In this study we aimed at clarifying the interactions between the rotavirus RNA polymerase VP1 and the nonstructural proteins NSP5 and NSP2. These two proteins, which are well known to interact with each other within cytoplasmic viroplasms (1
), have been proposed to actively participate in the replication of the viral genomic RNA (4
). NSP2, because of its ATPase activity, has been suggested to work as a molecular motor to recruit single-stranded RNA templates for the synthesis of the genomic dsRNA by the VP1/VP2 replication complex (43
The capacity of NSP5 to interact with VP1 and NSP2 in virus-infected cells has already been observed in coimmunoprecipitation assays from DSP-cross-linked extracts (1
). Here we provide a more detailed description of the nature of the interaction of VP1 with NSP5, with NSP2, or with both in the absence of other viral proteins or “true” viral RNA (the viral proteins are translated from transcripts of plasmids containing only cDNA comprising the coding regions and lacking the 5′ and 3′ untranslated regions).
Using extracts of cotransfected MA104 cells, we show by coimmunoprecipitation assays that NSP5 has a strong interaction with VP1, which is not altered by the presence of NSP2. On the other hand, interaction of NSP2 with VP1 appears to be much weaker and is possibly stabilized by NSP5 (observed in DSP-cross-linked extracts). Although it was not formally proven, the formation of a ternary complex of VP1, NSP5, and NSP2 is a possibility, since tag-VP1 was found in the VLS formed as a consequence of the interaction of the two others (Fig. ). An interaction between NSP2 and VP1 in rotavirus-infected cells has been reported previously (25
). When VP1 was coexpressed with each of the nonstructural proteins individually, it became apparent that NSP5 interacted more strongly with VP1 than NSP2. However, when the three proteins were coexpressed, NSP5 seemed to enhance the interaction of VP1 with NSP2, a conclusion delineated by data using DSP-cross-linked extracts. Interestingly, several attempted pull-down experiments with anti-NSP2 serum did not coimmunoprecipitate NSP5 (as has already been published [(1
]) or VP1. Thus, it is likely that most of the NSP2 molecules are buried within the cross-linked complex and therefore are not accessible to the precipitating antibody.
Using NSP5 deletion mutants, we found that deletion of the N-terminal 33-amino-acid region from NSP5 did not affect binding to VP1, while deletion of the last 18 amino acids from the C terminus substantially reduced the interaction, and that of the last 48 amino acids completely abolished it (Fig. ). Also, results with single-domain EGFP fusion chimeras allowed us to identify the 48 amino acids at the C terminus as those involved in the interaction with VP1. Since the last 18 C-terminal amino acids of NSP5 have previously been found to be involved in dimerization (13
), our data suggest that dimeric NSP5 is better suited for interaction and that the binding region is located just upstream of the C-terminal tail. The region of VP1 interacting with NSP5 remains to be determined.
In some experiments, we used in vivo cross-linking with DSP to stabilize the complexes. This was needed in particular when using the anti-NSP5 serum, possibly because the high-affinity hyperimmune polyclonal antibody has a dissociating activity. By contrast, the anti-tag monoclonal antibody, which binds to the N terminus of tag-VP1, allowed efficient coimmunoprecipitation of NSP5 or NSP2 or both from extracts that have not been cross-linked (Fig. ).
So far, NSP5 has been proposed to have a crucial structural role, together with NSP2, in the assembly of viroplasms (8
). However, from the results presented here, new possible functions are emerging. The strong interaction between NSP5 and VP1 suggests that NSP5 may act at the crossroad of important processes of virus morphogenesis, such as replication, carried out by VP1 in conjunction with the scaffold protein VP2 (35
), and packaging, for which NSP2 is a putative molecular motor (46
It has been reported that NSP5 interacts with VP2 (5
). We observed that NSP5 and VP2, either when coexpressed after transfection or in infected cells, were unable to coimmunoprecipitate from extracts, which had been cross-linked with DSP or not cross-linked. In spite of this lack of interaction, NSP5 hyperphosphorylation was considerably increased in the presence of VP2. This is a novel finding requiring further analysis.
In cotransfected cells, the presence of VP1 impaired the recognized ability of NSP2 to up-regulate NSP5 phosphorylation (1
), but in spite of this, interaction between VP1 and NSP5 was not altered. Further data for virus-infected cells indicate that this interaction is independent of the phosphorylation status of NSP5 (M. Campagna et al., unpublished data). While this is an interesting observation, at present there are insufficient data for a comprehensive interpretation of whether and how NSP5 hyperphosphorylation is linked to replication and packaging.
Some of the results of this work were obtained by using a VP1 derivative in which the 11-amino-acid-long SV5 tag was fused to the N terminus of VP1. The presence of a terminal tag allowed VP1 to be recognized by using immunoprecipitation or immunofluorescence assays. It also allowed us to overcome the problem of having only a poorly reactive antibody against the wild-type protein. The polyclonal antiserum against VP1 did not react in either immunoprecipitation or immunofluorescence experiments, but only in Western blots. It is possible that the antibody, which was made against the N-terminal 435 amino acids, did not recognize the folded full-length VP1 protein, while it was able to react with the denatured protein in Western blots. By contrast, the availability of a potent anti-tag monoclonal antibody allowed the interaction of VP1 with NSP5 and NSP2 to be studied. In doing so, it had to be considered that fusing tags to a protein may modify its structural and functional properties and may lead to artifacts and misinterpretations (7
). Therefore, in order to ensure the validity of using a tagged version of VP1, we analyzed the behavior of such a protein in the context of viral infection by using immunofluorescence and analysis of purified viral particles. We found that tag-VP1 was localized in viroplasms and VLS (Fig. and ) and also packaged into viral progeny (Fig. ). The incorporation efficiency was not high but was unequivocal and reproducible. Thus, only a few among the 12 molecules of the VP1 polymerase per viral particle (41
) would be represented by the tagged VP1. The degree of tag-VP1 packaging varied, possibly depending on the relative ratios of infectious units of vaccinia virus and rotavirus, as well as on differences in the maturation of DLPs into TLPs. Nonspecific attachment of tag-VP1 to TLPs was ruled out by EDTA treatment of TLPs, which did not remove tag-VP1, and also by mixing TLPs with extracts containing an excess of tag-VP1, not leading to an association of these two components (results not shown).
Success in packaging tag-VP1 strengthens the results obtained in this work and represents a technical novelty. The system mainly used so far to study interactions among the structural proteins and their role in rotavirus morphogenesis (9
) is based on the coexpression of various combinations of structural proteins or derivatives thereof from baculovirus vectors in insect cells, forming so-called virus-like-particles. Although virus-like-particles have the structural characteristics and some of the functional properties of rotavirus particles (54
), their assembly occurs outside the context of natural infection. The data provided here demonstrate that the incorporation of the exogenous protein into viral particles can take place during natural infection, indicating new possibilities for studying viral morphogenesis. While the improvement of an already developed reverse genetics system (26
) and the development of new ones are under investigation, inserting recombinant proteins into progeny particles during natural viral infection could help to further define the roles of viral proteins.