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. 2002 Aug 6;99(16):10683-8.
doi: 10.1073/pnas.162352299. Epub 2002 Jul 26.

Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-alpha

Frederick Y Wu  1 , Qi-Qun Tang, Honglin Chen, Colette ApRhys, Christopher Farrell, Jianmeng Chen, Masahiro Fujimuro, M Daniel Lane, Gary S Hayward
Affiliations

Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-alpha

Frederick Y Wu et al. Proc Natl Acad Sci U S A. .

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G(1) cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12-24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPalpha and p21(CIP-1) proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPalpha in vitro and stimulates C/EBPalpha-induced expression from both the C/EBPalpha and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPalpha and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G(1) to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPalpha or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPalpha. Therefore, C/EBPalpha is essential for the p21-mediated inhibition of G(1) to S-phase progression by RAP in KSHV-infected host cells.

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Figures

Fig 1.
Fig 1.
Correlation between RAP, C/EBPα, and p21 protein expression. (A) Western immunoblot showing the levels of endogenous C/EBPα protein in different human primary, tumor, and lymphoma cells. (B) Western immunoblot showing increased expression of the C/EBPα and p21 proteins in HeLa cells after transfection with the pSG5-RAP cDNA expression plasmid. (C–E) Western immunoblot analysis illustrating the levels of RAP, C/EBPα, p21, p27, and p16 protein in JSC-1 cells at 0, 24, 48, and 72 h after TPA induction.
Fig 2.
Fig 2.
Immunohistochemical evidence for induction of p21 and C/EBPα protein expression only in lytically induced BCBL-1 cells that expressed the KSHV RAP protein. (A) In uninduced cultures, expression of RAP (Vecta Stain-red chromogen) and p21 (DAKO-True Blue chromogen) expression could only be detected in very few spontaneously lytic cell nuclei by double staining. (B and C) Increased numbers of cells expressing both p21 (blue) and KSHV RAP (red) 48 h after lytic reactivation by TPA induction. (D) Double-staining depicting colocalization of nuclear RAP (red) and p21 (blue) in a large fraction of the cells after TPA induction. (E) Low expression of C/EBPα protein (blue) in uninduced latent PEL cell culture. (F) Increased expression of C/EBPα (blue) in lytically activated cells after TPA induction. (G) Double-staining for KSHV RAP and C/EBPα in TPA-treated cells, showing induced C/EBPα (blue) expression colocalized in the nucleus predominantly in the same cells that were also expressing RAP (red). (H) No p16 protein (blue) expression was detected even in RAP-positive (red) cells after TPA induction.
Fig 3.
Fig 3.
Inhibition of S-phase progression by the C/EBPα and KSHV RAP proteins as detected by double-label IFA. (A and B) C/EBPα-mediated cell cycle arrest occurs in 3T3-L1 cells transfected with the pMSV-C/EBPα expression plasmid as demonstrated by the absence of BrdUrd pulse-labeling of newly synthesized DNA (B, rhodamine, red), in all of the cells expressing the C/EBPα protein (A, FITC, green). (C) Merge of the two frames. (D and E) At 48 h after TPA treatment in the presence of PAA to inhibit viral DNA synthesis, 15% of the BCBL-1 cells expressed the lytically induced RAP protein (D, green), but BrdUrd incorporation (E, red) revealed that very few (less than 4%) of the RAP-positive cells were undergoing cellular S phase. PAA treatment (400 μg/ml) for 48 h had no effect on cellular DNA replication during S phase as shown by positive BrdUrd incorporation (E, red). (F) Merge of the two frames.
Fig 4.
Fig 4.
KSHV RAP expressed by an adenovirus vector (Ad-RAP) induces C/EBPα and p21 protein expression in HF cells. (A and B) Double-label IFA showing the absence of BrdUrd incorporation (rhodamine, red) in most cells expressing the wild-type RAP protein (FITC, green), and (C) DAPI nuclear staining of the whole cell population. (D and E) Double-label IFA showing normal BrdUrd incorporation and S-phase patterns (red) in Ad-RAP(Δ169–237) expressing cells (green), and (F) merge illustrating that S phase occurs in RAP(Δ169–237)-positive cells. (G and H) Double-label IFA showing the induced expression of wild-type RAP (green) and p21 (red) in the same cell population, and (I), merge showing that p21 is induced only in RAP positive cells. (J and K) Double-label IFA showing RAP(Δ169–237) protein expression (green) and the absence of induced p21 protein (red) in the same cell population, and (L) merge showing that p21 expression is not up-regulated in Ad-RAP(Δ169–237)-positive cells. (M, N) Double-label IFA illustrating expression of RAP (red) and up-regulation of C/EBPα (green) in the same cell population, and (O) merge illustrating coexpression of C/EBPα only in RAP-positive cells. (P and Q) Double-label IFA showing RAP(Δ169–237) expression (red) and the absence of enhanced C/EBPα expression (green) in the same cell population, and (R) merge illustrating that RAP(Δ169–237) does not induce C/EBPα expression.
Fig 5.
Fig 5.
Analytical fluorescence-activated cell sorting to demonstrate that the KSHV wild-type RAP protein but not mutant RAP causes G1/S cell cycle arrest in human HF cells. (A) RAP-positive cells in Ad-RAP (1–237) or Ad-RAP(Δ169–237) infected HF cell populations were sorted from the uninfected RAP-negative cells through an FITC-gated channel on the basis of the difference in their fluorescence signals. (B) Flow cytometric analysis showing that the wild-type RAP-expressing cells (FITC channel-gated) were predominantly arrested in G1 in comparison with either the uninfected cells from the same population, or the Ad-RAP(Δ169–237) mutant positive cells.
Fig 6.
Fig 6.
The KSHV RAP protein cannot efficiently up-regulate p21 expression in C/EBPα knockout mouse embryonic fibroblast MEF (−/−) cells. (A and B) Double-label IFA showing the expression of RAP protein (FITC, green) in Ad-RAP-infected cells and the absence of C/EBPα protein (rhodamine, red) in the same cells. (C) DAPI nuclear staining of the whole cell population. (D and E) Double-label IFA of RAP protein expression (green) and BrdUrd incorporation (red) in Ad-RAP-infected cells. (F) Merge illustrating that S phase still proceeds in RAP-positive MEF (−/−) cells that lack C/EBPα. (G and H) Double-label IFA showing RAP protein expression (green) and the absence of mouse p21 induction (red) in RAP-positive (−/−) MEF cells after Ad-RAP infection. (I) DAPI nuclear staining of the same whole cell population.
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