pH-Dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

Abstract Physiological ionic strength conditions prevented low pH-mediated destabilization of the adenovirion. A conformational change of the virion was induced at low pH as demonstrated by endoproteolytic cleavage of virions with dispase at pH 5.0. Hidden cleavage sites of the hexons were exposed and upon enzymatic digestion, virions still were intact as physical entities. Enzymatic cleavage of the hexon protein increased its hydrophobicity.     

Influenza virus proteins: preparation of a soluble M1 polypeptide by means of a stepwise deproteination of virions

Layer by layer uncoating of influenza A and B viruses with non-ionic detergent (NP-40) at fixed pH was developed. Treatment of virions with NP-40 at neutral or alkaline pH solubilized the lipoprotein envelope and the surface glycopolypeptides HA1 and HA2, but the internal core structures containing matrix protein M1 remained. Exposition of the cores in acidic media (pH 4,5 and lower) selectively solubilized protein M1 and released viral ribonucleoprotein (RNP). The resulting M1 sedimented in a glycerol gradient with a coefficient of 2.8 S and most probably exists as a monomer of 27,000 Da polypeptide. Neutralization of protein M1 with Tris-HC1 at pH 7.0 did not cause aggregation of M1 polypeptides. The described method of viron layer by layer uncoating with non-ionic detergent at fixed pH is suitable for isolation of subvirus structures and individual viral proteins.     

Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.     

Extraction and purification of the chief glycoprotein of the erythrocyte membrane

The major glycoprotein of the human erythrocyte membrane has been released from ghosts, by the non-ionic detergent Tween 20 at pH 8,5 and at low ionic strength and further purified by successive passages through columns of DEAE Sephadex at pH 6,8 and CM Sephadex at pH 5 or by hydroxyapatite chromatography at pH 6,8. The purified glycoprotein thus obtained represents about 1 % of the membrane proteins, and shows two major bands upon polyacrylamide gel electrophoresis. These bands designed as PAS 1 and PAS 2 are the dimer and the monomer of the glycoprotein. Several other minor bands can also appear on SDS gels, according to experimental conditions of solubilization and purification and are probably oligomers of PAS 1 and PAS 2. This glycoprotein possess inhibitory activity against various phytohemagglutinins.     

Functional Comparison of SCARB2 and PSGL1 as Receptors for Enterovirus 71

Human scavenger receptor class B, member 2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL1) have been identified to be the cellular receptors for enterovirus 71 (EV71). We compared the EV71 infection efficiencies of mouse L cells that expressed SCARB2 (L-SCARB2) and PSGL1 (L-PSGL1) and the abilities of SCARB2 and PSGL1 to bind to the virus. L-SCARB2 cells bound a reduced amount of EV71 compared to L-PSGL1 cells. However, EV71 could infect L-SCARB2 cells more efficiently than L-PSGL1 cells. The results suggested that the difference in the binding capacities of the two receptors was not the sole determinant of the infection efficiency and that SCARB2 plays an essential role after attaching to virions. Therefore, we examined the viral entry into L-SCARB2 cells and L-PSGL1 cells by immunofluorescence microscopy. In both cells, we detected internalized EV71 virions that colocalized with an early endosome marker. We then performed a sucrose density gradient centrifugation analysis to evaluate viral uncoating. After incubating the EV71 virion with L-SCARB2 cells or soluble SCARB2 under acidic conditions below pH 6.0, we observed that part of the native virion was converted into an empty capsid that lacked both genomic RNA and VP4 capsid proteins. The results suggested that the uncoating of EV71 requires both SCARB2 and an acidic environment and occurs after the internalization of the virus-receptor complex into endosomes. However, the empty capsid formation was not observed after incubation with L-PSGL1 cells or soluble PSGL1 under any of the tested pH conditions. These results indicated that SCARB2 is capable of viral binding, viral internalization, and viral uncoating and that the low infection efficiency of L-PSGL1 cells is due to the inability of PSGL1 to induce viral uncoating. The characterization of SCARB2 as an uncoating receptor greatly contributes to the understanding of the early steps of EV71 infection.     

