A Beta-Stranded Motif Drives Capsid Protein Oligomers of the Parvovirus Minute Virus of Mice into the Nucleus for Viral Assembly

Abstract

The determinants of nuclear import in the VP-1 and VP-2 capsid proteins of the parvovirus minute virus of mice strain i (MVMi) synthesized in human fibroblasts were sought by genetic analysis in an infectious plasmid. Immunofluorescence of transfected cells revealed that the two proteins were involved in cooperative cytoplasmic interactions for nuclear cotransport. However, while VP-1 translocated regardless of extension of deletions and did not form capsid epitopes by itself, VP-2 seemed to require cytoplasmic folding and the overall conformation for nuclear transport. The sequence 528 KGKLTMRAKLR 538 was found necessary for nuclear uptake of VP-2, even though it was not sufficient to confer a nuclear localization capacity on a heterologous protein. In the icosahaedral MVMi capsid, this sequence forms the carboxy end of the amphipathic beta-strand I (␤I), and all its basic residues are contiguously positioned at the face that in the unassembled subunit would be exposed to solvent. Mutations in singly expressed VP-2 that either decrease the net basic charge of the exposed face (K530N-R534T), perturb the hydrophobicity of the opposite face (L531E), or distort the ␤I conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by ␤I mutants indicated that the basic residues clustered at one face of ␤I drive VP oligomers into the nucleus preceding and uncoupled to assembly and that the nuclear environment is required for MVMi capsid formation in the infected cell. The degree of VP-1/VP-2 transport cooperativity suggests that VP trimers are the morphogenetic intermediates translocating through the nuclear pore. The results support a model in which nuclear transport signaling preserves the VP-1/VP-2 stoichiometry necessary for efficient intranuclear assembly and in which the betastranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.The successful multiplication of many viruses depends on their gaining access to the transcription and replication machineries confined in the nucleus of the eukaryotic cell. Viruses of different compositions and sizes enter the nucleus during their life cycles in the form of particles, complexes, or virion subunits (28). These viruses use molecular interactions connecting to the physiological nuclear transport pathways of the cellular proteins. In the eukaryotic cell, most karyophilic polypeptides actively transported through the nuclear pore complex (NPC) (65) harbor a nuclear localization signal (NLS) necessary for nuclear import. In the so-called classical form, the NLS consists of a short sequence of basic amino acids either in a single cluster, as originally described for the simian virus 40 large T antigen (33), or in two domains, as for the nucleoplasmin bipartite nuclear targeting sequence (51). The import pathway of classical NLS-bearing proteins into the nucleus proceeds by consecutive interactions comprising importins ␣ and ␤ (14, 15; reviewed in reference 42), and additional soluble factors to dock to the cytopla...

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

“…Virions were recovered at 72 h from pMM984-transfected monolayers and titrated in standard plaque assays. Immunofluorescence assays were performed as described (44,45), with minor modifications.…”

“…1) are intermediates in the assembly of MVM and other parvoviruses. (i) The number of intersubunit contacts within trimers is much higher than it is between trimers (33, 43); (ii) transport of VP2 of MVM to the cell nucleus may require the formation of trimeric intermediates (44,45); (iii) small amounts of trimers were detected in cells expressing canine parvovirus (CPV) proteins (42); and (iv) the presence of trimeric intermediates has been observed directly in cross-linking and sedimentation assays of a few MVM capsid mutants whose assembly was impaired by steric mutations at the intertrimer interfaces (L.R., J.R., M.G.M., and J.M.A., unpublished data). We have used the empty capsid of MVM for identifying at the intertrimer interfaces the molecular determinants of assembly and stability of the parvoviral capsid, one of the simplest models available for a spherical virus capsid or multimeric protein complex.…”

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

“…2 Such VLPs thus constitute very simple models to study the mechanisms of assembly, stability, and disassembly of parvoviruses and of large macromolecular assemblies in general. In vivo, assembly of autonomous parvoviruses may proceed through a trimeric intermediate (20,27,28) that is transported to the cell nucleus (27,28). Uncoating may involve acidification at the endosomes (29,30), but the disassembly pathway is also unclear.…”

“…Immunofluorescence Assays-NB324K cells were electroporated essentially as described (27) with plasmid pSVTK-VP1/VP2, or with the same plasmid containing point mutations or insertions. The unassembled VPs and the assembled capsids were, respectively, detected by standard in situ immunofluorescence assays (27,28) (26,31,32).…”

“…In vivo, assembly of autonomous parvoviruses may proceed through a trimeric intermediate (20,27,28) that is transported to the cell nucleus (27,28). Uncoating may involve acidification at the endosomes (29, 30), but the disassembly pathway is also unclear.…”