Immunoprecipitation of nsP1a-derived proteins expressed in BHK cells with pAb 5-6. (A) nsP1a/1b-derived sequences were expressed in BHK cells, and processing products were analyzed as described in the legend to Fig. 1A, except that labeling was done for 90 min and pAb 5-6 was used for immunoprecipitation. (B) Interpretation of the bands seen in panel A. Representation is as described in the legend to Fig. 1B. The epitope for pAb 5-6 is indicated by a thick black line.

Immunoprecipitation of His-tagged nsP1a-derived proteins expressed in BHK cells with MAb αHis. (A) Confluent BHK cells in six-well plates were infected with a recombinant vaccinia virus (multiplicity of infection, 10) encoding T7 DNA-dependent RNA polymerase. After 45 min, the infected BHK cells were transfected with 2 to 3 μg of plasmid DNA encoding nsP1a/1b-derived sequences under the control of a T7 DNA-dependent RNA polymerase promoter, or a control plasmid, by using Lipofectamine in accordance with the manufacturer's directions (Gibco-BRL, Gaithersburg, Md.). At 4 h posttransfection, the medium was removed, and the cells were starved in Met-Cys-free medium for 30 min and then labeled with 30 to 50 μCi of [³⁵S]Met-Cys (Tran³⁵S-label; ICN Biomedicals, Irvine, Calif.) per well for 15 min. Cell lysates were prepared, and nsP1a-specific products were immunoprecipitated with MAb αHis (Qiagen) as described previously (5). The immunoprecipitated products were separated on an SDS-10% polyacrylamide gel (11), and the radiolabeled bands were visualized by autoradiography. Prestained molecular mass standards (Rainbow Marker; Amersham Pharmacia Biotech, Piscataway, N.J.) were run in parallel, and their positions on the gel were transferred to the autoradiogram. Mock, mock-infected cells. (B) Interpretation of the bands seen in panel A. Open boxes represent the generic nsP1a and nsP1b encoded by ORF1a and ORF1b, respectively. nsP1b is translated as a fusion protein with nsP1a only after the occurrence of a −1 ribosomal frameshift. Conserved motifs are indicated by horizontal stripes (TM, transmembrane helices), vertical stripes (NLS, nuclear localization signal), diagonal stripes (prot, protease), and cross-hatching (Δ, 9-aa substitution in protease; see text). nsP1a-derived translation products expressed in BHK cells are depicted as lightly shaded boxes. Processing products identified on the gel are shown as darkly shaded boxes. The N-terminally linked His tag is indicated in black. Suggested cleavage sites are indicated by arrowheads.

pAb 5-6-precipitable processing products derived from nsP1a expressed in BHK cells or Caco-2 cells. nsP1a was expressed in BHK cells as described in the legend to Fig. 1A, except that labeling was followed by an 85-min cold chase. Caco-2 cells in six-well plates were infected with astrovirus at a multiplicity of infection of 4 or were mock infected. At 4 h postinfection, the cells were starved for 30 min in medium without Cys or Met and then metabolically labeled with 50 μCi of [³⁵S]Met-Cys for 30 min. Medium containing ³⁵S label was replaced with cold medium, and cell lysates were prepared after either a 40- or a 90-min chase. nsP1a-specific products were immunoprecipitated with pAb 5-6 and analyzed as described in the legend to Fig. 1A.