Entry of adenovirus 2 into HeLa cells

Adenovirus 2 (Ad2) uncoating was analyzed as the destabilization of virions which renders the parental genome sensitive to DNase treatment. This event demonstrated a strong temperature dependence, and an Arrhenius plot of initial uncoating rates revealed an inflection point at around 16 degrees C. Activation energies of 331 kJ/mol below and 88 kJ/mol above this temperature were obtained for the uncoating process. Penetration of Ad2 through the plasma membrane was completely inhibited by sodium azide, whereas uncoating was only slightly influenced. This indicated that uncoating had already taken place at the outside of the plasma membrane. Incubations of Ad2 with isolated plasma membranes and cell homogenates showed that intact and metabolizing cells were required for uncoating. We further suggest, based on the inhibitory patterns of EDTA, EGTA, dansylcadaverine, and dithiothreitol, that this destabilization of virions follows upon reorganization in the plasma membrane. In the electron microscope the involvement of coated vesicles was shown for the initial uptake of virions, possibly followed by the engagement of acidic vesicles as judged from the effects of lysosomotropic agents on gene expression. The vectorial transport of virions from the plasma membrane to the nucleus was not affected by reagents interfering with the cytoskeletal system. Consequently, we propose that Ad2 virions are internalized by adsorptive endocytosis.     

Virus-Associated Nucleases: Location and Properties of Deoxyribonucleases and Ribonucleases in Purified Frog Virus 3

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.     

Maturation-Associated Destabilization of Hepatitis B Virus Nucleocapsid

The mature nucleocapsid (NC) of hepatitis B virus containing the relaxed circular (RC) DNA genome can be secreted extracellularly as virions after envelopment with the viral surface proteins or, alternatively, can be disassembled to release RC DNA (i.e., uncoating) into the host cell nucleus to form the covalently closed circular (CCC) DNA, which sustains viral replication and persistence. In contrast, immature NCs containing the viral single-stranded DNA or the pregenomic RNA are incompetent for either envelopment or uncoating. Little is currently known about how mature NCs, and not the immature ones, are specifically selected for these processes. Here, we have carried out a biochemical analysis of the different NC populations upon their separation through sucrose gradient centrifugation. We have found that the maturation of NCs is associated with their destabilization, manifested as increased protease and nuclease sensitivity, altered sedimentation during sucrose gradient centrifugation, and retarded mobility during native agarose gel electrophoresis. Also, three distinct populations of intracellular mature NCs could be differentiated based on these characteristics. Furthermore, mature NCs generated in vitro under cell-free conditions acquired similar properties. These results have thus revealed significant structural changes associated with NC maturation that likely play a role in the selective uncoating of the mature NC for CCC DNA formation and/or its preferential envelopment for virion secretion.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.     

Addition of exogenous protease facilitates reovirus infection in many restrictive cells

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.     

Osmotic swelling allows fusion of sendai virions with membranes of desialyzed erythrocytes and chromaffin granules

Sendai virus particles are able to fuse with Pronase-neuraminidase-treated human erythrocyte membranes as well as with vesicles obtained from chromaffin granules of bovine medulla. Fusion is inferred either from electron microscopic studies or from the observation that incubation of fluorescently labeled (bearing octadecyl Rhodamine B chloride) virions, with right-side-out erythrocyte vesicles (ROV) or with chromaffin granule membrane vesicles (CGMV), resulted in fluorescence dequenching. Fusion of Sendai virions with virus receptor depleted ROV was observed only under hypotonic conditions. Fusion with virus receptor depleted ROV required the presence of the two viral envelope glycoproteins, namely, the HN and F polypeptides. A 3-fold increase in the degree of fluorescence dequenching (virus-membrane fusion) was also obtained upon incubation of Sendai virions with CGMV in medium of low osmotic strength. This increase was not observed with inactivated, unfusogenic Sendai virions. The results of the present work demonstrate that, under hypotonic conditions, fusion between Sendai virions and biological membranes does not require the presence of specific receptors. Such fusion is characterized by the same features as fusion with and infection by Sendai virions of living cultured cells.     

Zinc- and pH-dependent conformational transition in a putative interdomain linker region of the influenza virus matrix protein M1

