Interaction of rotavirus particles with liposomes.

We have studied the interactions of purified viral particles with liposomes as a model to understand the mechanism of entry of rotavirus into the cell. Liposomes, made from pure as well as mixed lipids, that contained encapsulated self-quenching concentrations of the fluorophore carboxyfluorescein (CF) were used. Rotavirus-liposome interactions were studied from the fluorescence dequenching of CF resulting from its release to the bulk solution. Purified infectious double-shelled virus particles induced a concentration- and temperature-dependent release of CF. The rate and extent of CF release was maximum between pH 7.3 and 7.6. The removal of outer structural proteins VP4 and VP7 from virus, which results in the formation of single-shelled particles, prevented virus interaction with liposomes. Rotavirus particles with uncleaved VP4 did not interact with liposomes, but treatment in situ of these particles with trypsin restored the interaction with the liposomes and resulted in CF dequenching. Our data support the view that rotavirus enters the cell through direct penetration of the plasma membrane. In contrast, adenovirus, the only other nonenveloped virus studied by this method, shows the optimum rate of marker release from liposomes at around pH 6 (R. Blumenthal, P. S. Seth, M. C. Willingham, and I. Pastan, Biochemistry 25:2231-2237, 1986). The interaction between rotavirus and liposomes is sensitive to specific divalent metal ions, unlike the adenovirus-liposome interaction, which is independent of them.     

Proteolytic enhancement of rotavirus infectivity: molecular mechanisms

The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specifically, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those of known structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).     

NS35 and not vp7 is the soluble rotavirus protein which binds to target cells.

Recent studies using radiolabeled rotavirus lysates have demonstrated a 35-kilodalton viral protein that binds specifically to the surface of MA104 cells (N. Fukuhara, O. Yoshie, S. Kitakoa, and T. Konno, J. Virol. 62:2209-2218, 1988; M. Sabara, J. Gilchrist, G.R. Hudson, and L.A. Babiuk, J. Virol. 53:58-66, 1985). The binding protein was identified as vp7, an outer capsid glycoprotein and the product of rotavirus gene 9. These studies concluded that vp7 mediated viral attachment to MA104 cells and that the binding of a soluble viral protein to a cell monolayer mirrored the attachment of infectious rotavirus to permissive tissue culture cells. In the process of determining which viral protein adheres to the in vivo target cell in rotavirus infection, the mammalian enterocyte, we found that a similar 35-kilodalton rhesus rotavirus (RRV) protein bound to both MA104 cells and murine enterocytes. However, further analysis of this protein by immunoprecipitation, inhibition of glycosylation, and partial proteolysis showed that it was not the RRV gene 9 product, vp7, but the gene 8 product, NS35. Similar results were obtained by using porcine rotavirus (OSU) and bovine rotavirus (NCDV) strains. Binding studies using the in vitro-expressed products of RRV genes 8 and 9 confirmed these results. Since double-shelled virions inhibited the binding of NS35 to cells, we looked for the presence of this protein in preparations of purified virus. Examination of density gradient-purified virus preparations revealed biochemical and immunological evidence that NS35 copurifies in small amounts with double-shelled virions. Thus, these studies clearly demonstrated that when rotavirus proteins are prepared in a soluble form from infected cells, NS35, and not vp7, binds to the surfaces of MA104 cells and murine enterocytes. The observations do not confirm previous experimental results which supported the hypothesis that vp7 was the viral attachment protein. They are consistent with but do not prove the hypothesis that NS35 functions as the rotavirus attachment protein.     

Rotavirus capsid protein VP5* permeabilizes membranes

Proteolytic cleavage of the VP4 outer capsid spike protein into VP8* and VP5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. In this study we addressed the function of the VP5* cleavage fragment in permeabilizing membranes. Expressed VP5* and truncated VP5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (LUVs) preloaded with carboxyfluorescein (CF). VP5* and VP5* truncations, but not VP4 or VP8*, permeabilized LUVs as measured by fluorescence dequenching of released CF. Similar to virus-induced CF release, VP5*-induced CF release was concentration and temperature dependent, with a pH optimum of 7.35 at 37 degrees C, but independent of the presence of divalent cations or cholesterol. VP5*-induced permeability was completely inhibited by VP5*-specific neutralizing monoclonal antibodies (2G4, M2, or M7) which recognize conformational epitopes on VP5* but was not inhibited by VP8*-specific neutralizing antibodies. In addition, N-terminal and C-terminal VP5* truncations including residues 265 to 474 are capable of permeabilizing LUVs. These findings demonstrate that VP5* permeabilizes membranes in the absence of other rotavirus proteins and that membrane-permeabilizing VP5* truncations contain the putative fusion region within predicted virion surface domains. The ability of recombinant expressed VP5* to permeabilize membranes should permit us to functionally define requirements for VP5*-membrane interactions. These findings indicate that VP5* is a specific membrane-permeabilizing capsid protein which is likely to play a role in the cellular entry of rotaviruses.     

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine.

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.     

Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.     

Heterogeneity of Raft-type membrane microdomains associated with VP4, the rotavirus spike protein, in Caco-2 and MA 104 cells

Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.     

The VP7 outer capsid protein of rotavirus induces polyclonal B-cell activation

The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection.     

Proteolysis of Monomeric Recombinant Rotavirus VP4 Yields an Oligomeric VP5* Core

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.     

Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER.

