�-GAGGTTCACTAGTGAAGCTGAACCGGCTTCAG-3�) and mutation B antisense (5�-CTGAAGCCGGTTCAGCTTCACTAGTGAACCTC-3�). Mutant A-B was constructed frommutant B by insertion of mutation A with the mutation A primer set describedabove. Mutant A-C was constructed with XbaI and SpeI to excise the 5� 437nucleotides, including 24 nucleotides of polylinker sequence, of the L/C S4 genecDNA in pCR2.1. These sequences were ligated into the BacPAK8 transfervector containing the S4 gene cDNA of PI 3-1. Nucleotide sequences of the�3-encoding regions of all S4 gene cDNAs in baculovirus transfer vectors wereconfirmed by using an ABI model 377 automated sequencer (PE-Applied Bio-systems, Norwalk, Conn.). Error-free S4 gene cDNAs were used to constructrecombinant �3-expressing baculoviruses.

Expression of recombinant �3 proteins. Third-passage recombinant baculo-viruses containing wild-type or mutant S4 gene cDNAs were used to infect Sf21insect cells (5 � 106) at a multiplicity of infection (MOI) of 2 PFU per cell. Afterincubation at 25°C for 72 h, cells were washed once with phosphate-bufferedsaline (PBS) and incubated on ice in 1 ml of cytoplasmic extraction buffer (10mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5mM phenylmethysulfonyl fluoride) containing an EDTA-free protease inhibitorcocktail (Roche, Indianapolis, Ind.). After 30 min of incubation, 25 �l of 10%Igepal CA-630 (Sigma-Aldrich) was added, and the mixture was thoroughlyvortexed. Cell nuclei and membranes were collected by centrifugation at 500 �g for 5 min. The supernatant was removed, and the cell pellet was incubated onice in 1 ml of nuclear extraction buffer (20 mM HEPES [pH 7.9], 0.42 M NaCl,1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA) for 30 min. Cellular debris wascollected by centrifugation at 22,000 � g for 10 min. The supernatant wasremoved, analyzed by electrophoresis, and used for recoating experiments.

SDS-PAGE of reovirus structural proteins. Discontinuous sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as pre-viously described (28). Viral particles were solubilized by incubation in samplebuffer (125 mM Tris, 2% 2-mercaptoethanol, 1% SDS, 0.01% bromophenolblue) at 65°C for 5 min. Samples were loaded into wells of 10% polyacrylamidegels and electrophoresed at a constant voltage of 200 V for 1 h. Followingelectrophoresis, gels were stained with Coomassie blue R-250 (Sigma-Aldrich)and dried between cellophane.

Recoating ISVPs with recombinant �3 proteins. ISVPs of strain T1L wereprepared by treating 5 � 1012 purified virions in virion storage buffer (150 mMNaCl, 10 mM MgCl2, 10 mM Tris [pH 7.4]) with 200 �g of N�-p-tosyl-L-lysinechloromethyl ketone-treated bovine �-chymotrypsin (Sigma-Aldrich) at 37°C for60 min. T1L ISVPs were purified by cesium chloride (CsCl) density centrifuga-tion as previously described (20) and dialyzed against virion storage buffer.Recombinant �3 protein in nuclear extracts of baculovirus-infected Sf21 cells (5� 106) was mixed with T1L ISVPs (5 � 1012 particles) in a volume of 1 ml andincubated at 25°C for 30 to 60 min. rISVPs were purified by CsCl densitycentrifugation and dialyzed against virion storage buffer. Infectious titers ofrISVP preparations were determined by plaque assay with L-cell monolayers(49).

Treatment of reovirus virions and rISVPS with cathepsin L and cathepsin D.Purified reovirus virions and rISVPS at a concentration of 4 � 1010 particles perml in reaction buffer L (100 mM NaCl, 15 mM MgCl2, 50 mM sodium acetate[pH 5.0]) were treated with 100 �g of purified, recombinant human cathepsin L(7) per ml in the presence of 5 mM dithiothreitol at 37°C for 0 to 16 h. Virionsand rISVPS at a concentration of 4 � 1010 particles per ml in reaction buffer D(5 mM MgCl2, 10 mM cysteine, 100 mM potassium acetate [pH 3.8]) weretreated with 100 �g of purified, bovine cathepsin D (Sigma-Aldrich) per ml at37°C for 0 to 6 h. Protease treatment was terminated by adding either 500 �ME64 (Sigma-Aldrich) to cathepsin L reactions or 100 mg of pepstatin A (Sigma-Aldrich) per ml to cathepsin D reactions and incubating on ice for 5 min.Reaction mixtures were analyzed by SDS-PAGE.

Densitometric analysis of reovirus structural proteins. Dried, Coomassie-stained gels were scanned with Adobe Photoshop 5.0 (Adobe Systems, San Jose,Calif.). Band densities in the scanned gels were quantitated with the programScion Image Beta 4 (Scion Corporation, Fredrick, Md.). �3 stoichiometry onrecoated particles was determined by comparing the densities of bands corre-sponding to �3 to those of the � protein of ISVPs, which is a virion-associatedcleavage fragment of �1C protein. Mean band densities corresponding to �3were divided by those corresponding to �1C (virions) or � (rISVPs) proteins todetermine stoichiometry. In studies of virion and rISVP proteolysis, mean den-sities were determined for bands corresponding to the � and �3 proteins for each

VOL. 76, 2002 CLEAVAGE SUSCEPTIBILITY OF REOVIRUS �3 PROTEIN 9833


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

interval of protease treatment. Densities of bands corresponding to �3 weredivided by those corresponding to the � proteins as a control for loading.

