Rotavirus NSP5: mapping phosphorylation sites and kinase activation and viroplasm localization domains

Abstract

Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione S-transferase-NSP5 mutants were produced in Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser(153), Ser(155), Ser(163), and Ser(165)) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.

FIG. 1.

Schematic representation of NSP5 and mutants. The dashed lines correspond to deleted regions. A, Ser→Ala mutations.

FIG. 2.

In vitro phosphorylation assay. Analysis of immunoprecipitates of in vitro-translated, [³⁵S]methionine-labeled mutant Δ1. (A) Substrate was incubated with cellular extracts from cells transfected with the indicated mutants; mock, extracts from cells transfected with the same plasmid without the insert. −, no addition. (B) λ-Ppase treatment as indicated (+, treated; −, untreated). (C) Purified His₆-Δ1/Δ3 was obtained by nickel column purification, and the same amount was used in lanes 5, 6, and 7. cell. extr., cellular extracts. The arrowheads indicate the unphosphorylated Δ1 substrate, and the vertical brackets indicate the positions of mobility-shifted phosphorylated forms. −, no addition.

FIG. 3.

Mapping phosphorylation sites of NSP5. SDS-PAGE analysis of immunoprecipitated, in vitro-phosphorylated GST-NSP5 mutant proteins (0.2 μg) incubated with (+) and without (−) cellular extract (cel. extr.) from Δ1/Δ3-transfected cells. GST protein negative control (−) (lane 17) was loaded without immunoprecipitation.

FIG. 4.

Characterization of Ser→Ala mutants. (A) Scheme of Δ1/Δ3 mutant showing positions of the four serines mutated to alanine within region 4. The residue numbers correspond to the wild-type protein. (B) Immunoprecipitates of in vivo ³²P_i-labeled MA104 cells transfected with nonmutated (−) or with the indicated mutant constructs. The numbers refer to mutated Ser residues. (C) Western immunoblot of λ-Ppase-treated (+) transfected Δ1/Δ3 mutant. (D) Phosphorylation with [γ-³²P]ATP of GST-Δ1/Δ3 and GST-Δ1/Δ3(S→A) as for Fig. 3.

FIG. 5.

NSP5 is a substrate of CKII. Shown is SDS-PAGE analysis of an in vitro CKII phosphorylation assay with [γ-³²P]ATP. (A) The indicated GST-NSP5 mutants were used as substrates. Positive and negative controls are shown in lanes 1, 7, 8, and 9. −, no substrate added. (B) Purified, in vitro-translated ³⁵S-His₆-Δ1 was used as a substrate in the presence or absence of CKII. The two panels show autoradiography of the same gel, detecting ³⁵S plus ³²P (left) and ³²P (right). The solid and open arrowheads indicate His₆-Δ1 precursor and hyperphosphorylated forms, respectively. Autophosphorylated GST-α and GST-β CKII subunits are indicated. +, present; −, absent.

FIG. 6.

Confocal immunofluorescence microscopy. MA104 cells were transfected with NSP5 mutants fused to EGFP followed by infection with rotavirus. Viroplasms were detected with an anti-NSP2 (red) antibody. The rightmost column is the superimposition of the two independently acquired images.