Several findings in the paper by Sen et al. entitled “Hyperphosphorylation of the rotavirus NSP5 protein is independent of serine 67 or NSP2, and the intrinsic insolubility of NSP5 is regulated by cellular phosphatases” (5) seem to contradict previous published findings from our laboratory. The authors also state that “… hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% SDS.” We were puzzled by the fact that not a single experiment in this paper was performed with the natural protein. In all cases NSP5 was fused with a His6-G, GAL4, or Myc tag, but the conclusions were extended to the behavior of wild-type NSP5 (wtNSP5). Based on previous results showing that the addition of a tag at the amino terminus of NSP5 drastically changes the characteristics of the protein (e.g., viroplasm-like structure formation without NSP2 [4; unpublished results]), we addressed this point by expressing various NSP5 constructs either untagged or N-terminally tagged with the SV5 peptide (3). As shown in Fig. Fig.1A1A and in agreement with previous results of this group (2, 3), the soluble fraction of wild-type NSP5 and its mutants NSP5a (Ser63, 65, 67Ala) and NSP5-Ser67Ala were little or not phosphorylated, whereas the mutation of Ser67 into aspartic acid (NSP5-S67D) was associated with hyperphosphorylation of NSP5. In contrast, tagging of NSP5 at the N terminus increased hyperphosphorylation of SV5-NSP5 and SV5-NSP5-S67D, whereas the effect on the mutants SV5-NSP5a and SV5-NSP5-S67A was reduced. Analysis of the insoluble fractions showed that the relative amounts of the nontagged NSP5 constructs were less than 10% of the total and did not show a phosphorylation pattern different from that of the soluble fraction (Fig. (Fig.1A).1A). By contrast, N-tagged constructs showed a pattern of increased phosphorylation in the insoluble fraction, yet this represented in all cases not more than 20% of the total. These results demonstrate that there is a substantial difference in the behavior of tagged versus nontagged NSP5 proteins. FIG. 1. Western blot analysis (anti-NSP5) of soluble and insoluble fractions of untagged or N-terminally tagged wild-type and mutant NSP5, expressed in MA104 cells. Cells were lysed as described previously (2). Aliquots corresponding to 10% of supernatants from ... Sen et al. further claim that NSP5 phosphorylation is independent of NSP2. We addressed this point as well by analyzing NSP5 phosphorylation when coexpressed with NSP2, both in the soluble and insoluble fractions. We found, in agreement with our previous results (1), that NSP2 was able to induce hyperphosphorylation of wtNSP5, with identical forms being present in the insoluble fraction but with the amount representing less than 10% (Fig. (Fig.1B).1B). Finally, and very importantly, at least 90% of the total amount of wtNSP5 derived from virus-infected cells was present in the soluble fraction (Fig. (Fig.1C).1C). Therefore, we can rule out the possibility that lack of analysis of the insoluble fractions could have led to misinterpretation of our previously published data. Our results clearly show that without the control of the natural protein, the use of tagged-NSP5 constructs can produce spurious and misleading results. Daring statements on biochemical properties of NSP5 seem to be premature when the data on which they are based are derived only from proteins modified by the addition of tags.