Growth of wild-type and PI virions, ISVPs, and rISVPs in the presence andabsence of inhibitors of viral disassembly. Monolayers of L cells (5 � 105) in24-well plates (Corning-Costar, Corning, N.Y.) were preincubated at 37°C for 4 hin untreated medium or for 4 h in medium supplemented to contain either 10mM ammonium chloride or 100 �M E64. The medium was removed, and cellswere adsorbed with virus particles at an MOI of 2 PFU per cell. After incubationat 25°C for 1 h, the inoculum was removed, cells were washed with PBS, and 1ml of fresh, untreated medium or medium supplemented with either ammoniumchloride or E64 was added. After incubation at 37°C for 24 h, cells were frozenand thawed twice, and viral titers in cell lysates were determined by plaque assay(49). Independent experiments were performed in duplicate.

Cryo-EM. Reovirus particles were prepared for cryo-EM by previously de-scribed techniques (17, 38). A 4-�l aliquot of each specimen (virions or rISVPs)was applied to one side of a holey carbon grid. The grid was then blotted andplunged into a bath of liquid ethane (180°C). The frozen-hydrated sample wastransferred to a precooled GATAN cryoholder (GATAN, Inc., Pleasanton, Cal-if.) and imaged with a JEOL 1200 transmission electron microscope (JEOL, Inc.,Peabody, Mass.) operated at 100 kV and maintained at a specimen temperatureof 163°C. Regions of interest were imaged at a magnification of �30,000 withan electron dose of 5 electrons/A2. From each region, a focal pair was recordedwith intended defocus values of 1 and 2 �m. The electron images were recordedwith a 1-s exposure on Kodak SO-163 film (Kodak, Rochester, N.Y.). Film wasdeveloped in Kodak D-19 developer at 21°C for 12 min and fixed in Kodak fixerat 21°C for 10 min.

Three-dimensional image reconstructions. Micrographs were selected basedon particle concentration, quality of ice, and appropriate defocus conditions.Images were digitized on a Zeiss SCAI microdensitometer (Carl Zeiss, Inc.,Englewood, Colo.) with a 7-�m step size. Pixels were averaged to give a 14-�mstep size that corresponded to 4.67 A per pixel in the object. Particles were boxedwith an area of 256 by 256 pixels. Determination of orientational parameters,refinement of these parameters, and three-dimensional reconstructions wereperformed by using the ICOS Toolkit software suite (29). The orientations of theparticles were determined by the common lines approach (11) and refined by thecross-common lines method (19). Three-dimensional image reconstructions froma set of particles that adequately represented the icosahedral asymmetric unitwere computed by cylindrical expansion methods (11). The further-from-focusmicrograph in each focal pair was processed first to obtain a low-resolutionreconstruction. This reconstruction then was used to determine the correctorientations of particles imaged in the corresponding closer-to-focus micrograph.

Image reconstructions were computed to a resolution within the first zero ofthe contrast transfer function (CTF) of the corresponding micrograph. Defocusvalues were determined from CTF ring positions in the sum-of-particle Fouriertransforms. Defocus values of various specimens in the closer-to-focus micro-graphs ranged from 1.2 to 1.4 �m. Image reconstructions were corrected for theeffects of the CTF by previously described procedures (55). The final resolutionfor each reconstruction was determined by Fourier ring correlation analysis (48).Image reconstructions were computed to an 24-A resolution, which was thelowest resolution among the various specimens, for comparative analysis. Con-tour levels in each reconstruction were chosen to represent equal volumes be-tween radii at 305 and 425 A. Reconstructions of various specimens wereradially scaled to match the radial extension of the innermost layer (�1 layer)with an assumption that mutations in �3 are unlikely to alter the radial dispo-sition of this capsid layer. Reconstructions were viewed on a Silicon GraphicsWorkstation (SGI, Mountain View, Calif.) with IRIS Explorer v3.5 software(Numerical Algorithms Group, Inc., Oxford, United Kingdom).

Fitting of X-ray structure into the cryo-EM reconstruction. The X-ray coor-dinates of �3 (42) were initially rigid-body fitted into its portion of the cryo-EMdensity map by visual inspection with the graphical program O (25). Subsequentrefinements were performed with the Situs program package, a rigid-body cor-relation fitting procedure that allows the fitting of X-ray structures into low-resolution cryo-EM density maps (54).

RESULTS

Expression of recombinant �3 protein. The deduced aminoacid sequences of PI virus �3 proteins contain from one tothree mutations (Table 1), only one of which, Y354H, has beenconsistently linked to viral growth in the presence of inhibitorsof reovirus disassembly (2, 53). To determine whether Y354H

is sufficient to confer resistance to ammonium chloride andE64 and to assess possible contributions of other mutationsselected in �3 during persistent infection, we engineeredrecombinant baculoviruses to express wild-type T3D �3protein and �3 proteins containing mutations observed in PIviruses. We also generated four additional recombinant bacu-loviruses by using either site-directed mutagenesis or exchangeof PI virus S4 gene cDNA sequences to express novel �3mutants (Table 1). Recombinant baculoviruses were used toinfect insect cells, and nuclear extracts were prepared. As

FIG. 1. (A) Expression of �3 protein in insect cells with recombi-nant baculoviruses. The expressed �3 proteins of wild-type T3D, threePI viruses, and four site-directed �3 mutants are shown. A band cor-responding to expressed �3 proteins is labeled, and T3D virions areincluded as a control. An empty vector control was loaded in the lanelabeled V, and molecular mass markers (in kilodaltons) were loaded inthe lane labeled M. (B) ISVPs recoated with wild-type and mutant �3proteins. ISVPs of reovirus strain T1L were incubated with insect cellnuclear extracts containing the �3 proteins shown to generate rISVPs.Equal numbers of rISVPs were analyzed by SDS-PAGE with T1Lvirions included as controls. Viral proteins are labeled. The ratio of �3band density to that of �1C protein (virions) or � protein (rISVPs) isindicated at the bottom of the gel.

