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 Phosphorylation Status of the Parvovirus Minute Virus of Mice Particle: Mapping and Biological Relevance of the Major Phosphorylation Sites

2000 ◽  
Vol 74 (23) ◽  
pp. 10892-10902 ◽  
Author(s):  
Beatriz Maroto ◽  
Juan C. Ramı́rez ◽  
José M. Almendral
Keyword(s):  
Amino Acids ◽  
Nuclear Translocation ◽  
Biological Significance ◽  
Dimensional Structure ◽  
Phosphorylation Sites ◽  
Phenotypic Effect ◽  
Minute Virus Of Mice ◽  
Terminal Domain ◽  
Capsid Shell ◽  
Minute Virus

ABSTRACT The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the32P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.

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 B1 Lymphocytes and Myeloid Dendritic Cells in Lymphoid Organs Are Preferential Extratumoral Sites of Parvovirus Minute Virus of Mice Prototype Strain Expression

2005 ◽  
Vol 79 (6) ◽  
pp. 3517-3524 ◽  
Author(s):  
Zahari Raykov ◽  
Larissa Savelyeva ◽  
Ginette Balboni ◽  
Thomas Giese ◽  
Jean Rommelaere ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Lymph Nodes ◽  
Therapeutic Potential ◽  
Prototype Strain ◽  
Lymphoid Organs ◽  
Lymphoid Tissues ◽  
Myeloid Dendritic Cells ◽  
Minute Virus Of Mice ◽  
Minute Virus

ABSTRACT Due to their oncolytic properties and apathogenicity, autonomous parvoviruses have attracted significant interest as possible anticancer agents. Recent preclinical studies provided evidence of the therapeutic potential of minute virus of mice prototype strain (MVMp) and its recombinant derivatives. In a murine model of hemangiosarcoma, positive therapeutic outcome correlated with high intratumoral expression of MVMp-encoded genes in tumors and lymphoid organs, especially in tumor-draining lymph nodes. The source and relevance of this extratumoral expression, which came as a surprise because of the known fibrotropism of MVMp, remained unclear. In the present study, we investigated (i) whether the observed expression pattern occurs in different tumor models, (ii) which cell population is targeted by the virus, and (iii) the immunological consequences of this infection. Significant MVMp gene expression was detected in lymphoid tissues from infected tumor-free as well as melanoma-, lymphoma-, and hemangiosarcoma-bearing mice. This expression was especially marked in lymph nodes draining virus-injected tumors. Fluorescent in situ hybridization analysis, multicolor fluorescence-activated cell sorting, and quantitative reverse transcription-PCR revealed that MVMp was expressed in rare subpopulations of CD11b (Mac1)-positive cells displaying CD11c+ (myeloid dendritic cells [MDC]) or CD45B (B220+ [B1 lymphocytes]) markers. Apart from the late deletion of cytotoxic memory cells (CD8+ CD44+ CD62L−), this infection did not lead to significant alteration of the immunological profile of cells populating lymphoid organs. However, subtle changes were detected in the production of specific proinflammatory cytokines in lymph nodes from virus-treated animals. Considering the role of B1 lymphocytes and MDC in cancer and immunological surveillance, the specific ability of these cell types to sustain parvovirus-driven gene expression may be exploited in gene therapy protocols.

scholarly journals Virulent Variants Emerging in Mice Infected with the Apathogenic Prototype Strain of the Parvovirus Minute Virus of Mice Exhibit a Capsid with Low Avidity for a Primary Receptor

2005 ◽  
Vol 79 (17) ◽  
pp. 11280-11290 ◽  
Author(s):  
Mari-Paz Rubio ◽  
Alberto López-Bueno ◽  
José M. Almendral
Keyword(s):  
Recombinant Virus ◽  
Prototype Strain ◽  
Wild Type ◽  
Minute Virus Of Mice ◽  
Lower Affinity ◽  
Large Plaque ◽  
Primary Receptor ◽  
Primary Mouse ◽  
Minute Virus

ABSTRACT The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of low affinity, which favors systemic infection, is a major evolutionary process in the adaptation of parvoviruses to new hosts and in the cause of disease.

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

1986 ◽  
Vol 59 (3) ◽  
pp. 564-573 ◽  
Author(s):  
C V Jongeneel ◽  
R Sahli ◽  
G K McMaster ◽  
B Hirt
Keyword(s):  
Minute Virus Of Mice ◽  
Minute Virus ◽  
Splice Junctions
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