Transcriptional coactivator HCF-1 couples the histone chaperone Asf1b to HSV-1 DNA replication components

Abstract

The cellular transcriptional coactivator HCF-1 interacts with numerous transcription factors as well as other coactivators and is a component of multiple chromatin modulation complexes. The protein is essential for the expression of the immediate early genes of both herpes simplex virus (HSV) and varicella zoster virus and functions, in part, by coupling chromatin modification components including the Set1 or MLL1 histone methyltransferases and the histone demethylase LSD1 to promote the installation of positive chromatin marks and the activation of viral immediately early gene transcription. Although studies have investigated the role of HCF-1 in both cellular and viral transcription, little is known about other processes that the protein may be involved in. Here we demonstrate that HCF-1 localizes to sites of HSV replication late in infection. HCF-1 interacts directly and simultaneously with both HSV DNA replication proteins and the cellular histone chaperone Asf1b, a protein that regulates the progression of cellular DNA replication forks via chromatin reorganization. Asf1b localizes with HCF-1 in viral replication foci and depletion of Asf1b results in significantly reduced viral DNA accumulation. The results support a model in which the transcriptional coactivator HCF-1 is a component of the HSV DNA replication assembly and promotes viral DNA replication by coupling Asf1b to DNA replication components. This coupling provides a novel function for HCF-1 and insights into the mechanisms of modulating chromatin during DNA replication.

Fig. 1.

Localization of HCF-1 to viral replication compartments. (A). CV-1 cells were mock infected or infected with HSV-1 (10 pfu/cell) and stained for HCF-1 and UL29 at the indicated times postinfection. UL29, the viral single stranded DNA binding protein was used as a marker for viral replication foci. (B). HCF-1/UL29 ROI colocalization analyses were done on viral replication factories and control areas outside of the factories.

Fig. 2.

Interaction of HCF-1 with viral replication components. (A). The domains of HCF-1 are shown with the regions used as Gal4-DNA binding fusion proteins in yeast two-hybrid assays delineated. Kelch (+), amino terminal region containing the predicted kelch domain (aa 1–380); MN-1 and MN-2, mid-amino terminal domains; PPD, containing HCF-1 processing repeats (Blue and Red Ovals); TA, transactivation domain; COOH, carboxyterminal domain containing fibronectin repeats (FN3); and the nuclear localization signal. (B). The results of two-hybrid analyses with HCF-1 domain-binding fusion proteins and HSV-1 DNA replication component activation domain fusion proteins. Control assays are in Fig. S1. C-Lam, negative control Lamin-C DNA binding domain fusion. (C). Extracts and V5 immunoprecipitates from uninfected CV-1 cells coexpressing V5-epitope-tagged HCF-1 and FLAG or HA-epitope-tagged HSV-1 UL30, UL52, UL9, or control UL42 and UL19 proteins were probed for V5 (HCF-1) and the indicated HSV-1 protein (UL). Coexpressed β-galactosidase was used as an internal control. The presence or absence of HCF-1-V5 is indicated at the top of the gel. (D). Extracts and HCF-1 or control IgG immunoprecipitates from cells expressing FLAG epitope-tagged UL30, UL52, or UL9 were probed for endogenous HCF-1 and FLAG (replication proteins).

Fig. 3.

Colocalization of Asf1b with HSV-1 single strand DNA binding protein in replication factories. (A). CV-1 cells were mock infected or infected with HSV-1 (10 pfu/cell) and stained for UL29 and Asf1b at the indicated times postinfection. (B). High-resolution zoom of an extensive nuclear replication factory illustrates the punctate colocalization of UL29 and Asf1b.

Fig. 4.

Interaction of HCF-1 with Asf1b. (A). Asf1b interacts with either of the HCF-1 two fibronectin repeats (FN-1, FN-1, or FN1 + 2) in two-hybrid analyses. The HCF-1 amino terminal region (HCF-1 aa 360–405) contains one set of sequences that mediate the HCF-1 amino-carboxyterminal subunit association (28). The activation domain vector plasmid (pGADT7) and DNA binding domain-Lamin C fusion were used as negative controls. Control assays are in Fig. S4. (B). Extracts and V5 immunoprecipitates from cells expressing V5-epitope-tagged wild-type HCF-1 (WT) or HCF-1 lacking the fibronectin repeats (ΔFN) and HA-Asf1b were probed for V5 (HCF-1) and HA (Asf1b). Control immunoprecipitates from cells expressing the HCF-1 proteins and UL52 are shown in the Right panel. (C). A schematic of HCF-1 indicating the interactions with Asf1b and the viral replication proteins is shown. The sequences required for HCF-1 amino/carboxyterminal subunit association are indicated (HCF N-C).

Fig. 5.

HCF-1 couples Asf1b to viral replication machinery. (A). Extracts and FLAG immunoprecipitates from uninfected cells expressing the indicated proteins were probed for V5 (HCF-1), FLAG (HSV-1 replication proteins), and HA (Asf1b). (B). Extracts of cells expressing the indicated proteins were immunoprecipitated with anti-FLAG, eluted with FLAG peptide (IP eluate), and reprecipitated with anti-V5 (HCF-1). (C). Extracts and HCF-1 immunoprecipitates from cells transfected with control or Asf1b specific shRNAs were probed for HCF-1 and histone H3. The depletion of Asf1b in these experiments was 98%.

Fig. 6.

Asf1b is required for efficient HSV-1 DNA replication. (A) CV-1 cells were transfected with control or Asf1b specific shRNAs and infected with HSV-1 (5 pfu/cell) for the indicated times. Extracts were probed for the expression of representative HSV-1 immediate early (IE), early (E), and late (L) proteins. Tubulin is a loading control. The extent of Asf1b depletion is shown (96%). (B). The quantity of HSV-1 viral DNA was determined in cells transfected with control or Asf1b shRNAs by qPCR, relative to a viral DNA standard. Actin DNA was used as a control to indicate equivalent levels of total DNA in control and Asf1b depleted samples. The 2 hr timepoint represents the viral genome input prior to replication. The results are from three experiments and error bars are SEM. (C) Control and Asf1b shRNA treated cells were infected with HSV-1 (1 pfu/cell) for 18 hr and viral yields were determined.

Fig. 7.

Model of HCF-1/Asf1b function in HSV DNA replication. The proposed function of the coactivator HCF-1 in coupling the histone chaperone Asf1b to viral replication components (UL52, UL30) during viral DNA replication is shown. The viral helicas-primase trimeric complex is represented by 52/8/5. In this model, Asf1b promotes disassembly and reassembly of nucleosomes proximal to the replication fork to allow efficient viral DNA replication. The interactions of HCF-1 with Asf1b and the origin helicase UL9 is not shown but the role is expected to be similar in mediating chromatin reorganization during the initiation of origin-dependent replication.