TABLE 1. Mutations in the deduced amino acid sequences of �3proteins used to define sequences that modulate susceptibility

of �3 to proteolysis

�3 protein Location and nature of mutation(s)

Mutant A............................................26, Y3CMutant B ............................................130, E3KPI 3-1 ..................................................354, Y3HMutant A-B........................................26, Y3C; 130, E3KMutant A-C........................................26, Y3C; 354, Y3HPI 2A1.................................................130, E3K; 354, Y3HL/C.......................................................26, Y3C; 130, E3K; 354, Y3H

9834 WILSON ET AL. J. VIROL.


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

demonstrated by SDS-PAGE (Fig. 1A), the major proteinband in nuclear extracts migrated with the electrophoretic mo-bility of native �3. Thus, insect cells infected with recombinantbaculoviruses produce �3 protein, as reported previously (24).

Recoating ISVPs with wild-type and mutant �3 proteins. Togenerate rISVPS with recombinant �3 proteins, T1L ISVPswere incubated with the expressed �3 protein isolated fromSf21 cells. Although T3D would have been an appropriatestrain for �3 recoating studies, it was not selected due to thereduced infectivity of T3D ISVPs, resulting from cleavage ofthe T3D �1 protein during virion-to-ISVP conversion in vitrowith chymotrypsin (10, 39). After incubation, rISVPs wereisolated by CsCl gradient centrifugation and analyzed by SDS-PAGE (Fig. 1B). Each of the recombinant �3 proteins wascapable of recoating T1L ISVPs, resulting in the generation of

rT3D, r3-1, rL/C, r2A1, rA, rB, rA-B, and rA-C. To assess thestoichiometry of �3 protein on these recoated particles, thedensities of bands corresponding to �3 were compared to thoseof the � protein of ISVPs, which is a virion-associated cleavagefragment of �1C protein. For each rISVP species, the �3/�ratio approximated the �3/�1C ratio determined for virions.These results demonstrate that expressed �3 proteins recoatISVPs from a different reovirus strain with native stoichiome-try.

Treatment of virions and rISVPs with endocytic proteases.To identify mutations in �3 protein that influence the suscep-tibility of �3 to proteolytic disassembly in vitro, virions ofwild-type and PI viruses and rISVPs containing expressed wild-type and mutant �3 proteins were treated with either of theendocytic proteases cathepsin L or cathepsin D. Cathepsin L is

FIG. 2. Electrophoretic analysis of viral proteins of wild-type (wt) and PI virions and rISVPs following treatment with cathepsin L. (A to D)Purified virions of the particles shown were treated with human cathepsin L (pH 5.0). Equal numbers of viral particles were analyzed bySDS-PAGE. Molecular mass markers (in kilodaltons) were loaded in lanes labeled M. Positions of viral proteins are indicated. (E and F) For eachinterval of protease treatment, the densities of bands corresponding to �3 were divided by those corresponding to the � proteins as a control forloading. Shown are the mean �3:� ratios for two or three independent experiments. Error bars indicate the standard errors of the means.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

a cysteine protease that is capable of producing ISVPs whenused to treat reovirus virions (3, 18, 18a). Cathepsin D is anaspartic protease that is incapable of producing ISVPs (18, 26).Virions and rISVPs were treated with purified, recombinant,human cathepsin L (7) for 0 to 16 h, and proteolysis of viralouter-capsid proteins was monitored by SDS-PAGE (Fig. 2).Treatment of wild-type T3D and PI 3-1 virions with cathepsinL resulted in proteolysis of outer-capsid proteins indicative ofISVP formation with degradation of �3 and cleavage of �1C toform �. In comparison to T3D, proteolysis of PI 3-1 duringcathepsin L treatment occurred with substantially faster kinet-ics, consistent with previous results obtained with the intestinalprotease chymotrypsin (53). Treatment of rISVPS with cathep-sin L resulted in degradation of �3 alone, since conversion of�1C to � had occurred during ISVP preparation prior to re-

coating. In comparison to rT3D, proteolysis of rL/C, r2A1,r3-1, and rA-C with cathepsin L occurred with faster kineticsand was similar to that of PI 3-1 virions. For virions andrISVPS, the densities of bands corresponding to �3 were com-pared to those of the � proteins as a control for loading.Analysis of �3/� band intensities for PI 3-1 virions and rL/C,r2A1, r3-1, and rA-C particles demonstrated that �90% of �3was removed from the viral outer capsid by 2 h (Fig. 2). Insharp contrast, proteolysis of wild-type T3D virions and rT3D,rA, rB, and rA-B particles was incomplete after 16 h (digestionof rA and rB was equivalent to that of rA-B [data not shown]).The �3 proteins of virions and rISVPS demonstrating fasterkinetics of �3 proteolysis after treatment with cathepsin Lcontain a common mutation, Y354H.

To determine whether mutations in �3 protein correlate

FIG. 3. Electrophoretic analysis of viral proteins of wild-type (wt) and PI virions and rISVPs following treatment with cathepsin D. (A to D)Purified virions of the particles shown were treated with bovine cathepsin D (pH 3.8). Equal numbers of viral particles were analyzed bySDS-PAGE. Molecular mass markers (in kilodaltons) were loaded in lanes labeled M. Positions of viral proteins are indicated. (E and F) For eachinterval of protease treatment, densities of bands corresponding to �3 were divided by those corresponding to the � proteins as a control forloading. Shown are the mean �3:� ratios for two or three independent experiments. Error bars indicate the standard errors of the means.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

