PMC

Gene 1 structure and anti-Hel antibodies. The organization of the MHV genome is shown, with the location of gene 1, the organization of ORF 1a and ORF 1b, and the polymerase and Hel domains shown to scale. The region of the B1 antiserum directed against the B1 fusion protein (white box) and the 96.8 antiserum (black bar) are shown. The entire sequence of the 96.8 peptide is shown, and the boundaries of the B1 fusion protein are indicated, as described in Materials and Methods.

Colocalization of Hel and N in MHV-infected cells. MHV-infected DBT cells were fixed in 100% methanol at 5.5 h p.i. and prepared for confocal immunofluorescence with the anti-N MAb and the anti-Hel rabbit polyclonal serum B1. Hel was imaged at 647 nm (far red), and N was imaged at 488 nm (green). For colocalization (Hel and N), the images were merged with an “and” function requiring the presence of white pixels in both N and Hel images for the white pixels to be seen in the merged image (NIH Image 1.62). Thus, the white pixels in this image were colocalized in the N and Hel images. For the mock Hel-N image, the Hel and N signals were overlapped without filtering, to show the maximum signal from both channels in the merged image.

Immunoblotting of MHV virion proteins by structural and gene 1 protein antibodies. MHV virions were purified by sucrose gradient centrifugation. Virions were lysed in SDS buffer, separated on an SDS–15% acrylamide gel, transferred to polyvinylidene difluoride, and incubated with antibodies directed against the proteins as indicated above the lanes. Equivalent amounts of virion proteins were present in each lane. The anti-M and anti-N mouse MAbs J.1.3 and J.3.3, obtained from John Fleming, were used as primary antibodies at a 1:1,000 dilution. The rabbit polyclonal antibodies directed against 3CLpro (SP9) and Hel (B1) were used as primary antibodies at a 1:100 dilution.

Confocal immunofluorescence detection of Hel in MHV-infected DBT cells. Monolayers of DBT cells were infected with MHV-A59 for 6 h and then fixed and processed for immunofluorescence as described in Materials and Methods. The B1 antibody was used for detection of Hel. Imaging was performed on a Zeiss LSM 410 laser confocal microscope, with a 488-nm laser to excite the Cy-2 dye. The images were obtained with a 63× objective. Phase-contrast images were obtained with a Nomarski polarizer. Separate fluorescent and transmitted images were obtained and merged with Photoshop 4.0. (A) Fluorescent images of mock-infected cells (mock) and two different fields of the infected-cell monolayer (infected), showing individual cells and virus-induced syncytia. (B) Superimposition of transmitted light and fluorescent images. The fluorescent images from panel A were merged with transmitted light images.

Detection of Hel and viral RNA in membrane fractions of MHV-infected cells. MHV-infected DBT cell monolayers were homogenized in the absence of detergent, and differential centrifugation was performed to separate cellular membranes and cytosol, including a final spin at 100,000 rpm with a TLA 20.2 rotor in a Beckman TLX Optima centrifuge. The combined membrane pellets (memb.) and the post-100,000-rpm cytosol were assessed for Hel by immunoprecipitation with the B1 antibody followed by fluorography and densitometric analysis (NIH Image 1.62) with a calculation of area in pixels × the mean density of the pixels. Quantitation of new viral RNA was performed by determination of TCA-precipitable [³H]uridine in the presence of actinomycin D. Mock-infected cells were used as controls.

Detection of RNA and Hel in MHV-infected DBT cells. MHV-infected or mock-infected DBT cells were metabolically labeled with BrUTP and processed for confocal immunofluorescence as described in Materials and Methods. (A) Transfection of infected cells in the absence of actinomycin D, to allow for incorporation into both viral and cellular RNA. Cells were imaged for BrUTP incorporation (Cy-2, green, RNA column) and helicase (Cy-5, red, Hel column). The images were merged (“RNA/helicase”), with yellow pixels representing areas of signal colocalization. The white brackets in the “RNA/helicase” panel indicate the area of enlargement in the second row of panels. (B) Mock-infected cells were labeled with BrUTP in the absence of actinomycin D and imaged for RNA and Hel as described for panel A. Only the merged image is shown. (C) Transfection of infected cells in the presence of actinomycin D. Imaging and processing were performed as described for panel A. The small white brackets in the merged image indicate the area of enlargement in the second row. (D) Mock-infected cells were labeled with BrUTP in the presence of actinomycin D and imaged for RNA and Hel as described for panel A. Only the merged image is shown.

