Transcriptional inhibition of the parvovirus minute virus of mice by constitutive expression of an antisense RNA targeted against the NS-1 transactivator protein

Ramírez, Juan Camilo; Santarén, Juán F.; Almendral, José M.

doi:10.1016/s0042-6822(95)80019-0

Cited by 25 publications

(24 citation statements)

References 53 publications

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“…Site-directed mutagenesis of the VP1/ VP2 gene of MVM (strain p) was carried out using the QuikChange system (Stratagene) on recombinant plasmid pSVtk-VP1/2 originally provided by J. M. Almendral (Centro de Biología Molecular, Madrid, Spain) (45). The mutations were introduced by subcloning in an MVMp infectious clone originally provided by P. Tattersall (Yale University Medical School, New Haven, CT) (46) and then modified (45). The presence of the mutations was confirmed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

Manipulation of the mechanical properties of a virus by protein engineering

Carrasco¹,

Castellanos

Pablo³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

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In a previous study, we showed that the DNA molecule within a spherical virus (the minute virus of mice) plays an architectural role by anisotropically increasing the mechanical stiffness of the virus. A finite element model predicted that this mechanical reinforcement is a consequence of the interaction between crystallographically visible, short DNA patches and the inner capsid wall. We have now tested this model by using protein engineering. Selected amino acid side chains have been truncated to specifically remove major interactions between the capsid and the visible DNA patches, and the effect of the mutations on the stiffness of virus particles has been measured using atomic force microscopy. The mutations do not affect the stiffness of the empty capsid; however, they significantly reduce the difference in stiffness between the DNA-filled virion and the empty capsid. The results (i) reveal that intermolecular interactions between individual chemical groups contribute to the mechanical properties of a supramolecular assembly and (ii) identify specific protein-DNA interactions as the origin of the anisotropic increase in the rigidity of a virus. This study also demonstrates that it is possible to control the mechanical properties of a protein nanoparticle by the rational application of protein engineering based on a mechanical model. atomic force microscopy ͉ nanomechanics ͉ protein-DNA interactions

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Section: Methodsmentioning

confidence: 99%

Manipulation of the mechanical properties of a virus by protein engineering

Carrasco¹,

Castellanos

Pablo³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

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“…Site-directed mutagenesis of the VP2 gene of MVM strain p (MVMp) was carried out by using the QuikChange system (Stratagene) on recombinant plasmids pSVtk-VP1͞2 [which included the VP1͞VP2 gene of MVMp and was derived from a molecular clone of the MVMp genome (46)] and pFB1-VP2 (47). The mutations were confirmed by automated sequencing.…”

Section: Methodsmentioning

confidence: 99%

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

Reguera

Carreira

Riolobos

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

126

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Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles. P rotein-protein recognition mediates many fundamental biological processes. A detailed knowledge of these processes requires the determination of the structural, energetic, and functional roles of individual amino acid residues and interactions in protein-protein interfaces. These studies have been generally undertaken by using small protein-ligand complexes or oligomeric proteins of moderate size (reviewed in ref. 1; see also refs. 2-4). In contrast, for multimeric protein complexes, such as viral capsids (5, 6) or large cellular assemblies, little is known about the specific molecular determinants of protein association and stability. Mutational studies of virus capsids, generally focused on a few specific amino acid residues, have provided important insights (7-22). However, exhaustive experimental studies on the relative importance of residues and molecular interactions in viral capsid assembly, disassembly, and͞or stability are still very limited. These studies contribute also to the understanding of protein structure-function relationships and evolution under conflictive selective constraints (22-27), and they could be exploited possibly in the design of thermostable vaccines and antiviral agents promoting capsid disassembly or interfering with assembly (23, 28-31).Many viruses, including viruses of medical or veterinary significance, have capsids of icosahedral symmetry. The icosahedral T ϭ 1 capsids of parvoviruses (32-37) are formed by 60 protein subunits that are contributed by three nonidentical polypeptide chains (VP1, VP2, and VP3). These polypeptides derive, however, from a single gene and show identical fold and core sequence (Fig. 1). In the minut...

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“…Recombinant Plasmids-Plasmid pSVTK-VP1/VP2 included the VP1/VP2 gene of MVMp and was derived from a molecular clone of the MVMp genome (51). The VP2-coding segment was amplified, using PCR and oligonucleotides with flanking BamHI and XhoI sites.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Disassembly of a Parvovirus Capsid and Effect on Capsid Stability of Heterologous Peptide Insertions in Surface Loops

Carreira

Menéndez

Reguera

et al. 2004

Journal of Biological Chemistry

126

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We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.The study of the folding, association, and disassembly of large multimeric proteins is complicated by their size, the general irreversibility of the reactions involved, and the frequent occurrence of off-pathway intermediates. However, the significant advances already made hold promise for a detailed understanding of these processes (1). Spherical virus capsids are large, multimeric proteins (2-5) and constitute attractive models for the study of the association, stability, and disassembly of very large protein complexes (for reviews see Refs. 4 and 6 -13). In addition, viral capsids are highly dynamic entities and have evolved unique structural solutions in response to the diverse, sometimes conflicting functions they must perform during the virus life cycle (4, 7, 11, 14 -16). Thus, they provide good opportunities to understand finely tuned structure-function relationships in proteins and to develop new antiviral approaches based on the inhibition of assembly or uncoating (15,17, 18).The icosahedral T ϭ 1 capsids of parvoviruses (19 -24) are formed by 60 protein subunits contributed by three nonidentical polypeptide chains that show, however, identical -fold and core sequence. VP2 is the major capsid protein and can selfassemble into empty (DNA-free) capsids (viral-like particles or VLPs).1 VP1, a minor component of natural capsids, includes...

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Transcriptional inhibition of the parvovirus minute virus of mice by constitutive expression of an antisense RNA targeted against the NS-1 transactivator protein

Cited by 25 publications

References 53 publications

Manipulation of the mechanical properties of a virus by protein engineering

Manipulation of the mechanical properties of a virus by protein engineering

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

In Vitro Disassembly of a Parvovirus Capsid and Effect on Capsid Stability of Heterologous Peptide Insertions in Surface Loops

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