with altered susceptibility to proteolysis by an endocytic pro-tease incapable of generating ISVPs, virions and rISVPS weretreated with purified bovine cathepsin D for 0 to 6 h, andproteolysis of viral outer-capsid proteins was monitored bySDS-PAGE (Fig. 3). Cathepsin D treatment of PI 3-1 virionsand rL/C, r2A1, r3-1, and rA-C particles resulted in degrada-tion of �3 with little proteolysis of other viral proteins, exceptat the 6-h time point. However, degradation of �3 duringcathepsin D treatment of rT3D, rA, rB, and rA-B particles(digestion of rA and rB was equivalent to that of rA-B [datanot shown]) was minimal. Digestion of wild-type T3D virionsby cathepsin D was intermediate to that of rT3D and r3-1particles. These findings were confirmed by densitometric anal-ysis of �3/� band intensities, which demonstrated that �90%of �3 was removed from PI 3-1 virions and rL/C, r2A1, r3-1,and rA-C particles by 1 h (Fig. 3). Similar to our findings withcathepsin L, the �3 proteins of virions and rISVPS demon-strating specific cleavage of �3 with cathepsin D containY354H. These results indicate that �3 proteins containing anisolated mutation, Y354H, are more susceptible to cleavage byeither cathepsin L, which is capable of mediating reovirusdisassembly (3, 18, 18a), or cathepsin D, which is not (18, 26).

Growth of rISVPS in the presence and absence of inhibitorsof viral disassembly. To identify mutations in �3 protein thatconfer viral growth in the presence of inhibitors of viral disas-sembly, virions and rISVPs were tested for the capacity to growin the presence of either the acidification inhibitor ammoniumchloride or the protease inhibitor E64. L cells were infectedwith wild-type viruses, T1L or T3D, T1L ISVPs, PI 3-1, or

rISVPs generated by using the mutant �3 proteins shown inTable 1 in the presence or absence of ammonium chloride orE64. After 24 h of growth under each condition, viral titers incell lysates were determined by plaque assay (Fig. 4). Each ofthe particles tested produced approximately equivalent yieldsafter 24 h of growth in the absence of inhibitors of disassembly.However, in the presence of ammonium chloride, yields of PI3-1 virions and r3-1, rL/C, r2A1, and rA-C particles were ap-proximately 10- to 20-fold greater than those of wild-type T1Land T3D virions and rA, rB, and rA-B particles. Virions andrISVPs with greater yields in the presence of ammonium chlo-ride each contain �3 proteins with Y354H. Similarly, in thepresence of E64, yields of PI 3-1 virions and r3-1, rL/C, r2A1,and rA-C particles were approximately 10- to 30-fold greaterthan those of wild-type T1L and T3D virions and rA, rB, andrA-B particles. These results indicate that �3 proteins contain-ing Y354H are resistant to inhibitors of acid-dependent andprotease-dependent steps that occur during viral disassembly.

Structural analysis of virions of wild-type and PI reovirusesand rISVPs. To gain insight into the structural basis for thealterations in kinetics of viral disassembly and resistance todisassembly inhibitors associated with �3-Y354H, we usedcryo-EM and three-dimensional image analysis to compare thestructures of wild-type and PI virions and rISVPs. Cryo-imagesof unstained, frozen-hydrated wild-type virions, PI virions, andrISVPs are shown in Fig. 5. These images indicate that PIvirions and rISVPs have morphological characteristics that aresimilar to those of wild-type virions. Reconstructions of nativevirions and their corresponding rISVPs were computed from

FIG. 4. Growth of reovirus virions, ISVPs, and rISVPs in the presence and absence of inhibitors of viral disassembly. Monolayers of L cells (5� 105) were preincubated for 4 h in untreated medium or for 4 h in medium supplemented with either 10 mM ammonium chloride or 100 �ME64. The medium was removed, and cells were infected with the particles shown at an MOI of 2 PFU per cell. After 1 h of adsorption, the inoculumwas removed, fresh medium with or without either ammonium chloride or E64 was added, and cells were incubated for 24 h. Viral titers in celllysates were determined by plaque assay. The results are presented as mean viral yields, calculated by dividing the titer at 24 h by the titer at 0 hfor each condition, for two to three independent experiments. Error bars indicate the standard error of the means.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

their respective cryo-images. These reconstructions revealedthat both wild-type and PI virions have a similar structuralorganization. The structures of wild-type T3D (computed to a24-A resolution with 183 particles) and PI virus L/C (computedto a 24-A resolution with 496 particles) are shown in Fig. 6. Inall reconstructions, the fingerlike projections of �3 protein areorganized on an incomplete T�13 icosahedral lattice with six�3 subunits at the local sixfold axis and an incomplete ring offour �3 subunits adjacent to the fivefold axis surrounding the�2 turret. Three-dimensional image reconstructions of rT3D,rL/C, and r3-1 (each computed to a 24-A resolution with 113,90, and 121 particle images, respectively) revealed identicalstructural organizations, both with respect to the �3 proteinand the remainder of the structure (Fig. 6 and data not shown).These results demonstrate that the overall arrangements of �3proteins in the outer capsid of wild-type and PI virions andrISVPs are similar and that interactions of �3 with other struc-tural proteins on rISVPs recapitulate those of native reovirusparticles.

Analysis of conformational changes in �3 protein. To iden-tify structural differences in �3 proteins of wild-type and PIviruses, we compared three-dimensional image reconstructionsof wild-type and PI virions (Fig. 7). In this analysis, we foundtwo significant differences. First, the radial extension of the �3protein of L/C virions was less than that of the other PI virusesand wild-type T3D (Fig. 7C), which were equivalent. Second,T3D virions contained a cleft in the �3 density (Fig. 7A and C)that was filled in reconstructions of PI virions and rISVPscontaining �3-Y354H (Fig. 7B and C and data not shown). Toensure that these features were reproducible, a minimum oftwo reconstructions with independent sets of particles weregenerated. Altered �3 density in the region of the T3D cleftwas consistently observed for all PI viruses. These results dem-

onstrate that PI viruses have an alteration in �3 protein den-sity, which likely indicates a conformational change associatedwith Y354H.

