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

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, the 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of the 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 and in 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 vivo during 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 the 11-kDa abolished its capability to reduce ERK activity, and accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding an shRNA targeting Grb2 decreased the expression of Grb2 as well as the level of ERK phosphorylation, which results in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.SIGNIFICANCE Human parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of non-immunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins VP1/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 the 11-kDa 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 at a 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 the 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 through interacting with a host protein that is predominately expressed in the cytoplasm.