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.

HCF-1 is an essential cellular transcriptional coactivator that interacts with members of numerous transcription factor families (CREB/ATF, Krupple, Ets, E2F, THAP) as well as other transcriptional coactivators (PGC, PRC, FHL2) (1). More recently, HCF-1 has been shown to be a component of multiple chromatin modification complexes that contain histone methyltransferase (Set1, MLL family members) (2 –4), demethylase (LSD1) (5), acetyltransferase (ATAC/STAGA, MOF) (6 –8), and deacetylase (sin3a/HDAC) (2) activities. Thus, HCF-1 functions to bridge transcription factors and chromatin modulation machinery.

HCF-1 was originally identified and characterized as an essential component of the α-herpesvirus [herpes simplex virus (HSV)-1 and varicella zoster virus (VZV)] immediate early gene enhancer complex along with the viral-encoded immediate early (IE) transcriptional activators (VP16 for HSV; ORF10 and IE62 for VZV) and the cellular POU domain protein Oct-1 (1). However, even in the absence of the viral activators, HCF-1 can mediate IE gene expression via factors such as Sp1 and GA-binding protein (GABP), suggesting that the protein plays a central role in regulating IE gene expression through multiple mechanisms (9). At the initiation of viral infection, an HCF-1 complex is recruited to the viral IE promoters that contains the histone methyltransferase Set1 or MLL1 and the histone demethylase LSD1 (5, 10). This HCF-1 dependent complex thus couples modification activities that counter the accumulation of repressive histone H3K9 methylation and promote the installation of positive H3K4-trimethylation marks to enhance the efficiency of IE gene transcription (5, 10, 11).

In addition to its role in initiation of lytic infection, HCF-1 may also play a role in the initial stages of viral reactivation from latency. The protein is sequestered in the cytoplasm of unstimulated sensory neurons where these viruses establish latency. Upon stimulation, HCF-1 is rapidly transported to the nucleus and recruited to the viral IE promoters during the initiation of viral reactivation (12 –14). As repressive and activating chromatin modifications correlate with latency and reactivation, respectively (15 –20), it has been hypothesized that HCF-1 modification complexes may play a central role in this process, in a manner analogous to its role in the initiation of viral lytic infection.

The requirement and role of HCF-1 in the regulation of viral and cellular gene expression has been well characterized. However, the role(s) of HCF-1 in functions other than transcriptional regulation have not been identified. Here we describe a unique HCF-1 function in HSV-1 DNA replication where HCF-1 couples the histone chaperone Asf1b to viral DNA replication components. During cellular DNA replication, the Asf1 histone H3/H4 chaperones modulate the flow of histones during nucleosome disassembly and reassembly at the DNA replication fork to allow proper fork progression and stability (21 –23). For HSV, the virus encodes its own major DNA replication components including the UL5-8-52 trimeric helicase/primase, replicative polymerase UL30, polymerase accessory/processivity factor UL42, origin binding helicase UL9, and single stranded DNA binding protein UL29 (24, 25). HSV DNA replication is complex and progresses through multiple stages and replicative mechanisms (25). Whereas significant work has been done on the enzymology of HSV DNA replication, the mechanisms involved in the integration of chromatin modulation components remain unclear. Coupling Asf1 to the viral replication machinery via the transcriptional coactivator HCF-1 provides a unique function for this coactivator and insights into chromatin control during viral infection.

Results

Accumulation of the Coactivator HCF-1 in HSV-1 Viral Replication Factories.

The cellular transcriptional coactivator HCF-1 mediates the basal and viral induced transcription of the immediate early genes and is essential for the initiation of the α-herpesvirus infectious cycle. However, HCF-1 also accumulates in viral replication foci and factories at later times of infection (Fig. 1 A), in contrast to control chromatin associated factors (HDAC5 and NCoR, Fig. S1A and B). HCF-1 colocalized in early replication foci (4 hr postinfection) and late stage viral replication factories (6 hr postinfection) with the UL29 single stranded DNA binding protein, a marker for viral DNA replication. Region of interest (ROI) analysis indicated a high degree of colocalization with UL29 at punctate locations in the replication factories (Fig. 1 B). In contrast, TATA-binding protein was incorporated into viral replication factories but did not significantly colocalize with UL29 foci (Fig. S1C and D).

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.

Interaction of HCF-1 with Viral Replication Machinery.

During the initiation of infection, HCF-1 is recruited to the viral IE gene promoter-enhancer domains by interactions with the viral IE activators. The colocalization of HCF-1 with UL29 in viral replication factories suggested that the protein might play a role in viral DNA replication, in addition to its function in the regulation of viral IE gene expression. To determine if the accumulation of HCF-1 in the viral replication factories represented specific HCF-1 interactions with HSV-1 DNA replication components, domains of HCF-1 (Fig. 2 A) were tested against the 7 major HSV-1 encoded DNA replication proteins in yeast two-hybrid analyses. Strikingly, the amino-terminal region containing the HCF-1 kelch domain scored positive when coexpressed with the UL9 origin binding helicase, UL30 viral DNA polymerase, and the UL52 subunit of the viral helicase-primase (Fig. 2 B and Fig. S2). In contrast, no HCF-1 interactions were detected with the other DNA replication components and conversely, no interactions of the replication proteins with other HCF-1 domains.