Maturation of rotavirus occurs in the ER. The virus transiently acquires an ER-derived membrane surrounding the virus particle before the eventual formation of double-shelled particles. The maturation process includes the retention and selective loss of specific viral protein(s) as well as the ER-derived membrane during formation of the outer capsid of the mature virus. When infected cells were depleted of Ca++ by use of the ionophore A23187 in calcium-free medium, membrane-enveloped intermediates were seen to accumulate. When Mn++, an efficient Ca++ competitor, was used to replace Ca++ in the medium, the accumulation of the enveloped intermediate was again observed, pointing to an absolute requirement of Ca++ in the maturation process. It was previously demonstrated in this laboratory that a hetero-oligomeric complex of NS28, VP7, and VP4 exists which may participate in the budding of the single-shelled particle into the ER (Maass, D. R., and P. H. Atkinson, 1990. J. Virol. 64:2632-2641). The present study demonstrates that either in the absence of Ca++ or in the presence of tunicamycin, a glycosylation inhibitor, VP7 is excluded from these hetero-oligomers. In the presence of Mn++, VP4 was blocked in forming a hetero-oligomeric complex with NS28 and VP7. The electrophoretic mobility of the viral glycoproteins synthesized in the presence of the ionophore were found to be altered. This size difference was attributed to altered N-linked glycosylation and carbohydrate processing of the viral glycoproteins. These results imply a major role for calcium and the state of glycosylation of NS28 in the assembly and acquisition of specific viral protein conformations necessary for the correct association of proteins during virus maturation in the ER.     

The concentration of Ca2+ that solubilizes outer capsid proteins from rotavirus particles is dependent on the strain.

It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.     

Rotavirus protein rearrangements in purified membrane-enveloped intermediate particles.

Rotavirus, a double-shelled nonenveloped member of the REoviridae family, becomes transiently membrane enveloped during its maturation process, as single-shelled particles bud from cytoplasmic viroplasm structures into the adjacent endoplasmic reticulum. The present study describes the isolation of these membrane-enveloped viral intermediates from rotavirus SA11-infected Ma104 cells. The enveloped intermediates comprised the proteins VP1, VP2, VP4, VP6, VP7, and NS28 and small amounts of NS35 and NS34. VP7 in the intermediate particles was recognized by either a polyclonal antibody to VP7, which previous studies had shown recognizes the membrane-associated form of VP7, or a monoclonal antibody which recognizes VP7 on mature virus. NS28, VP7, and VP4 could be complexed to a higher-molecular-weight form when the membrane-permeable cross-linker dithiobis(succinimidylproprionate) was used. However, when an impermeable cross-linker was used, the structural proteins, including VP7, were not accessible to cross-linking. Velocity sedimentation of cross-linked immunoisolated enveloped virus particles showed that VP7 and VP4 were located in the same fractions only when the membrane-permeable cross-linker was used, implying their heterooligomeric association during outer capsid formation. When intermediate enveloped virus particles were treated with protease, VP6 and VP7 were protected, but not in the presence of detergent. Taken together, these results support the idea that in the membrane-enveloped intermediate, VP7 is repositioned from its location in the endoplasmic reticulum lumen back across the viral membrane envelope to the inferior of the virus particle during the maturation process.     

Role for actin in the polarized release of rotavirus

Rotaviruses are characterized by polarized release from the apical side of infected enterocytes, and the rotavirus VP4 spike protein specifically binds to the actin network at the apical pole of differentiated enterocytic cells. To determine the functional consequences of this VP4-actin interaction, fluorescence recovery after photobleaching experiments were carried out to measure the diffusional mobility of VP4 associated with the microfilaments. Results show that VP4 binds to barbed ends of microfilaments by using actin treadmilling. Actin treadmilling inhibition results in the loss of rotavirus apical preferential release, suggesting a major role for actin in polarized rotavirus release.     

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.     

Rotavirus spike structure and polypeptide composition.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.     

Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.     

Selective depletion of stored calcium by thapsigargin blocks rotavirus maturation but not the cytopathic effect.

Rotavirus matures inside the endoplasmic reticulum (ER), a site of intracellular calcium storage. Total cell Ca2+ depletion has been shown to impair virus maturation, arresting this process at the membrane-enveloped intermediate form following its budding into the ER. On the other hand, rotavirus infection leads to an increase in the internal Ca2+ concentration ([Ca2+]i) and sequestered Ca2+ pools. We have used thapsigargin, an inhibitor of the Ca(2+)-ATPase of the ER, to release stored Ca2+ and to study its role in rotavirus morphogenesis and cytopathic effect. Thapsigargin (0.1 to 1 microM) released stored Ca2+ from MA-104 cells, as measured by chlorotetracycline fluorescence. The concentration of cytoplasmic Ca2+, measured with fura2, increased in infected cells whether treated or not with thapsigargin. Infectivity was decreased dose dependently by thapsigargin (3 log units at 0.25 to 1 microM). In infected cells treated with thapsigargin, glycosylation of VP7 and NS28 was inhibited. Electron microscopy of infected cells treated with thapsigargin showed normal synthesis of viroplasm. However, only membrane-enveloped, not double-shelled, particles could be observed within the ER. The conformation of VP7 in infected cells treated with thapsigargin appeared to be altered, as suggested by decreased immunofluorescence reactivity with monoclonal antibodies to highly conformation-dependent VP7 epitopes. The progression of cell death in infected cells, as measured by penetration of ethidium bromide, was not affected by thapsigargin. These results indicate that rotavirus maturation depends on a high sequestered [Ca2+], specifically in the ER. Cell death is the result of the accumulation of a viral product and is not related to the production of infective particles. This viral product(s) may be responsible for the increase in [Ca2+]i, which in turn leads to cell death.