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 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.

A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection

2013 ◽  
Vol 455 (1) ◽  
pp. 87-94 ◽  
Author(s):  
Milagros Castellanos ◽  
Rebeca Pérez ◽  
Alicia Rodríguez-Huete ◽  
Esther Grueso ◽  
José M. Almendral ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Viral Capsid ◽  
Minute Virus Of Mice ◽  
Vp2 Protein ◽  
Terminal Domain ◽  
Minute Virus

Very small residues at a glycine-rich tract in the capsid protein of the minute virus of mice are required to allow the translocation of a peptide segment through narrow pores in the viral capsid, which is needed for infection.

scholarly journals MOLECULAR AND STRUCTURAL BASIS OF THE EVOLUTION OF PARVOVIRUS TROPISM

1999 ◽  
Vol 47 (3) ◽  
pp. 379-394 ◽  
Author(s):  
P. Tijssen
Keyword(s):  
Amino Acids ◽  
Structural Basis ◽  
Threefold Axis ◽  
Minute Virus Of Mice ◽  
Feline Panleukopenia Virus ◽  
Infectious Clones ◽  
Minute Virus ◽  
Feline Parvovirus ◽  
Enteritis Virus ◽  
Aleutian Disease Virus

Parvoviruses have small genomes and, consequently, are highly dependent on their host for various functions in their reproduction. Since these viruses generally use ubiquitous receptors, restrictions are usually intracellularly regulated. A lack of mitosis, and hence absence of enzymes required for DNA replication, is a powerful block of virus infection. Allotropic determinants have been identified for several parvoviruses: porcine parvovirus, canine parvovirus (CPV), feline parvovirus (feline panleukopenia virus), minute virus of mice, Aleutian disease virus, andGmDNV (an insect parvovirus). Invariably, these identifications involved the use of infectious clones of these viruses and the exchange of restriction fragments to create chimeric viruses, of which the resulting phenotype was then established by transfection in appropriate cell lines. The tropism of these viruses was found to be governed by minimal changes in the sequence of the capsid proteins and, often, only 2 or 3 critical amino acids are responsible for a given tropism. These amino acids are usually located on the outside of the capsid near or on the spike of the threefold axis for the vertebrate parvoviruses and on loops 2 or 3 for the insect parvoviruses. This tropism is not mediated via specific cellular receptors but by interactions with intracellular factors. The nature of these factors is unknown but most data point to a stage beyond the conversion of the single-stranded DNA genome by host cell DNA polymerase into monomeric duplex intermediates of the replicative form. The sudden and devastating emergence of mink enteritis virus (MEV) and CPV in the last 50 years, and the possibility of more future outbreaks, demonstrates the importance of understanding parvovirus tropism.

scholarly journals Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains

1999 ◽  
Vol 19 (4) ◽  
pp. 2465-2474 ◽  
Author(s):  
Rongtuan Lin ◽  
Yael Mamane ◽  
John Hiscott
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Virus Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Interferon Regulatory Factor ◽  
Phosphorylation Sites ◽  
Transactivation Domain ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 3

ABSTRACT The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.

scholarly journals Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

Virology ◽  
2015 ◽  
Vol 476 ◽  
pp. 61-71 ◽  
Author(s):  
Sunil K. Tewary ◽  
Lingfei Liang ◽  
Zihan Lin ◽  
Annie Lynn ◽  
Susan F. Cotmore ◽  
...  
Keyword(s):  
Minute Virus Of Mice ◽  
Initiator Protein ◽  
Terminal Domain ◽  
Replication Initiator Protein ◽  
Dna Nicking ◽  
Minute Virus ◽  
Replication Initiator

scholarly journals Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription

2018 ◽  
Vol 92 (21) ◽  
Author(s):  
Bersabeh Tigabu ◽  
Palaniappan Ramanathan ◽  
Andrey Ivanov ◽  
Xionghao Lin ◽  
Philipp A. Ilinykh ◽  
...  
Keyword(s):  
Amino Acids ◽  
Small Molecule ◽  
Case Fatality ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Dimensional Structure ◽  
Regulation Of Transcription ◽  
Phosphorylation Sites ◽  
Content Type

ABSTRACTThe filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds.IMPORTANCEThe largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member ofFiloviridaefamily, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.

scholarly journals Initiation of Minute Virus of Mice DNA Replication Is Regulated at the Level of Origin Unwinding by Atypical Protein Kinase C Phosphorylation of NS1

2001 ◽  
Vol 75 (13) ◽  
pp. 5730-5739 ◽  
Author(s):  
Jürg P. F. Nüesch ◽  
Jesper Christensen ◽  
Jean Rommelaere
Keyword(s):  
Protein Kinase C ◽  
Dna Replication ◽  
Protein Kinase ◽  
Regulatory Elements ◽  
Esterification Reaction ◽  
Phosphorylation Sites ◽  
Minute Virus Of Mice ◽  
Atypical Pkc ◽  
Kinase C ◽  
Minute Virus

ABSTRACT Minute virus of mice nonstructural protein NS1 is a multifunctional protein that is involved in many processes necessary for virus propagation. To perform its distinct activities in timely coordinated manner, NS1 was suggested to be regulated by posttranslational modifications, in particular phosphorylation. In fact, NS1 replicative functions are dependent on protein kinase C (PKC) phosphorylation, most likely due to alteration of the biochemical profile of the viral product as determined by comparing native NS1 with its dephosphorylated counterpart. Through the characterization of NS1 mutants at individual PKC consensus phosphorylation sites for their biochemical activities and nickase function, we were able to identify two target atypical PKC phosphorylation sites, T435 and S473, serving as regulatory elements for the initiation of viral DNA replication. Furthermore, by dissociating the energy-dependent helicase activity from the ATPase-independent trans esterification reaction using partially single-stranded substrates, we could demonstrate that atypical PKC regulation of NS1 nickase activity occurs at the level of origin unwinding prior to trans esterification.

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