Interaction of Rotavirus Polymerase VP1 with Nonstructural Protein NSP5 Is Stronger than That with NSP2

Abstract

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.Rotavirus is a major etiologic agent of severe gastroenteritis in infants and young children worldwide (20,21,33). The virion (defined as a triple-layered particle [TLP]) contains a genome consisting of 11 segments of double-stranded RNA (dsRNA) and is made up of three concentric layers of proteins: the outer layer consists of the two proteins VP7 and VP4, the intermediate layer of VP6, and the internal (core) layer of VP2, with VP1 and VP3 attached at its inside as minor components (29, 41). After entry into the host cell, the virion loses the outer layer to become a double-layered particle (DLP), which is active in transcription of viral mRNAs from the dsRNA genome. The viral RNA-dependent RNA polymerase (RdRp) acts as both the transcriptase and the replicase. Several lines of evidence indicate that VP1 is the viral RdRp: (i) VP1 contains sequence motifs that are shared by RdRps of other RNA viruses (31); (ii) VP1 has NTP-binding activity and, when cross-linked with the nucleotide analog 8-azido-ATP, inhibits RNA transcription (50); (iii) VP1 specifically recognizes the 3Ј end of viral mRNAs (35); and (iv) recombinant VP1 can direct template-dependent minus-strand synthesis in vitro in the presence of VP2 (37, 54).Despite partial characterization of rotavirus replication intermediates (3,19,36,37,54), molecular details of viral genome replication and of the different steps of viral morphogenesis still remain to be elucidated. It has been shown that VP1 and VP2, the scaffolding protein of viral cores, are...

Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5′ GG Sequence for RTPase Activity

“…Along with NSP5, it is essential for the formation of viroplasms, sites of rotavirus genome replication and DLP assembly. As one of the components of early replication intermediates, NSP2 is suggested to directly interact with VP1 and partially replicated viral RNA (2,3,12,13). Biochemical studies with recombinant NSP2 show that it possesses several properties that are consistent with its intricate role in genome replication/ packaging.…”

“…Each VP1-VP3 heterodimeric complex inside the DLPs may associate with one segment to allow simultaneous transcription of dsRNA segments (15,26,36). Based on previous biochemical studies, which strongly suggest the possibility of direct interaction between NSP2 and VP1 (3,12), one model that can be envisioned is that the specific recognition of 5= CS of (Ϫ)RNA and sequenceindependent binding of the 3= end of the (ϩ)RNA of the duplex as it emerges from VP1 allows for proper structural organization of the genome segment around VP1. This might then allow for subsequent packaging into assembling core particles such that each segment is associated with one VP1 molecule inside the assembled particles, which then would be conducive for simultaneous transcription of the genome segments during endogenous transcription.…”

Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

“…11) envisages that NSP2 and NSP5 recruit LD proteins and lipids and establish an amphipathic assembly complex, which could then interact with VP1 (2) before or after VP1 binds rotavirus single-stranded RNA. The building blocks for rotavirus precores (2,25,45) may be assembled on the surface of the phospholipid LD monolayer (Fig. 11).…”

“…The bovine rotavirus RF strain (G6P6 [1]) and the porcine rotavirus OSU strain (G5P9 [7]) were used and replicated in MA-104 cells. Rotavirus grown in large amounts was concentrated by ultracentrifugation, treated with Freon, and TLPs and DLPs were purified by repeated CsCl gradient ultracentrifugation as described elsewhere (2). DLPs in CsCl solution were deionized by passage over a G25 Sephadex column.…”

“…Western blotting followed established procedures (2). Full-range protein rainbow markers (Amersham) were used for calibration.…”