Complementary Roles of Multiple Nuclear Targeting Signals in the Capsid Proteins of the Parvovirus Minute Virus of Mice during Assembly and Onset of Infection

Abstract

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T‫1؍‬ icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.The nuclear membrane offers a second barrier to those viruses that, upon specific cell surface recognition and internalization, need components of the replication and transcription machinery of the host cells for their multiplication. Indeed the structural components of karyophilic viruses reach the nucleus at two stages of the life cycle, first when the incoming particle delivers the genome and late in the infection during the nuclear accumulation of viral components leading to the biogenesis of the virions. As for the cellular components, the nuclear import of viral macromolecules must proceed across the central aqueous channel of the nuclear pore complex (NPC) (18, 58), a large structure with an eightfold rotational symmetry built from proteins called nucleoporins. Cytoplasmic-nuclear transport is directed by the interac...

Manipulation of the mechanical properties of a virus by protein engineering

“…NB324K cells were electroporated with the MVM infectious plasmid carrying the appropriate mutations (47). Virions were recovered from transfected monolayers and titrated in plaque assays.…”

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.…”

Nuclear and Mitochondrial Localization Signals Overlap within Bovine Herpesvirus 1 Tegument Protein VP22

“…Small proteins like VP22-(130 -232)-YFP can diffuse across the nuclear envelope, and their accumulation would require either constitutively active import rendered by nuclear transport signals, retention in the nucleus by nuclear retention signals, or both. Efficient nuclear accumulation involving both active import and nuclear retention has been demonstrated for other proteins (29,30). We previously reported the interaction of full-length VP22 with histones by far-Western blot analysis (6), an observation that makes the retention mechanism attractive.…”

“…Mitochondrial membrane permeability can be induced during the onset of apoptosis resulting in leakage of apoptogenic factors into the cytoplasm from the mitochondrial intermembrane space leading to a cascade of activated caspases, nuclear damage, and cell death (18,29). Virus infection triggers numerous cellular defense responses to limit viral replication; one cellular defense mechanism is apoptosis of the host cells.…”