scholarly journals Mutagenesis of Tyrosine 24 in the VPg Protein Is Lethal for Feline Calicivirus

2004 ◽  
Vol 78 (9) ◽  
pp. 4931-4935 ◽  
Author(s):  
Tanaji Mitra ◽  
Stanislav V. Sosnovtsev ◽  
Kim Y. Green
Keyword(s):  
Amino Acids ◽  
Cdna Clone ◽  
Mutational Analysis ◽  
Covalent Bond ◽  
Feline Calicivirus ◽  
Tyrosine Residues ◽  
Positive Sense ◽  
Growth Properties ◽  
Calculated Mass ◽  
Covalently Linked

ABSTRACT The genome of feline calicivirus (FCV) is an ∼7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3′ end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5′ end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2134 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of theCaliciviridaefamily of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP) moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

2016 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notable feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double stranded RNA intermediate in the cytoplasm of the infected cell which is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5’ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterisation of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

2016 ◽  
Author(s):  
Allan Olspert ◽  
Myra Hosmillo ◽  
Yasmin Chaudhry ◽  
Lauri Peil ◽  
Erkki Truve ◽  
...  
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Serine Phosphorylation ◽  
Viral Rna ◽  
Feline Calicivirus ◽  
Genome Replication ◽  
Murine Norovirus ◽  
Wide Range ◽  
Positive Sense ◽  
Covalently Linked

Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notable feline calicivirus (FCV) and murine norovirus (MNV). These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double stranded RNA intermediate in the cytoplasm of the infected cell which is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5’ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterisation of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

scholarly journals In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽  
1998 ◽  
Vol 149 (4) ◽  
pp. 1649-1663
Author(s):  
Oliver Z Nanassy ◽  
Kelly T Hughes
Keyword(s):  
Amino Acids ◽  
Biochemical Analysis ◽  
Mutational Analysis ◽  
Recombination Reaction ◽  
Recombination Site ◽  
Dna Inversion ◽  
Hin Recombinase ◽  
One Step ◽  
A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

scholarly journals Infectious Transcripts from Cloned Genome-Length cDNA of Porcine Reproductive and Respiratory Syndrome Virus

1998 ◽  
Vol 72 (1) ◽  
pp. 380-387 ◽  
Author(s):  
J. J. M. Meulenberg ◽  
J. N. A. Bos-de Ruijter ◽  
R. van de Graaf ◽  
G. Wensvoort ◽  
R. J. M. Moormann
Keyword(s):  
Cdna Clone ◽  
De Novo ◽  
Infectious Clone ◽  
Full Length ◽  
Respiratory Syndrome Virus ◽  
Cdna Clones ◽  
Genome Length ◽  
Full Length Cdna ◽  
Lung Macrophages ◽  
Growth Properties

ABSTRACT The 5′-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5′-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

scholarly journals Tyrosine phosphorylation of P160 BCR by P210 BCR-ABL

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1257-1263 ◽  
Author(s):  
D Lu ◽  
J Liu ◽  
M Campbell ◽  
JQ Guo ◽  
N Heisterkamp ◽  
...  
Keyword(s):  
Amino Acids ◽  
Tyrosine Phosphorylation ◽  
Deletion Mutant ◽  
Physical Interaction ◽  
Tyrosine Residues ◽  
Ph Chromosome ◽  
Pathologic Processes ◽  
Simultaneous Expression

Abstract It is well established that the chimeric BCR-ABL gene formed by joining parts of the BCR and ABL genes plays a key role in the pathogenesis of Philadelphia (Ph) chromosome-positive leukemias. We report that simultaneous expression of P210 BCR-ABL and P160 BCR in simian COS-1 cells yielded stable complexes of these two proteins, and induced phosphorylation of P160 BCR on tyrosine residues in vivo. Tyrosine phosphorylation of a deletion mutant encoding 553 amino acids of BCR N- terminal sequences was also detected when it was coexpressed with P210 BCR-ABL. We propose that tyrosine phosphorylation of P160 BCR by P210 BCR-ABL and their stable physical interaction may perturb normal BCR functions and that these alterations are directly involved in the pathologic processes found in Ph chromosome-associated leukemias.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

scholarly journals Novel RNA Virus Genome Discovered in Ghost Ants (Tapinoma melanocephalum) from Hawaii

2017 ◽  
Vol 5 (30) ◽  
Author(s):  
Laura E. Brettell ◽  
Gideon J. Mordecai ◽  
Purnima Pachori ◽  
Stephen J. Martin
Keyword(s):  
Amino Acids ◽  
Genome Sequence ◽  
Rna Virus ◽  
Virus Genome ◽  
Full Genome Sequence ◽  
Full Genome ◽  
Tapinoma Melanocephalum ◽  
Positive Sense

ABSTRACT Here, we report the full-genome sequence of Milolii virus, a novel single-stranded (positive-sense) RNA virus discovered from Tapinoma melanocephalum ants in Hawaii. The genome is 10,475 nucleotides long, encoding a polyprotein of 3,304 amino acids.

scholarly journals Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

2010 ◽  
Vol 84 (11) ◽  
pp. 5550-5564 ◽  
Author(s):  
Robert J. Ossiboff ◽  
Yi Zhou ◽  
Patrick J. Lightfoot ◽  
B. V. Venkataram Prasad ◽  
John S. L. Parker
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Structural Changes ◽  
Feline Calicivirus ◽  
Soluble Receptor ◽  
Viral Capsids ◽  
Dimer Interface ◽  
Metastable Structures ◽  
Growth Properties ◽  
Functional Receptor

ABSTRACT Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37°C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-Å structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37°C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

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