Identification of a 67-kDa protein by anti-Hel antibodies in MHV-infected DBT cells. (A) Cells were mock infected (mock) or infected (inf.) as described in Materials and Methods. Cells were incubated with actinomycin D for 1 h beginning at 5 h p.i., followed by addition of [³⁵S]methionine for 2 h. Following cell lysis, proteins were immunoprecipitated with immune serum (B1 or 96.8) or preimmune serum (inf. pre). Molecular mass markers (in kilodaltons) are to the left of the gel, and the locations of the 67-kDa protein and nucleocapsid (N) are shown. (B) Radiolabeling of infected cells was performed in the absence (−) or presence (+) of the proteinase inhibitor E64d (400 μg/ml). Mass markers (in kilodaltons) are to the right of the gel.

Three-dimensional sectioning of MHV-infected cells and characterization of Hel-containing inclusions. Cells were imaged for Hel with the 488-nm laser to detect Cy-2. (A) Sagittal (xy) sections of the cells were obtained by z sectioning at 0.2-μm intervals, with resolution in the x, y, and z planes of 0.12 μm/pixel. The total z distance of 12 μm was imaged with 58 sections. The sections were transformed into a stack with NIH Image 1.62b. Section 1 was at the level of the coverslip, and section 58 was at the “top” of the cell most distant from the coverslip. The numbers in each panel are the numbers of the sections: 11 indicates 11 of 58. (B) Coronal (xz) reconstruction of a cell. The cell on the right of the panels in panel A was isolated, and the z stack was used to reconstruct the cell in the xz (coronal) plane with NIH Image 1.62b. Section 29 was used as the reference because it is in the middle of the cell in z dimension and shows the complexes clearly. The levels of the xz slices are shown by white lines 1 through 6, and the panels to the right show the respective slices. The viewpoint is indicated by the arrow, and the orientation of the left and right sides of the respective images is shown. The white line through the panels shows the level of section 29. (C) Axial (yz) reconstruction of the cell. This was performed as described for panel B, except in the yz plane.

Pulse-label and pulse-chase translation of p67 Hel in MHV-infected DBT cells. (A) TCA-precipitable counts per minute of [³⁵S]Met during pulse-label and pulse-chase translation. MHV-infected DBT cells were labeled in suspension beginning at 5.5 h p.i., and samples were taken at the time indicated in duplicate for TCA precipitation and scintillation counting. At 60 min, the suspended cells were split and the chase cells were rinsed twice, followed by resuspension in chase medium and sampling for TCA-precipitable counts at the times indicated. Data points are the means of two samples with standard deviations shown. (B) Pulse-label translation. At 5.5 h p.i., replicate plates of MHV-infected DBT cells were incubated in medium containing 200 mM excess NaCl for 30 min, then the medium was changed to medium containing normal concentrations of NaCl with added [³⁵S]Methionine, and plates were harvested for lysis and immunoprecipitation at the times in minutes shown above the lanes. Proteins were immunoprecipitated with the 96.8 antiserum, followed by electrophoresis on an SDS–5 to 18% acrylamide gradient gel and fluorography. Molecular mass markers (m) (in kilodaltons) are to the left of the gel, and the locations of p67 Hel and N are indicated to the right. (C) Pulse-chase translation. Replicate plates were infected and radiolabeled concurrently with the pulse-label plates. Cells were all pulsed with [³⁵S]methionine for 30 min (see panel A, 30 min) followed by chase in medium containing 10-fold excess unlabeled l-methionine for the times in minutes indicated, prior to lysis, immunoprecipitation, and electrophoresis. Numbers at right are molecular masses in kilodaltons.