The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

Abstract

Lytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection andin vitro. We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD) value of 18.13 nM.In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence,in vivoduring infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of 11-kDa abolished its capability to reduce ERK activity and, accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding a short hairpin RNA (shRNA) targetingGrb2decreased the expression of Grb2 as well as the level of ERK phosphorylation, which resulted in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa protein interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.IMPORTANCEHuman parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of nonimmunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins, VP1 and VP2, and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here, we elucidated a mechanism underlying 11-kDa protein-regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection.In vitro, 11-kDa interacts with Grb2 with high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication by interacting with a host protein that is predominately expressed in the cytoplasm.

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers

“…In addition, Southern blot analysis of Hirt DNA extracted from transfected cells showed that silent mutations of ISE3 in infectious clone M20 significantly decreased B19V DNA replication (by ϳ2.5-fold for the monomer replicative form [mRF] DNA) compared with the results seen with the counterpart WT M20 clone ( Fig. 8H and I), which was again in line with the previous observation that 11-kDa plays a role in viral DNA replication (38).…”

“…NS1 is the only viral protein that is essential for B19V DNA replication (35)(36)(37). However, the small nonstructural protein 11-kDa, which is localized predominantly in the cytoplasm and plays an enhancement role in viral DNA replication, is unique among parvoviruses (38). Mechanistically, the 11-kDa protein interacts with Grb2 (growth factor receptor bound protein 2) through the three SH3-binding motifs of the 11-kDa protein.…”

“…Mechanistically, the 11-kDa protein interacts with Grb2 (growth factor receptor bound protein 2) through the three SH3-binding motifs of the 11-kDa protein. This interaction disrupts the extracellular signal-regulated kinase-1 signaling and upregulates viral DNA replication (38).…”

Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression

“…pET30a-NP1 was constructed by inserting an NP1 ORF into the pET30a(ϩ) vector (EMD Millipore) through the NdeI and XhoI sites. pMBP-His was constructed by fusing 6ϫ histidines (His) to the maltose binding protein (MBP) ORF at the C terminus in pMAL-c5X (New England Biolabs) through the SacI site, as described previously (43).…”

Genetic Basis of Myocarditis: Myth or Reality?

“…Variants in RBM38 gene have not been reported so far. The RBM38 protein (coded by the RBM38 gene, 20q13.31, MIM * 612428) interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression that regulates the 11-kDa protein-mediated augmentation of B19V replication [62]. The B19V-antibody complex enters the cells through an endocytosis process mediated by the direct interaction of antibody-bound complement factor C1q with its receptor CD93 on the cell surface [63].…”