Human parvovirus B19 nonstructural protein (NS1) induces cell cycle arrest at G(1) phase

Abstract

Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G(2) arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G(2) arrest but also G(1) arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G(2) arrest but not G(1) arrest. Furthermore, treatment with caffeine, a G(2) checkpoint inhibitor, abrogated the B19 virus-induced G(2) arrest despite expression of NS1. These results suggest that the B19 virus-induced G(2) arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G(1) phase. These results indicate that NS1 expression plays a critical role in G(1) arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G(1) arrest. Thus, G(1) arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.

FIG. 1.

B19 virus infection induces both G₁ and G₂ arrests. (A) UT7/Epo-S1 cells were infected with 20-fold-diluted B19 virus or mock infected, and at 24 h postinfection they were left treated or treated with a mitotic inhibitor, paclitaxel, for 24 h. Subsequently, they were stained with PI for detection of DNA content and then loaded onto a fluorescence-activated cell sorting (FACS) caliber apparatus. (B) UT7/Epo-S1 cells were infected with various doses of B19 virus or mock infected and then treated with paclitaxel and PI in a manner similar to that for panel A. The percentages of G₀/G₁- and G₂/M-phase cells were calculated.

FIG. 2.

UV-irradiated virus can induce G₂ arrest but not G₁ arrest. B19 viruses in human serum were irradiated with various doses of UV and then infected to UT7/Epo-S1 cells. After 24 h postinfection, the cells were treated (A) or not treated (B) with paclitaxel for 24 h. They were then stained with PI for detection of DNA content and loaded on a FACS caliber. Percentages of the cells at G₀/G₁ phase (A) and G₂/M phase (B) were plotted. A part of the cells treated with paclitaxel in panel B were lysed in whole-cell extraction buffer and separated by SDS-PAGE. (C) After membrane transfer, proteins were detected by anti-NS1 and anti-α-tubulin MAbs.

FIG. 3.

G₂ checkpoint inhibitor (caffeine) treatment abrogates B19 virus-induced G₂ arrest. B19 virus-infected, mock-infected, or X-ray (10 Gy)-irradiated UT7/Epo-S1 cells were treated with or without various doses of caffeine. (A) After 48 h of cultivation, the cells were stained with PI, and the percentages of G₂/M-phase cells were measured by a FACS caliber. The remainder cells were lysed in whole-cell extraction buffer and separated by SDS-PAGE. (B) After membrane transfer, proteins were detected by using anti-NS1 and anti-α-tubulin MAbs. (C) Immunoblotted bands of NS1 in panel B were measured with a densitometer, and their densities were represented as relative amounts.

FIG. 4.

NS1 expression induces cell cycle arrest at the G₁ phase. (A) Schematic structure of NS1 expressing and control vector constructs. UT7/Epo-S1 (B) or 293T cells (D) were transfected with pMP6 (left panels) or pMP6-NS1 (right panels) and incubated for 24 h. They were then treated (upper panels) or untreated (lower panels) with a mitotic inhibitor, paclitaxel, for 24 h. Subsequently, they were stained with PI for detection of DNA content and loaded onto a FACS caliber. (C) The percentages of UT7/Epo-S1 cells at the G₁ phase were plotted after transfection of pMP6 and pMP6-NS1 plasmids at various ratios, and their NS1 expression levels were detected by Western blotting. (E) The transfected UT7/Epo-S1 cells with pMP6 or pMP6-NS1 plasmids were assayed for [³H]thymidine incorporation (upper panel). UT7/Epo-S1 cells were transiently transfected with 5 μg of the pMP6-NS1 or pMP6 plasmid by electroporation, and then the cells (10⁵ per well) were cultured for 24 h and assayed for [³H]thymidine incorporation for the last 4 h of cultivation. The expression levels of NS1 and α-tubulin were detected by Western blotting with their specific antibodies (lower panels).

FIG. 5.

Both B19 virus infection and NS11 transfection induce expression of p21/WAF1. Expression levels of p21/WAF1 and NS1 were analyzed sequentially in 293T cells transfected with pMP6 or pMP6-NS1 plasmids (A) and in UT7/Epo-S1 cells infected with B19 virus (B) by Western blotting with anti-NS1 and anti-p21/WAF1 MAbs, respectively.