DISCUSSION

In this study, we show that a single mutation in the reovirus�3 protein enhances the kinetics of viral disassembly, mediatesresistance to inhibitors of endocytic acidification and proteol-ysis, and produces structural alterations in �3. Y354H ispresent in the �3 proteins of PI viruses L/C, PI 2A1, and PI 3-1,which were isolated from independent persistently infectedL-cell cultures (1, 13). A Y354H mutation also is found inE64-adapted variant viruses selected by serial passage of wild-type T3D in L cells treated with E64 (18). By using rISVPscontaining individual or combinations of �3 mutations foundin PI viruses, we found that Y26C and E130K do not contrib-ute to altered disassembly phenotypes. Although it is possiblethat these mutations play other roles during persistent reovirusinfection, these effects are not apparent from the experimentsreported here. Three-dimensional image analysis of PI virusesindicates that �3 proteins with Y354H have a common struc-tural alteration. We propose that this mutation alters proteaseaccess to a �3 cleavage site and enhances the susceptibility of�3 to proteolysis.

Proteolytic removal of �3 is the initial step in the reovirusdisassembly cascade that results in formation of ISVPs. Threelines of evidence support a central role for �3 cleavage inreovirus disassembly. First, pharmacologic inhibitors of eitherendocytic acidification (33, 46) or proteolysis (2, 18) block �3cleavage, which leads to an arrest of reovirus disassembly andinhibition of the virus replication cycle. Second, PI virusesgrow in the presence of these inhibitors (2, 13, 53) and exhibitaccelerated proteolysis of �3 according to in vitro assays of

FIG. 5. Electron cryo-micrographs of wild-type and PI virions and rISVPs embedded in a thin layer of vitreous ice. (A) Wild-type T3D, (B) L/C,(C) PI 2A1, (D) PI 3-1, (E) rT3D, (F) rL/C, (G) r2A1, and (H) r3-1. Scale bar in panel A, 1,000 A.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

viral disassembly (53). Third, reovirus variants selected specif-ically for resistance to protease inhibitor E64 have mutations in�3 protein (18). The findings reported here extend those re-ported previously and demonstrate that Y354H in an otherwise

isogenic background accelerates �3 cleavage and confers re-sistance to viral disassembly inhibitors. These results supportthe conclusion that �3 plays an essential role in the regulationof reovirus disassembly.

FIG. 6. Three-dimensional image reconstructions of wild-type and PI virions and rISVPs. Reconstructions viewed along the icosahedralthreefold axis of wild-type T3D (A), L/C (B), rT3D (C), and rL/C (D) are shown. Closeup views of the hexameric rings of �3 proteins from thereconstructions of recoated particles (E) rT3D and (F) rL/C are shown to indicate that the assembly of �3 in rISVPs is native.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

Growth of PI viruses in the presence of ammonium chloridesegregates with either the �1-encoding S1 gene (L/C) or the�3-encoding S4 gene (PI 2A1 and PI 3-1) (53), whereas growthof PI viruses in the presence of E64 segregates exclusively withthe S4 gene (2). Using rISVPs containing wild-type and mutant�3 proteins, we found that ISVPs recoated with �3 proteinscontaining Y354H were capable of growth in the presence ofeither ammonium chloride or E64. No other mutation in �3contributed to these phenotypes. Interestingly, rISVPs con-taining the �3 protein of L/C were capable of growth in thepresence of ammonium chloride, although this phenotype seg-regates with the L/C S1 gene (53). It is possible that interac-tions between �1 and �3 in rISVPs differ from those in nativevirions, which might obviate a requirement for �1 mutations inconferring resistance to ammonium chloride of rISVPs con-taining the L/C �3 protein. In support of this possibility, the �1protein undergoes a dramatic conformational change from aretracted to an extended form during ISVP generation (16,20). Mutations in the L/C �1 protein that segregate with am-monium chloride resistance might influence this conforma-tional change and confer acid-independent viral disassembly inthe absence of mutations in �3. Such effects might not beoperant for rISVPs, in which any conformational changes in �1presumably would have occurred prior to recoating. Nonethe-less, our results clearly demonstrate that mutations in �3 alone

can confer growth in the presence of ammonium chloride. Aprecise delineation of the role of �1 and �3 mutations inconferring resistance to ammonium chloride will require anal-ysis of core particles recoated with various combinations ofwild-type and PI virus outer-capsid proteins (8).

How might a Y354H mutation enhance the susceptibility of�3 to proteolysis? The crystallographic structure of T3D �3reveals a bilobed molecule consisting of a virion-proximalsmall lobe and a virion-distal large lobe (42). Independentimage reconstructions of L/C, PI 2A1, and PI 3-1 demonstratea bulge in �3 density in the same region of wild-type T3D thatcontains a cleft between the two lobes. These findings indicatethat Y354H alters the structure of �3. By virtue of a histidineat residue 354, L/C, PI 2A1, and PI 3-1 �3 are anticipated to bemore sensitive than wild-type �3 to decreases in pH withinendosomes that would ultimately result in alterations in inter-actions between amino acid side chains in the vicinity of resi-due 354. We predict that such alterations modulate access topotential �3 cleavage sites.

