Functional interaction of nuclear domain 10 and its components with cytomegalovirus after infections: cross-species host cells versus native cells

Abstract

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection.

Figure 1. Immunofluorescent assay to show IE1 and ND10 in CMV cross-species infection.

A–C: After HCMV infection in NIH3T3 cells for 5 hour, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (A); anti-IE1 antibody (mouse) was used to show IE1 (in green) (B); the merged picture is shown in C. D–F: After MCMV infection in Mrc-5 cells for 5 hour, cells were stained with anti-PML antibody (Rabbit) to show ND10 (in red) (D); anti-IE1 antibody (mouse) was used to show IE1 (in green) (E); the merged picture is shown in F.

Figure 2. Immunofluorescent assay to show cytomegalovirus infection and ND10.

A–C: After MCMV infection in NIH3T3 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (A); anti-IE1 antibody (mouse) was used to show IE1 (in green) (B); the merged picture is shown in C. D–F: After MCMV infection in Mrc-5 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (D); anti-IE1 antibody (mouse) was used to show IE1 (in green) (E); the merged picture is shown in F. G–H: After HCMV infection in NIH3T3 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (G); anti-IE1 antibody (mouse) was used to show IE1 (in green) (H); the merged picture was shown in I. J–L: After HCMV infection in Mrc-5 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (J); anti-IE1 antibody (mouse) was used to show IE1 (in green) (K); the merged picture is shown in L.

Figure 3. Immunofluorescent assay to show IE1 and IE3 in murine cytomegalovirus-infected human and mouse cells.

Upper: MCMVIE1/3gfp-infected NIH3T3 cells to show distribution of IE1 (left) and IE1_IE3 (right). Lower: MCMVIE1/3gfp-infected Mrc-5 cells to show distribution of IE1 (left) and IE1_IE3 (right).

Figure 4. Detection of HCMV proteins and DNA after infection in NIH3T3 and Mrc-5 cells.

A. Western blot assay to detect HCMV protein production: Whole-cell lysates were prepared at 48 hr after the infection of different strains of HCMV in MRC-5 cells (left) and NIH3T3 cells (right); after being run on PAGE gels, the proteins were transferred and detected with specific antibodies. Detected HMCV proteins are indicated on the right. B. PCR to detect HCMV DNA replication: Using a modified Hirt method (7), viral DNA was prepared from MRC-5 cells (left) or NIH3T3 cells (right) at the indicated post-infection times. PCR was performed using the primers (described in the Material and Methods) and PCR products were run on an agarose gel in order to visualize the DNA (using a UV light).

Figure 5. Immunofluorescent assay to detect HCMV proteins after infection in mouse cells.

A–C: Detection of HCMV IE2 in red (A), DAPI to show total cells (B), and the two merged in C. D–F: Detection of UL112/113 in red (D), DAPI to show total cells (E), and the two merged in F. G–I: Detection of UL58 in red (G), DAPI to show total cells (H), and the two merged in I.

Figure 6. Western blot assay to detect the effects of ND10 proteins (Daxx, PML, and SP100) on MCMV protein production.

A. siRNA to knockdown the ND10 protein. One hundred pmol of siRNA (the sequences are shown in Materials and Methods) was transfected into HEK293 cells for 24 hours, the whole-cell lysates were run on PAGE gels and probed with antibodies to the targeted proteins, as indicated. Tubulin was used as a house-keeping gene for sample-loading control. siRNA-Luc was used as control. B. One hundred pmol of siRNA was used to transfect HEK293 cells in a 24-well plate; 12 hours after transfection, the cells were infected with MCMVIE1/3gfp at an MOI of 1; the cells were harvested at the time indicted, and the whole-cell lysates were used to detect viral-protein production.

Figure 7. Western blot assay to detect the effects of ND10 proteins (Daxx, PML, and SP100) on HCMV protein production in mouse cells.

A. siRNA to knockdown the ND10 protein. One hundred pmol of siRNA (the sequences are shown in Materials and Methods) was transfected into NIH3T3 cells for 24 hours, the whole-cell lysates were run on PAGE gels and probed with antibodies to the targeted proteins, as indicated. Tubulin was used as a house-keeping gene for sample-loading control. siRNA-Luc was used as control. B. One hundred pmol of siRNA was used to transfect NIH3T3 cells in a 24-well plate; 12 hours after transfection, the cells were infected with repaired AD169 at an MOI of 1, the cells were harvested at the time indicted, and the whole-cell lysates were used to detect viral-protein production.

Figure 8. Detection of HCMV DNA after infection in NIH3T3 and Mrc-5 cells.

siRNA was used to transfect NIH3T3 cells; 12 hours after transfection, the cells were infected with repaired AD169 at an MOI of 1; using a modified Hirt method (7), viral DNA was prepared from MRC-5 cells (left) or NIH3T3 cells (right) at the indicated post-infection times. PCR was performed using the primers (described in the Material and Methods) and PCR products were run on an agarose gel to visualize the DNA (using a UV light).