The matrix protein M1 of influenza A virus forms a shell beneath the viral envelope and sustains the virion architecture by interacting with other viral components. A structural change of M1 upon acidification of the virion interior in an early stage of virus infection is considered to be a key step to virus uncoating. We examined the structure of a 28-mer peptide (M1Lnk) representing a putative linker region between the N- and C-terminal domains of M1 by using circular dichroism, Raman, and absorption spectroscopy. M1Lnk assumes an alpha-helical structure in a mildly hydrophobic environment irrespective of pH, being consistent with the X-ray crystal structures of an N-terminal fragment of M1 at pH 7 and 4. In the presence of Zn(2+), on the other hand, M1Lnk takes a partially unfolded conformation at neutral pH with a tetrahedral coordination of two Cys residues and two His residues to a Zn(2+) ion in the central part of the peptide. Upon acidification, the peptide releases the Zn(2+) ion and refolds into the alpha-helix-rich structure with a midpoint of transition at pH 5.9. The pH-dependent conformational transition of M1Lnk strongly suggests that the interdomain linker region of M1 also undergoes a pH-dependent unfolding-refolding transition in the presence of Zn(2+). A small but significant portion of the M1 protein is bound to Zn(2+) in the virion, and the Zn(2+)-bound M1 molecule may play a special role in virus uncoating by changing the disposition of the N- and C-terminal domains upon acidification of the virion interior.     

Solubilisation of human erythrocyte band 4.1 protein in the non-ionic detergent Tween 20

Extraction of spectrin-depleted erythrocyte membranes with the non-ionic detergent Tween 20, in a 0.1 M glycine-NaOH buffer (pH 9.8) leads to the solubilization of band 4.1 and the sialoglycoproteins. The comigration of band 4.1 with the sialoglycoproteins in gel filtration and detergent-free electrophoresis indicated that these proteins may be associated as complexes of high molecular weight. Although treatment of intact membranes with Tween 20 under the same conditions does not lead to direct solubilization of proteins, severe disruption of the membranes was observed under phase contrast microscopy. Suspension of the treated membranes in 5 mM phosphate buffer (pH 8.0) leads to the solubilization of band 4.1, spectrin, actin and the sialoglycoproteins. High molecular weight complexes of band 4.1 and the sialoglycoproteins were isolated from these extracts, suggesting a possible interaction between band 4.1 and sialoglycoproteins which may be important for linking the cytoskeleton to the membrane.     

Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1.

Recent studies have shown that ICP4, one of the major immediate-early proteins of herpes simplex virus type 1 is present within the tegument region of the virion (F. Yao and R. J. Courtney, J. Virol. 63:3338-3344, 1989). With monoclonal antibodies to two additional immediate-early proteins, ICP0 and ICP27, and Western blot (immunoblot) analysis, ICP0, but not ICP27, was also found to be associated with purified virus particles. In an effort to localize the ICP0 within the virion, purified virions were treated with trypsin in the presence and absence of detergent. The data suggest that ICP0 is located within the tegument region of the virion and is not localized in the envelope or within the nucleocapsid. The number of molecules of ICP0 per virion was estimated to be approximately 150.     

Conformational changes in Sindbis virus induced by decreased pH are revealed by small-angle neutron scattering

Alphaviruses, such as Sindbis virus, undergo dramatic changes in three-dimensional structure upon exposure to low pH, and such exposure can establish conditions allowing fusion of the virus membrane with a cell plasma membrane upon return to neutral pH. While exposure to low pH is not required for entry of Sindbis virus into vertebrate or invertebrate cells, the conformational changes occurring at low pH may mimic those occurring upon virus-receptor interaction. Here, we employed small-angle neutron scattering with contrast variation to probe how the structure of a mammalian-grown Sindbis virus responds to moderately acidic pH. Several changes took place throughout the virion structure when the pH decreased from 7.2 to 6.4. Specifically, the RNA in the virion core underwent a conformational change. Additionally, the protein was redistributed. A significant amount of protein moved from the layer containing the lipid bilayer to the exterior of the virion. The results improve our understanding of the pH-driven alteration of Sindbis virus structure.     

Conformational changes in Sindbis virus envelope proteins accompanying exposure to low pH.

The attachment of high multiplicities of Sindbis virus to tissue-cultured cells followed by brief treatment at low pH has been shown to produce cell fusion (fusion from without). In this report, experiments to determine the effects of low pH on the physical and biological properties of Sindbis virus are described. Exposure of purified Sindbis virions to mildly acidic conditions resulted in a rapid and irreversible alteration in particle density and sedimentation characteristics, followed by a slower loss of infectivity. Infectivity was not restored by a return to neutral pH; rather, the loss of virus infectivity seemed to be initiated by exposure to low pH but continued at neutral pH. The formation of a virus-cell complex in which virions were attached to the cell surface protected the particles from low-pH inactivation, although low pH could still expose virus functions responsible for cell fusion. Low pH was found to induce a conformational change in the E2 polypeptide of the intact virion. These results are discussed with respect to the process of Sindbis virus infection of tissue-cultured cells.