Tyr 354 in T3D �3 lies at the end of an amino acid sequenceconnecting an �-helix in the virion-proximal small lobe to twoC-terminal -strands in the virion-distal large lobe of the mol-ecule (Fig. 8). Contact amino acids in this network include thecarbonyl oxygen of Tyr 354 with the ε-amino nitrogen of Lys234 (42). In addition, the Tyr 354 side chain faces a hydropho-

FIG. 7. Structural alterations in �3 protein. (A) A thin slice of density representing two of the hexameric �3 proteins, represented as a mesh,from two independent wild-type T3D reconstructions (shown in yellow and red) superimposed. An arrow indicates the position of the cleft in �3density. The atomic structure of �3 (in white) is docked into one of the �3 densities (right). The ribbon diagram of �3 in the same orientation isshown (inset) with an arrow indicating the location of the cleft. Also shown (inset) are mutations found in PI virus �3 protein: Y26C (purple),E130K (red), and Y354H (green). (B) Density maps from two independent reconstructions of PI virus L/C are superimposed in cyan and green.(C) Density maps for wild-type T3D (red) and PI virus L/C (cyan) are shown superimposed. The cleft seen in the wild-type T3D reconstructionis absent in L/C.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

FIG. 8. Atomic structure of T3D �3 with mutations. (A) Ribbon diagram of the carbon tracing of the 1.8-A crystal structure of the T3D �3protein (42). Mutations found in PI virus �3 protein are Y26C (purple), E130K (red), and Y354H (green). The area of increased density foundin PI virus �3 containing His 354 visualized by cryo-EM is marked by an asterisk. The C terminus and N terminus are marked with arrows. (B) TheC-terminal inset in panel A (dashed lines) is rotated approximately 90o to highlight residues that potentially interact with Y354H. In panel B1, ahydrophobic pocket is created by residues Y223, Y225, L228, Y238, L242, V243, and F349. In panel B2, the charged residues are D224, E227, andK234. The C terminus is indicated by the letter “C.”



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

bic pocket (Tyr 223, Tyr 225, Leu 228, Tyr 238, Leu 242, Val243, and Phe 349) in the vicinity of two acidic residues (Asp224 and Glu 227), the carboxyl side chains of which are di-rected away from the hydrophobic pocket (Fig. 8B). In virionscontaining Tyr 354 in �3, the predominate interactions be-tween the side chains would be hydrophobic, and access to thisregion by solvent and other molecules would be hindered.However, His 354 in �3 is likely repulsed from the hydrophobicpocket and possibly forms salt bridges with either Asp 224 orGlu 227 located nearby. Such interactions might alter �3 struc-ture, resulting in the density changes observed by cryo-EMassociated with His 354 in �3. Since Tyr 354 is among theresidues joining the small and large lobes of the protein, con-formational alterations in �3 associated with His 354 might betransmitted to a lower region of the protein. Furthermore,structural changes in �3 proteins containing His 354 also wouldallow enhanced access to residues 238 to 250 within the hydro-phobic pocket, which form a protease-sensitive region identi-fied in T1L and T3D �3 (18a, 23). Therefore, we propose thatC-terminal residues in �3 act as a latch to protect �3 cleavagesites and that His 354 may act as a hinge, which becomesactivated for optimal movement as pH decreases from neutralto acidic within endosomes. Thus, His 354 in �3 may mimic alower-pH �3 disassembly intermediate in which the densityshift seen in PI viruses corresponds to an open C-terminallatch. To test these predictions, high-resolution structural stud-ies of PI 3-1 �3 are in progress.

Nonenveloped viruses must maintain a balance between ex-tracellular stability and the capacity to disassemble followinginternalization into host cells. Reovirus virions are remarkablyresistant to various inactivating agents (14, 15, 50, 52), and �3is hypothesized to provide enhanced environmental stability ofvirions compared to that of ISVPs (40, 45). While mutations in�3 selected during persistent infection enhance viral disassem-bly, these mutations actually attenuate viral virulence (36).Therefore, enhanced susceptibility to proteolytic disassemblycomes at a cost in the virus-host interaction. The findingsreported here identify the molecular basis for susceptibility of�3 to proteolytic cleavage and lead to an improved under-standing of mechanisms by which �3 regulates reovirus stabil-ity and disassembly.

ACKNOWLEDGMENTS

We express our appreciation to Louisa Craddock for administrativeassistance and to Angela Billingsley, Howard Price, Deloris Radcliff,and Brenda Starks for technical support. We also thank Jim Chappelland Tim Peters for careful review of the manuscript and John Mort forproviding cathepsin L.

This work was supported by Public Health Service award AI32539from the National Institute of Allergy and Infectious Diseases and theElizabeth B. Lamb Center for Pediatric Research. Additional supportwas provided by Public Health Service awards CA68485 for theVanderbilt Cancer Center and DK20593 for the Vanderbilt DiabetesResearch and Training Center.

REFERENCES

1. Ahmed, R., W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, andB. N. Fields. 1981. Role of the host cell in persistent viral infection: coevo-lution of L cells and reovirus during persistent infection. Cell 25:325–332.

2. Baer, G. S., and T. S. Dermody. 1997. Mutations in reovirus outer-capsidprotein �3 selected during persistent infections of L cells confer resistance toprotease inhibitor E64. J. Virol. 71:4921–4928.

3. Baer, G. S., D. H. Ebert, C. J. Chung, A. H. Erickson, and T. S. Dermody.1999. Mutant cells selected during persistent reovirus infection do not ex-

press mature cathepsin L and do not support reovirus disassembly. J. Virol.73:9532–9543.

4. Barrett, A. J., A. A. Kembhavi, M. A. Brown, H. Kirschke, C. G. Knight, M.Tamai, and K. Hanada. 1982. L-trans-Epoxysuccinyl-leucylamido(4-guanidi-no)butane (E-64) and its analogues as inhibitors of cysteine proteinasesincluding cathepsins B, H and L. Biochem. J. 201:189–198.

5. Borsa, J., B. D. Morash, M. D. Sargent, T. P. Copps, P. A. Lievaart, and J. G.Szekely. 1979. Two modes of entry of reovirus particles into L cells. J. Gen.Virol. 45:161–170.

6. Borsa, J., M. D. Sargent, P. A. Lievaart, and T. P. Copps. 1981. Reovirus:evidence for a second step in the intracellular uncoating and transcriptaseactivation process. Virology 111:191–200.

7. Carmona, E., E. Dufour, C. Plouffe, S. Takebe, P. Mason, J. S. Mort, and R.Menard. 1996. Potency and selectivity of the cathepsin L propeptide as aninhibitor of cysteine proteases. Biochemistry 35:8149–8157.

