Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40 (original) (raw)
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Importance of Calcium-Binding Site 2 in Simian Virus 40 Infection
Journal of Virology, 2007
The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calciumbinding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.
BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol
Nature Cell Biology, 2011
How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane. Simian virus 40 (SV40) is a member of the polyomaviruses, a family of small, non-enveloped DNA viruses, of which some are human pathogens 1-3. The icosahedral capsid with a diameter of 45 nm is composed of 72 VP1 pentamers 4 stabilized by a network of interpentameric disulphide bonds 5,6. In an internal cavity, each pentamer carries another structural protein, either VP2 or VP3, which are hydrophobic 7 and not exposed on the virus surface. VP2 and VP3 differ in their N terminus in that VP2 contains 118 additional amino acids and an N-terminal myristyl group 8. The DNA genome is circular and located inside the viral capsid associated with nucleosomes 9. To infect cells, SV40 binds through the VP1 pentamers to the carbohydrate moiety of ganglioside GM1 at the plasma membrane, and is transported by endosomes to the lumen of the ER (refs 10-14). After isomerization of interpentameric disulphide bonds by the oxidoreductase ERp57 (ref. 6), the virus penetrates the ER membrane to access the cytosol from which it is imported into the nucleus 15-17. The membrane penetration step is not understood, nor is it known in which form the virus exits the ER. Here, we studied this process and found that SV40 capsids undergo a major conformational change in the lumen of the ER. To cross the membrane, SV40 takes advantage of ERAD machinery dedicated to the identification and removal of aberrant membrane proteins 18-23. RESULTS SV40 undergoes a conformational change in the ER Electron micrographs indicated that SV40 underwent a structural change after reaching the ER of CV-1 cells. Whereas particles present
GM1 structure determines SV40-induced membrane invagination and infection
Nature Cell Biology, 2010
Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.
Virology, 1997
Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and VP3, in Sf9 cells using baculovirus expression vectors. Coproduction of the capsid proteins VP1, VP2, and VP3 was achieved by infecting Sf9 cells with the three recombinant baculovirus species at equal multiplicities. All three proteins were found to be localized in the nuclear fraction. Electron microscopy of nuclear extracts of the infected cells showed an abundance of SV40-like capsid structures and heterogeneous aggregates of variable size, mostly 20-45 nm. Under the same staining conditions wild-type SV40 virions are 45 nm. The capsid-like particles sedimented in glycerol gradients similarly to authentic wild-type SV40 virions. Pentamers of the major capsid protein VP1 were also seen. Protein analysis on sucrose gradients demonstrated that the capsid-like particles can be disrupted by treatment with the reducing agent dithiothreitol and the calcium chelator EGTA. The capsid-like particles were found to be significantly less stable than SV40 virions and were partially stabilized by calcium ions. Understanding the complex interactions between the capsid proteins is important for the development of an efficient in vitro packaging system for SV40 virions and pseudovirions. ᭧ 1997 Academic Press
Journal of Virology, 2003
For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsid structures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40) mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and in combination. Our study provides evidence that calcium ions mediate not only virion assembly but also the initial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/or Glu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216R suggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected for further analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330K could not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed to enter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the seven acidic side chains at the two calcium-binding sites-Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), and Glu216 (both sites)-are important for SV40 infection. We propose that calcium coordination imparts not only stability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sites to control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in the cytoplasm early during infection.
SV40 Assembly In Vivo and In Vitro
Computational and Mathematical Methods in Medicine, 2008
The Simian virus 40 (SV40) capsid is aT = 7dicosahedral lattice ∼45 nm in diameter surrounding the ∼5 kb circular minichromosome. The outer shell is composed of 360 monomers of the major capsid protein VP1, tightly bound in 72 pentamers. VP1 is a jellyroll β-barrel, with extending N- and C-terminal arms. The N-terminal arms bind DNA and face the interior of the capsid. The flexible C-arms tie together the 72 pentamers in three distinct kinds of interactions, thus facilitating the formation of aT = 7 icosahedron from identical pentameric building blocks. Assemblyin vivowas shown to occur by addition of capsomers around the DNA. We apply a combination of biochemical and genetic approaches to study SV40 assembly. Ourin vivoandin vitrostudies suggest the following model: one or two capsomers bind at a high affinity toses, the viral DNA encapsidation signal, forming the nucleation centre for assembly. Next, multiple capsomers attach concomitantly, at lower affinity, around the minichromo...
Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells
Biochemical and Biophysical Research Communications, 2011
Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.
High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly
PLOS One, 2007
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ,6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility. Citation: Mukherjee S, Abd-El-Latif M, Bronstein M, Ben-nun-Shaul O, Kler S, et al (2007) High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly. PLoS ONE 2(8): e765.
Journal of Virology, 2001
The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indic...
The synthesis and transport of SV40 structural proteins
Virology, 1986
The kinetics of the synthesis and transport of viral structural proteins, Vpl and Vp3, and of actin in SV40 infected TC7 cells were studied. The newly synthesized proteins were found in the NP-40-soluble (Sol) fraction of the cell cytoplasm. The majority of newly synthesized viral structural proteins, destined for the cell nucleus for virion assembly, were transported to the cell nucleus (NW) between 10 and 30 min after synthesis, whereas the majority of newly synthesized actin remained in the Sol fraction of the cytoplasm, suggesting that some specific mechanism exists for selecting the proper sites for transport. The synthesis and transport of both Vpl and Vp3 throughout infected cells were similar. However, there is a difference in the transport properties of these two proteins. Once Vpl was synthesized, the mature Vpl was transported to both the cytoskeletal (Csk) and the Nut fractions in the absence of further protein synthesis, whereas the movement of Vp3 from the Sol to the Csk, but not to the Nut fraction, was partially inhibited in the absence of protein synthesis. Modification of Vpl occurred in the cell cytoplasm before transport to the cell nucleus. Its modification pattern suggests that the Csk is the site for the modification of Vpl. The efficiency of viral protein transport to the cell nucleus was diminished after 47 hr of infection. This trend was preceded by a decrease in the ability to incorporate label into actin 12 hr earlier in infection. Thus, some marking event appears to have occurred prior to the actual decrease in transport efficiency and the integrity of the cytoarchitecture appears to be important for viral protein transport.