Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication

Abstract

Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms, which form during virus infection. These processes are orchestrated by yet-to-be-understood complex networks of interactions involving nonstructural proteins (NSPs) 2, 5, and 6 and structural proteins (VPs) 1, 2, 3, and 6. The multifunctional enzyme NSP2, an octamer with RNA binding activity, is critical for viroplasm formation with its binding partner, NSP5, and for genome replication/packaging through its interactions with replicating RNA, the viral polymerase VP1, and the inner core protein VP2. Using isothermal calorimetry, biolayer interferometry, and peptide array screening, we examined the interactions between NSP2, VP1, VP2, NSP5, and NSP6. These studies provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from reciprocal peptide arrays were found to be in close proximity to the RNA template entry and double-stranded RNA (dsRNA) exit tunnels of VP1 and near the catalytic cleft and RNA-binding grooves of NSP2; these sites are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1. Peptide screening of VP2 identified NSP2-binding sites in the regions close to the intersubunit junctions, suggesting that NSP2 binding could be a regulatory mechanism for preventing the premature self-assembly of VP2. The binding sites on NSP2 for NSP6 were found to overlap that of VP1, and the NSP5-binding sites overlap those of VP2 and VP1, suggesting that interaction of these proteins with NSP2 is likely spatially and/or temporally regulated. IMPORTANCE Replication and packaging of the rotavirus genome occur in cytoplasmic compartments called viroplasms that form during virus infection and are orchestrated by complex networks of interactions involving nonstructural proteins (NSPs) and structural proteins (VPs).A multifunctional RNA-binding NSP2 octamer with nucleotidyl phosphatase activity is central to viroplasm formation and RNA replication. Here we provide the first evidence that NSP2 can directly bind to VP1, VP2, and NSP6, in addition to the previously known binding to NSP5. The interacting sites identified from peptide arrays are consistent with the proposed role of NSP2 in facilitating dsRNA synthesis by VP1 and also point to NSP2's possible role in preventing the premature self-assembly of VP2 cores. Our findings lead us to propose that the NSP2 octamer with multiple enzymatic activities is a principal regulator of viroplasm formation, recruitment of viral proteins into the viroplasms, and possibly genome replication.

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

“…While it is not directly involved in catalysis, the N-terminal domain is essential for VP1 activity because it helps create a channel for the entry of single-stranded RNA templates into the catalytic interior and is involved in sequence-specific recognition of viral RNAs (14,29). The large surface-exposed area of the N-terminal domain may also provide a platform for the binding of regulatory cofactor proteins (e.g., VP2, NSP2, and NSP5), in turn orchestrating VP1 localization and function (15,17). A leucine at position 138 in the N-terminal domain may be critical for maintaining such intra-and intermolecular protein interactions.…”

“…This region of VP1 helps form a template RNA entry tunnel, and it may also aid in polymerase regulation by serving as a binding platform for other viral proteins. VP1 is known to engage at least three other viral proteins (VP2, NSP2, and NSP5) during the RV life cycle, though the interaction interfaces are poorly defined (16)(17)(18)(19). Binding of VP1 by NSP2 and/or NSP5 localizes the polymerase to viroplasms, sites of genome replication and early particle assembly in the cell cytosol (20,21).…”

<b>Ocorrência e caracterização molecular de coronavírus e rotavírus do grupo A em quirópteros do Estado de São Paulo</b>

“…Especula-se que seja a principal reguladora na formação dos viroplamas, recrutando as proteínas virais para o viroplasma (FABBRETTI et al, 1999;VISKOVSKA et al, 2014 (DEO et al, 2002).…”

“…Especula-se que seja a principal reguladora na formação dos viroplamas, recrutando as proteínas virais para o viroplasma (FABBRETTI et al, 1999;VISKOVSKA et al, 2014 O segmento 10 possui 751 nucleotídeos e codifica para a proteína NSP4 que apresenta 20 KDa (DESSELBERGER, 2014). Essa proteína possui múltiplas funções na patogenia e replicação dos rotavírus (BALL et al, 1996;ZHANG et al, 1998).…”

“…O papel dessas duas proteínas ainda não foi muito bem estabelecido. A NSP5 atua em conjunto com a NSP2 na formação dos viroplasmas (FABBRETTI et al, 1999), embora sua função não esteja totalmente esclarecida, especula-se que esteja envolvida na replicação do genoma e na montagem viral (VISKOVSKA et al, 2014). Pouco se sabe sobre a NSP6: também é encontrada no viroplasma, porém é degradada em poucas horas após sua síntese (HU et al, 2012).…”