8. Chandran, K., S. B. Walker, Y. Chen, C. M. Contreras, L. A. Schiff, T. S.Baker, and M. L. Nibert. 1999. In vitro recoating of reovirus cores withbaculovirus-expressed outer-capsid proteins �1 and �3. J. Virol. 73:3941–3950.

9. Chang, C. T., and H. J. Zweerink. 1971. Fate of parental reovirus in infectedcell. Virology 46:544–555.

10. Chappell, J. D., E. S. Barton, T. H. Smith, G. S. Baer, D. T. Duong, M. L.Nibert, and T. S. Dermody. 1998. Cleavage susceptibility of reovirus attach-ment protein �1 during proteolytic disassembly of virions is determined by asequence polymorphism in the �1 neck. J. Virol. 72:8205–8213.

11. Crowther, R. A. 1971. Procedures for three-dimensional reconstruction ofspherical viruses by Fourier synthesis from electron micrographs. Philos.Trans. R. Soc. London B Biol. Sci. 261:221–230.

12. Dermody, T. S. 1998. Molecular mechanisms of persistent infection by reo-virus. Curr. Top. Microbiol. Immunol. 233:1–22.

13. Dermody, T. S., M. L. Nibert, J. D. Wetzel, X. Tong, and B. N. Fields. 1993.Cells and viruses with mutations affecting viral entry are selected duringpersistent infections of L cells with mammalian reoviruses. J. Virol. 67:2055–2063.

14. Drayna, D., and B. N. Fields. 1982. Biochemical studies on the mechanism ofchemical and physical inactivation of reovirus. J. Gen. Virol. 63:161–170.

15. Drayna, D., and B. N. Fields. 1982. Genetic studies on the mechanism ofchemical and physical inactivation of reovirus. J. Gen. Virol. 63:149–160.

16. Dryden, K. A., G. Wang, M. Yeager, M. L. Nibert, K. M. Coombs, D. B.Furlong, B. N. Fields, and T. S. Baker. 1993. Early steps in reovirus infectionare associated with dramatic changes in supramolecular structure and pro-tein conformation: analysis of virions and subviral particles by cryoelectronmicroscopy and image reconstruction. J. Cell Biol. 122:1023–1041.

17. Dubochet, J., M. Adrian, J. J. Chang, J. C. Homo, J. Lepault, A. W. Mc-Dowall, and P. Schultz. 1988. Cryo-electron microscopy of vitrified speci-mens. Q. Rev. Biophys. 21:129–228.

18. Ebert, D. H., J. D. Wetzel, D. E. Brumbaugh, S. R. Chance, L. E. Stobie, G. S.Baer, and T. S. Dermody. 2001. Adaptation of reovirus to growth in thepresence of protease inhibitor E64 segregates with a mutation in the carboxyterminus of viral outer-capsid protein �3. J. Virol. 75:3197–3206.

18a.Ebert, D. H., J. Deussing, C. Peters, and T. S. Dermody. 2002. Cathepsin Land cathepsin B mediate reovirus disassembly in murine fibroblast cells.J. Biol. Chem. 277:24609–24617.

19. Fuller, S. D. 1987. The T�4 envelope of Sindbis virus is organized byinteractions with a complementary T�3 capsid. Cell 48:923–934.

20. Furlong, D. B., M. L. Nibert, and B. N. Fields. 1988. Sigma 1 protein ofmammalian reoviruses extends from the surfaces of viral particles. J. Virol.62:246–256.

21. Hooper, J. W., and B. N. Fields. 1996. Monoclonal antibodies to reovirus �1and �1 proteins inhibit chromium release from mouse L cells. J. Virol.70:672–677.

22. Hooper, J. W., and B. N. Fields. 1996. Role of the �1 protein in reovirusstability and capacity to cause chromium release from host cells. J. Virol.70:459–467.

23. Jane-Valbuena, J., L. A. Breun, L. A. Schiff, and M. L. Nibert. 2002. Sites anddeterminants of early cleavages in the proteolytic processing pathway ofreovirus surface protein �3. J. Virol. 76:5184–5197.

24. Jane-Valbuena, J., M. L. Nibert, S. M. Spencer, S. B. Walker, T. S. Baker, Y.Chen, V. E. Centonze, and L. A. Schiff. 1999. Reovirus virion-like particlesobtained by recoating infectious subvirion particles with baculovirus-ex-pressed �3 protein: an approach for analyzing �3 functions during virusentry. J. Virol. 73:2963–2973.

25. Jones, T. A., J. Y. Zhou, S. W. Cowan, and M. Kjeldgaard. 1991. Improvedmethods for building protein models in electron density maps and the loca-tion of errors in these models. Acta Crystallogr. A47:110–119.

26. Kothandaraman, S., M. C. Hebert, R. T. Raines, and M. L. Nibert. 1998. Norole for pepstatin-A-sensitive acidic proteinases in reovirus infections of L orMDCK cells. Virology 251:264–272.

27. Kowalik, T. F., Y.-Y. Yang, and J. K.-K. Li. 1990. Molecular cloning andcomparative sequence analyses of bluetongue virus S1 segments by selectivesynthesis of specific full-length DNA copies of dsRNA genes. Virology 177:820–823.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

28. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

29. Lawton, J. A., and B. V. Venkataram Prasad. 1996. Automated softwarepackage for icosahedral virus reconstruction. J. Struct. Biol. 116:209–215.

30. Lee, P. W., E. C. Hayes, and W. K. Joklik. 1981. Protein �1 is the reovirus cellattachment protein. Virology 108:156–163.

31. Lucia-Jandris, P., J. W. Hooper, and B. N. Fields. 1993. Reovirus M2 geneis associated with chromium release from mouse L cells. J. Virol. 67:5339–5345.

