Cellular transcription factor Sp1 recruits simian virus 40 capsid proteins to the viral packaging signal, ses.
ABSTRACT: Simian virus 40 (SV40) capsid assembly occurs in the nucleus. All three capsid proteins bind DNA nonspecifically, raising the dilemma of how they attain specificity to the SV40 minichromosome in the presence of a large excess of genomic DNA. The SV40 packaging signal, ses, which is required for assembly, is composed of multiple DNA elements that bind transcription factor Sp1. Our previous studies showed that Sp1 participates in SV40 assembly and that it cooperates in DNA binding with VP2/3. We hypothesized that Sp1 recruits the capsid proteins to the viral minichromosome, conferring upon them specific DNA recognition. Here, we have tested the hypothesis. Computer analysis showed that the combination of six tandem GC boxes at ses is not found at cellular promoters and therefore is unique to SV40. Cooperativity in DNA binding between Sp1 and VP2/3 was not abolished at even a 1,000-fold excess of cellular DNA, providing strong support for the recruitment hypothesis. Sp1 also binds VP1 and cooperates with VP1 in DNA binding. VP1 pentamers (VP1(5)) avidly interact with VP2/3, utilizing the same VP2/3 domain as described for polyomavirus. We conclude that VP1(5)-VP2/3 building blocks are recruited by Sp1 to ses, where they form the nucleation center for capsid assembly. By this mechanism the virus ensures that capsid formation is initiated at a single site around its minichromosome. Sp1 enhances the formation of SV40 pseudovirions in vitro, providing additional support for the model. Analyses of Sp1 and VP3 deletion mutants showed that Sp1 and VP2/3 bind one another and cooperate in DNA binding through their DNA-binding domains, with additional contacts outside these domains. VP1 contacts Sp1 at residues outside the Sp1 DNA-binding domain. These and additional data allowed us to propose a molecular model for the VP1(5)-VP2/3-DNA-Sp1 complex.
Project description: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 approximately 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.
Project description:The nonenveloped polyomavirus simian virus 40 (SV40) is taken up into cells by a caveola-mediated endocytic process that delivers the virus to the endoplasmic reticulum (ER). Within the ER lumen, the capsid undergoes partial disassembly, which exposes its internal capsid proteins VP2 and VP3 to immunostaining with antibodies. We demonstrate here that the SV40 genome does not become accessible to detection while the virus is in the ER. Instead, the genome becomes accessible two distinct detection procedures, one using anti-bromodeoxyuridine antibodies and the other using a 5-ethynyl-2-deoxyuridine-based chemical reaction, only after the emergence of partially disassembled SV40 particles in the cytoplasm. These cytoplasmic particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome. Thus, SV40 particles undergo discrete disassembly steps during entry that are separated temporally and topologically. First, a partial disassembly of the particles occurs in the ER, which exposes internal capsid proteins VP2 and VP3. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection.
Project description:Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.
Project description:Two groups of temperature-sensitive (ts) mutants, termed ts B and ts C, have mutations in the major capsid protein of SV40, Vp1. These mutants have virion assembly defects at the nonpermissive temperature, but can complement one another when two mutants, one from each group, coinfect a cell. A third group of mutants, termed ts BC, have related phenotypes, but do not complement other mutants. We found that the mutations fall into two structural and functional classes. All ts C and one ts BC mutations map to the region close to the Ca2+ binding sites, and are predicted to disrupt the insertion of the distal part of the C-terminal invading arm (C-arm) into the receiving clamp. They share a severe defect in assembly at the nonpermissive temperature, with few capsid proteins attached to the viral minichromosome. By contrast, all ts B and most ts BC mutations map to a contiguous region including acceptor sites for the proximal part of the C-arm and intrapentamer contacts. These mutants form assembly intermediates that carry substantial capsid proteins on the minichromosome. Thus, accurate virion assembly is prevented by mutations that disrupt interactions between the receiving pentamer and both the proximal and distal parts of the C-arms, with the latter having a greater effect. The distinct spatial localization and assembly defects of the two classes of mutants provide a rationale for their intracistronic complementation and suggest models of capsid assembly.
Project description:The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.
Project description:Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human polyomaviruses. The SV40-GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.
Project description:Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.
Project description:The major capsid protein of norovirus VP1 assembles to form an icosahedral viral particle. Despite evidence that the Norwalk virus (NV) minor structural protein VP2 is present in infectious virions, the available crystallographic and electron cryomicroscopy structures of NV have not revealed the location of VP2. In this study, we determined that VP1 associates with VP2 at the interior surface of the capsid, specifically with the shell (S) domain of VP1. We mapped the interaction site to amino acid 52 of VP1, an isoleucine located within a sequence motif IDPWI in the S domain that is highly conserved across norovirus genogroups. Mutation of this isoleucine abrogated VP2 incorporation into virus-like particles without affecting the ability for VP1 to dimerize and form particles. The highly basic nature of VP2 and its location interior to the viral particle are consistent with its potential role in assisting capsid assembly and genome encapsidation.
Project description:The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.
Project description:Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ?25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.