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

2002 ◽  
Vol 76 (14) ◽  
pp. 7049-7059 ◽  
Author(s):  
Eleuterio Lombardo ◽  
Juan C. Ramírez ◽  
Javier Garcia ◽  
José M. Almendral
Keyword(s):  
Nuclear Localization ◽  
Nuclear Transport ◽  
Degradation Pathway ◽  
Capsid Proteins ◽  
Nuclear Targeting ◽  
Nuclear Entry ◽  
Minute Virus Of Mice ◽  
Evolutionarily Conserved ◽  
Ubiquitin Proteasome ◽  
Minute Virus

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 T=1 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.

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

2000 ◽  
Vol 74 (8) ◽  
pp. 3804-3814 ◽  
Author(s):  
Eleuterio Lombardo ◽  
Juan C. Ramírez ◽  
Mavis Agbandje-McKenna ◽  
José M. Almendral
Keyword(s):  
Nuclear Localization ◽  
Nuclear Transport ◽  
Human Fibroblasts ◽  
Nuclear Pore ◽  
Minute Virus Of Mice ◽  
Capsid Formation ◽  
The Face ◽  
Minute Virus ◽  
Nuclear Environment ◽  
Viral Morphogenesis

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 sequence528KGKLTMRAKLR538 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 (βI), 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 βI conformation (G529P) produced cytoplasmic subviral oligomers. Particle formation by βI mutants indicated that the basic residues clustered at one face of βI 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 beta-stranded VP-2 nuclear localization motif contributes to the quality control of viral morphogenesis.

scholarly journals Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

2002 ◽  
Vol 76 (24) ◽  
pp. 12634-12645 ◽  
Author(s):  
Carlos Ros ◽  
Christoph J. Burckhardt ◽  
Christoph Kempf
Keyword(s):  
Nuclear Translocation ◽  
Brefeldin A ◽  
Prototype Strain ◽  
Endosomal Escape ◽  
Cellular Functions ◽  
Minute Virus Of Mice ◽  
Chloromethyl Ketone ◽  
Late Endosomes ◽  
Ubiquitin Proteasome ◽  
Minute Virus

ABSTRACT The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A 1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.

scholarly journals Developmental regulation of the heat shock response by nuclear transport factor karyopherin-α3

Development ◽  
2001 ◽  
Vol 128 (17) ◽  
pp. 3349-3358 ◽  
Author(s):  
Xiang-dong Fang ◽  
Tianxin Chen ◽  
Kim Tran ◽  
Carl S. Parker
Keyword(s):  
Heat Shock ◽  
Nuclear Localization ◽  
Heat Shock Response ◽  
Nuclear Transport ◽  
Early Embryo ◽  
Nuclear Localization Sequence ◽  
Nuclear Targeting ◽  
Transport Factor ◽  
Shock Response ◽  
Hsp70 Induction

During early stages of Drosophila development the heat-shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a Drosophila nuclear transport protein karyopherin-α3 (dKap-α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF’s nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations, which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3. Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 633-640 ◽  
Author(s):  
Philippe Collas ◽  
Peter Aleström
Keyword(s):  
Nuclear Localization ◽  
Plasmid Dna ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Transport ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
Expression Frequency ◽  
Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

scholarly journals The Infectivity and Lytic Activity of Minute Virus of Mice Wild-Type and Derived Vector Particles Are Strikingly Different

2005 ◽  
Vol 79 (1) ◽  
pp. 289-298 ◽  
Author(s):  
Susanne I. Lang ◽  
Stephanie Boelz ◽  
Alexandra Y. Stroh-Dege ◽  
Jean Rommelaere ◽  
Christiane Dinsart ◽  
...  
Keyword(s):  
Gene Expression ◽  
Marker Gene ◽  
Lytic Activity ◽  
Capsid Proteins ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Vector Particles

ABSTRACT Gene therapy vectors have been developed from autonomous rodent parvoviruses that carry a therapeutic gene or a marker gene in place of the genes encoding the capsid proteins. These vectors are currently evaluated in preclinical experiments. The infectivity of the vector particles deriving from the fibroblastic strain of minute virus of mice (MVMp) (produced by transfection in human cells) was found to be far less (approximately 50-fold-less) infectious than that of wild-type virus particles routinely produced by infection of A9 mouse fibroblasts. Similarly, wild-type MVMp produced by transfection also had a low infectivity in mouse cells, indicating that the method and producer cells influence the infectivity of the virus produced. Interestingly, producer cells made as many full vector particles as wild-type particles, arguing against deficient packaging being responsible for the low infectivity of viruses recovered from transfected cells. The hurdle to infection with full particles produced through transfection was found to take place at an early step following entry and limiting viral DNA replication and gene expression. Infections with transfection or infection-derived virus stocks normalized for their replication ability yielded similar monomer and dimer DNA amplification and gene expression levels. Surprisingly, at equivalent replication units, the capacity of parvovirus vectors to kill tumor cells was lower than that of the parental wild-type virus produced under the same transfection conditions, suggesting that beside the viral nonstructural proteins, the capsid proteins, assembled capsids, or the corresponding coding region contribute to the lytic activity of these viruses.

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