32. Marsh, M., and A. Pelchen-Matthews. 1994. The endocytic pathway andvirus entry, p. 215–240. In E. Wimmer (ed.), Cellular receptors for animalviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

33. Martinez, C. G., R. Guinea, J. Benavente, and L. Carrasco. 1996. The entryof reovirus into L cells is dependent on vacuolar proton-ATPase activity.J. Virol. 70:576–579.

34. Maxfield, F. R. 1982. Weak bases and ionophores rapidly and reversibly raisethe pH in endocytic vesicles in cultured mouse fibroblasts. J. Cell Biol.95:676–681.

35. McCrae, M. A., and W. K. Joklik. 1978. The nature of the polypeptideencoded by each of the ten double-stranded RNA segments of reovirus type3. Virology 89:578–593.

36. Morrison, L. A., B. N. Fields, and T. S. Dermody. 1993. Prolonged replica-tion in the mouse central nervous system of reoviruses isolated from persis-tently infected cell cultures. J. Virol. 67:3019–3026.

37. Mustoe, T. A., R. F. Ramig, A. H. Sharpe, and B. N. Fields. 1978. Geneticsof reovirus: identification of the dsRNA segments encoding the polypeptidesof the � and � size classes. Virology 89:594–604.

38. Nason, E. L., J. D. Wetzel, S. K. Mukherjee, E. S. Barton, B. V. VenkataramPrasad, and T. S. Dermody. 2001. A monoclonal antibody specific for reo-virus outer-capsid protein �3 inhibits �1-mediated hemagglutination bysteric hinderance. J. Virol. 75:6625–6634.

39. Nibert, M. L., J. D. Chappell, and T. S. Dermody. 1995. Infectious subvirionparticles of reovirus type 3 Dearing exhibit a loss in infectivity and contain acleaved �1 protein. J. Virol. 69:5057–5067.

40. Nibert, M. L., D. B. Furlong, and B. N. Fields. 1991. Mechanisms of viralpathogenesis: distinct forms of reoviruses and their roles during replicationin cells and host. J. Clin. Investig. 88:727–734.

41. Ohkuma, S., and B. Poole. 1978. Fluorescence probe measurement of theintralysosomal pH in living cells and the perturbation of pH by variousagents. Proc. Natl. Acad. Sci. USA 75:3327–3331.

42. Olland, A. M., J. Jane-Valbuena, L. A. Schiff, M. L. Nibert, and S. C.

Harrison. 2001. Structure of the reovirus outer capsid and dsRNA-bindingprotein �3 at 1.8 A resolution. EMBO J. 20:979–989.

43. Rubin, D. H., D. B. Weiner, C. Dworkin, M. I. Greene, G. G. Maul, and W. V.Williams. 1992. Receptor utilization by reovirus type 3: distinct binding siteson thymoma and fibroblast cell lines result in differential compartmentaliza-tion of virions. Microb. Pathog. 12:351–365.

44. Silverstein, S. C., C. Astell, D. H. Levin, M. Schonberg, and G. Acs. 1972.The mechanism of reovirus uncoating and gene activation in vivo. Virology47:797–806.

45. Spendlove, R. S., and F. L. Schaffer. 1965. Enzymatic enhancement of in-fectivity of reovirus. J. Bacteriol. 89:597–602.

46. Sturzenbecker, L. J., M. Nibert, D. Furlong, and B. N. Fields. 1987. Intra-cellular digestion of reovirus particles requires a low pH and is an essentialstep in the viral infectious cycle. J. Virol. 61:2351–2361.

47. Tosteson, M. T., M. L. Nibert, and B. N. Fields. 1993. Ion channels inducedin lipid bilayers by subvirion particles of the nonenveloped mammalianreoviruses. Proc. Natl. Acad. Sci. USA 90:10549–10552.

48. van Heel, M. 1987. Similarity measures between images. Ultramicroscopy21:456–469.

49. Virgin, H. W., IV, R. Bassel-Duby, B. N. Fields, and K. L. Tyler. 1988.Antibody protects against lethal infection with the neurally spreading reovi-rus type 3 (Dearing). J. Virol. 62:4594–4604.

50. Wallis, C., K. O. Smith, and J. L. Melnick. 1964. Reovirus activation byheating and inactivation by cooling in MgCl2 solution. Virology 22:608–619.

51. Weiner, H. L., K. A. Ault, and B. N. Fields. 1980. Interaction of reovirus withcell surface receptors. I. Murine and human lymphocytes have a receptor forthe hemagglutinin of reovirus type 3. J. Immunol. 124:2143–2148.

52. Wessner, D. R., and B. N. Fields. 1993. Isolation and genetic characterizationof ethanol-resistant reovirus mutants. J. Virol. 67:2442–2447.

53. Wetzel, J. D., G. J. Wilson, G. S. Baer, L. R. Dunnigan, J. P. Wright, D. S. H.Tang, and T. S. Dermody. 1997. Reovirus variants selected during persistentinfections of L cells contain mutations in the viral S1 and S4 genes and arealtered in viral disassembly. J. Virol. 71:1362–1369.

54. Wriggers, W., R. A. Milligan, and J. A. McCammon. 1999. Situs: a packagefor docking crystal structures into low-resolution maps from electron micros-copy. J. Struct. Biol. 125:185–195.

55. Zhou, Z. H., B. V. Venkataram Prasad, J. Jakana, F. J. Rixon, and W. Chiu.1994. Protein subunit structures in the herpes simplex virus capsid deter-mined from 400-kV spot-scan electron cryomicroscopy. J. Mol. Biol. 242:456–469.



http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

A Single Mutation in the Carboxy Terminus of Reovirus ...jvi.asm.org/content/76/19/9832.full.pdf ·...

Documents

Transcript of A Single Mutation in the Carboxy Terminus of Reovirus ...jvi.asm.org/content/76/19/9832.full.pdf ·...