ABSTRACT
Human parvovirus B19 (B19V) is the causative agent of erythema infectiosum in humans. B19 infection also causes severe disease manifestations, such as chronic anemia in immunocompromised patients, aplastic crisis in patients with a high turnover rate of red blood cells, and hydrops fetalis in pregnant women. Although a secreted phospholipase A2 (PLA2) motif has been identified in the unique region of the B19V minor capsid protein VP1(VP1u), the determinants for its enzyme activity and its influences on host cells are not well understood. The purpose of this study was to investigate the contribution of the PLA2 motif and other regions of the VP1u to the PLA2 activity, to determine the cellular localization of the VP1u protein, and to examine the effects of VP1u on cellular cytokines. We found that in addition to the critical conserved and non-conserved amino acids within the VP1u PLA2 motif, amino acid residues outside the VP1u PLA2 motif are also important for the PLA2 activity. VP1u and various mutants all revealed a nucleo-cytoplasmic distribution. UT7-Epo cells treated with prokaryotic expressed VP1u or mutant proteins with PLA2 activity released a large amount of free fatty acid (FFA), and the cell morphological change occurred dramatically. However, neither free fatty acid nor cell morphology change occurred for cells treated with the mutants without PLA2 activity. The wild type and the VP1u mutants with the PLA2 activity also activated TNF-α promoter and upregulated the transcription activity of NF-κB in transfected cells. In addition, we found that the amino acids outside the PLA2 domain are critical for the viral PLA2 activity, and that these tested VP1u mutants did not affect the localization of the VP1u protein.
Subject(s)
Parvovirus B19, Human/enzymology , Phospholipases A2/metabolism , Amino Acid Motifs , Amino Acid Substitution , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Membrane/metabolism , Cells, Cultured , Consensus Sequence , Enzyme Activation/genetics , Erythema Infectiosum , Female , Humans , Models, Molecular , Mutation , NF-kappa B/metabolism , Parvovirus B19, Human/genetics , Phospholipases A2/chemistry , Phospholipases A2/genetics , Pregnancy , Protein Conformation , Protein Transport , Signal Transduction , Transcriptional Activation , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: Activity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage. METHODS: To investigate the roles of B19-VP1u in response to macrophage, phospholipase A2 activity, cell migration assay, phagocytosis activity, metalloproteinase assay, RT-PCR and immunoblotting were performed. RESULTS: In the present study, we report that migration, phagocytosis, IL-6, IL-1beta mRNA, and MMP9 activity are significantly increased in RAW264.7 cells by B19-VP1u protein with sPLA2 activity, but not by B19-VP1uD175A protein that is mutated and lacks sPLA2 activity. Additionally, significant increases of phosphorylated ERK1/2 and JNK proteins were detected in macrophages that were treated with B19-VP1u protein, but not when they were treated with B19-VP1uD175A protein. CONCLUSION: Taken together, our experimental results suggest that B19-VP1u with sPLA2 activity affects production of IL-6, IL-1beta mRNA, and MMP9 activity, possibly through the involvement of ERK1/2 and JNK signaling pathways. These findings could provide clues in understanding the role of B19-VP1u and its sPLA2 enzymatic activity in B19 infection and B19-related diseases.
Subject(s)
Macrophages/physiology , Parvovirus B19, Human/enzymology , Parvovirus B19, Human/immunology , Phospholipases A/metabolism , Animals , Cell Line , Cell Movement/physiology , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , JNK Mitogen-Activated Protein Kinases/genetics , JNK Mitogen-Activated Protein Kinases/metabolism , Macrophages/cytology , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Parvovirus B19, Human/genetics , Phagocytosis/physiology , Phospholipases A/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Three full-length genomic clones (pB19-M20, pB19-FL and pB19-HG1) of parvovirus B19 were produced in different laboratories. pB19-M20 was shown to produce infectious virus. To determine the differences in infectivity, all three plasmids were tested by transfection and infection assays. All three clones were similar in viral DNA replication, RNA transcription, and viral capsid protein production. However, only pB19-M20 and pB19-HG1 produced infectious virus. Comparison of viral sequences showed no significant differences in ITR or NS regions. In the capsid region, there was a nucleotide sequence difference conferring an amino acid substitution (E176K) in the phospholipase A2-like motif of the VP1-unique (VP1u) region. The recombinant VP1u with the E176K mutation had no catalytic activity as compared with the wild-type. When this mutation was introduced into pB19-M20, infectivity was significantly attenuated, confirming the critical role of this motif. Investigation of the original serum from which pB19-FL was cloned confirmed that the phospholipase mutation was present in the native B19 virus.
Subject(s)
Capsid Proteins/metabolism , Cloning, Molecular , Genome, Viral , Parvovirus B19, Human/pathogenicity , Phospholipases A2/metabolism , Amino Acid Sequence , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cell Line , Child , Humans , Molecular Sequence Data , Mutation , Parvovirus B19, Human/enzymology , Parvovirus B19, Human/genetics , Phospholipases A2/chemistry , Phospholipases A2/genetics , Sequence Analysis, DNAABSTRACT
Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A2 (PLA2) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA2 enzymatic activity and how these critical residues contribute to its PLA2 activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca2+-dependent secreted PLA2-like (sPLA2) activity, which was inhibited by sPLA2-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA2 activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA2-like enzymatic activity, and these residues are crucial for its sPLA2-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.
Subject(s)
Bocavirus/enzymology , Phospholipases A2/metabolism , Viral Proteins/metabolism , Acetophenones/pharmacology , Amino Acid Motifs , Amino Acid Sequence , Calcium/metabolism , Cloning, Molecular , Humans , Molecular Sequence Data , Mutation , Parvoviridae Infections/metabolism , Parvovirus B19, Human/enzymology , Phospholipase A2 Inhibitors , Phospholipases A2/genetics , Terpenes/pharmacology , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca(2+) dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region.
