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.

FIG. 1.

Coimmunoprecipitation of VP1 and NSP5. (A) Immunoprecipitation (ip) with anti-NSP5 serum of DSP-cross-linked extracts of [³⁵S]methionine-labeled cells transiently transfected with the indicated genes. (B) Western blot analysis of DSP-cross-linked cellular extracts (lanes 1 to 4) or immunoprecipitates (lanes 5 to 8) derived from cells cotransfected with VP1 and NSP5. After separation by PAGE and Western blotting, the upper and lower parts of the blot were reacted with specific anti-VP1 or anti-NSP5 antibodies, respectively.

FIG. 2.

Analysis of coimmunoprecipitation of VP2 with NSP5. Western blot analysis of cellular extracts (lanes 1 to 8, DSP cross-linked in lanes 1 to 5 and non-cross-linked in lanes 6 to 8) or immunoprecipitates (lanes 9 to 16, DSP cross-linked in lanes 9 to 13 and non-cross-linked in lanes 14 to 16) derived from cells infected with rotavirus (lanes 1 and 2, 9 and 10) or cotransfected with VP2 and NSP5 (lanes 3 to 8, 11 to 16). After separation by PAGE and Western blotting, the upper and lower parts of the blots were reacted with specific anti-VP2 or anti-NSP5 antibodies, respectively.

FIG. 3.

Coimmunoprecipitation of NSP5 and tagged VP1. (A) Western blot of immunoprecipitates (ip) of DSP-cross-linked extracts from MA104 cells cotransfected with NSP5 and a VP1 derivative, N-terminally modified with the SV5 tag. Immunoprecipitates were obtained with an anti-NSP5 serum (anti-NSP5) and the anti-SV5 monoclonal antibody (anti-tag) and upper and lower parts of the blots developed with either of them, as described in legend to Fig. 1. (B) Western blot of immunoprecipitates obtained with anti-NSP5 or anti-tag antibody from both extracts of non-cross-linked or DSP-cross-linked cells and developed with the indicated antibodies. (C) Western blot of immunoprecipitates obtained with anti-NSP5 from extracts of non-DSP-cross-linked cells, treated with RNase or not treated.

FIG. 4.

Packaging of tag-VP1. Rotavirus particles, obtained from infected cells expressing tag-VP1 and purified by CsCl gradient ultracentrifugation, were analyzed by Western blotting with anti-tag, anti-VP1, or anti-VP7 antibody, as indicated. vTF7.3 was used to drive expression of tag-VP1. (A) Rotavirus particles (T, TLPs; D, DLPs; E, EPs) obtained from cells transfected with tag-VP1 (lanes 1 to 3) or nontransfected (lanes 4 to 6) were analyzed by Western blotting as indicated. (B) Western blot of total cellular extracts. Lane 9 corresponds to the conditions under which viral particles shown in lanes 1 to 3 were obtained. Of note, tag-VP1 (SA11 strain; lane 8) migrated faster than the untagged VP1 (RF strain; lane 7). (C) Western blot with anti-tag, anti-VP1, and anti-VP7 antibodies of TLPs, DLPs, and EPs obtained as for panel A (lanes 10 to 12, respectively). Tag-VP1 TLPs and control TLPs were treated with EDTA as described (T_+E; lanes 13 and 15, respectively) or mock treated (lanes 14 and 16, respectively). The traces of VP7 in the DLP preparations (lanes 2 and 11) are probably due to contamination with small amounts of TLPs.

FIG. 5.

Colocalization of tag-VP1 in viroplasms. Confocal immunofluorescence of tag-VP1 (green) and NSP5 (red) in MA104 cells transfected with the tag-VP1-expressing plasmid and infected with SA-11 rotavirus. The individual and merged patterns are shown.

FIG. 6.

Localization of tag-VP1 in VLS. (A) Immunofluorescence of tag-VP1 (green), NSP2 (red), and NSP5 (red) in cells cotransfected with and expressing the indicated genes. (B) Colocalization of tag-VP1 and NSP5 in VLS formed in cells coexpressing NSP5, NSP2, and tag-VP1, as shown by confocal microscopy with anti-NSP5 (red) and anti-tag (green).

FIG. 7.

Coimmunoprecipitation of NSP2, NSP5, and tag-VP1. (A) Western blot of cellular extracts (lanes 1 to 3) or anti-NSP5 immunoprecipitates (lanes 4 to 6) derived from cells transfected with NSP2, NSP5, and tag-VP1 and DSP cross-linked. (B) Western blot of cellular extracts (lanes 1 to 6) or anti-tag immunoprecipitates (lanes 7 to 12) derived from cells transfected with NSP2, NSP5, and tag-VP1, DSP cross-linked (lanes 4 to 6, 10 to 12) or non-cross-linked (lanes 1 to 3, 7 to 9). Upper, middle, and lower parts of the blots were cut and reacted with anti-tag, anti-NSP2, or anti-NSP5 antibodies, respectively.

FIG. 8.

Effects of DSP cross-linking and tag-VP1 on NSP5 hyperphosphorylation. (A) Western blot (WB) analysis of the soluble (sol.) (left panel, lanes 1 to 3) and insoluble (insol.) (right panel, lanes 4 to 6) fractions derived from extracts of DSP-cross-linked cells transfected with and expressing the genes, as indicated. (B) Western blot of the soluble fractions of non-cross-linked cells. Upper, middle, and lower parts of the blots were cut and reacted with anti-tag, anti-NSP2, or anti-NSP5 antibodies, respectively.

FIG. 9.

Interaction of VP1 with NSP5 mutants. (A) Diagram of NSP5 mutant constructs used. (B to D) Western blots of DSP-cross-linked cellular extracts or immunoprecipitates of cells cotransfected with wild-type (wt) VP1 (B) or tag-VP1 (C and D) and NSP5 or NSP5 mutants, as indicated. (E) Western blot of non-DSP-cross-linked cellular extracts or immunoprecipitates derived from cells cotransfected with tag-VP1 and either wt NSP5 or the mutant ΔC48.