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).

To confirm the interactions of HCF-1 with these viral replication components, CV-1 cells were transfected with plasmids expressing FLAG or HA-tagged UL30, UL52, or UL9, and control protein β-Galactosidase or were cotransfected with a construct encoding a V5 epitope-tagged HCF-1. Plasmids expressing FLAG-UL42 (polymerase accessory factor) and a nonreplication related HSV-1 protein (UL19, HSV major capsid protein) were used as additional controls. Extracts and V5 immunoprecipitates were probed for V5 (HCF-1) and FLAG or HA (replication proteins and controls) (Fig. 2 C). In each case, HCF-1-V5 efficiently immunoprecipitated the anticipated replication component in contrast to the β-gal, UL42, or UL19 negative control proteins. No protein was immunoprecipitated from extracts expressing the epitope-tagged replication proteins in the absence of HCF-1-V5. In an analogous manner, endogenous HCF-1 efficiently coimmunoprecipitated UL30, UL52, and UL9 from HSV-1 infected cells expressing the epitope-tagged replication proteins (Fig. 2 D).

The interaction of HCF-1 with each replication protein was further defined by two-hybrid analyses (Fig. S3). For UL30, HCF-1 interacted strongly with the amino terminus consisting of the “preN, N1, exonuclease, and N2” subdomains and more weakly with a smaller 365 amino acid region consisting of only the preN and N1 regions. Given the structure of the polymerase, this weak interaction may reflect the requirement for the additional domains for appropriate folding and stability of the fusion protein. In UL52 and UL9, HCF-1 interacted with a 70 amino acid region adjacent to the primase motif and a 75 amino acid region between helicase motifs III and IV, respectively. Conversely, each of the replication proteins interacted with a region of HCF-1 (aa 348–455) that overlaps the junction of the HCF-1 kelch and mid-amino terminal domain. Together, the localization of HCF-1 in viral replication factories and its interactions with viral DNA replication components suggested that the coactivator is an important component of this stage in the viral infectious cycle.

The Histone Chaperone Asf-1b Interacts with HCF-1 and Colocalizes to Viral Replication Factories.

In contrast to its roles in HSV IE gene regulation, the role that HCF-1 might play in replication was not apparent. However, the histone H3/H4 chaperone Asf1b had previously been isolated in two-hybrid screens for HCF-1 cellular interaction partners. Recent data have demonstrated that Asf1 proteins play critical roles in cellular DNA replication and repair processes, in part by modulating histone modification and dynamics at the replication fork or site of repair.

To determine if Asf1b might be involved in HSV-1 DNA replication, cells were mock infected or infected for 4–6 hr and stained for the viral DNA single strand binding protein UL29 and Asf1b. As shown in Fig. 3, the pattern of Asf1b accumulation in the early viral replication foci and late viral replication factories parallels that of HCF-1 (refer to Fig. 1). High-resolution confocal images of extensive replication factories clearly shown a punctate colocalization of Asf1b and UL29 (Fig. 3 B) indicating that Asf1b localizes to sites of viral replication.

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.

HCF-1 Couples Asf-1b to Viral Replication Components.

HCF-1 is a family of polypeptides derived from a common 230 kD precursor by site-specific proteolysis at a series of 20 amino acid reiterations located in the central proteolytic processing domain (PPD) (26, 27). Cleavage results in amino and carboxyterminal subunits that do not segregate but remain tightly but noncovalently associated. Asf1b was initially isolated as an HCF-1 binding partner by interaction with the HCF-1 carboxy terminal domain that contains two fibronectin repeats and the protein’s nuclear localization signal. The fibronectin repeats contain one of the two sets of sequences that mediate the association of the HCF-1 amino and carboxterminal subunits (28). As shown in Fig. 4 A and Fig. S4, the amino terminal domain (aa 1–154) of Asf1b interacted with each of the HCF-1 fibronectin repeats in yeast two-hybrid assays. In contrast, a fusion protein containing the HCF-1 amino terminal sequence that mediates the HCF-1 amino-carboxyterminal association interacted only with the first repeat (FN-1). Additionally, Asf1b could be efficiently coimmunoprecipitated from cells by V5 epitope-tagged wild-type HCF-1 but not from cells expressing an HCF-1 mutant lacking the FN repeats, thus confirming that the HCF-1 FN repeats are important Asf1b interaction determinants (Fig. 4 B). In contrast, UL52, which interacts with HCF-1’s amino terminal domain, was efficiently immunoprecipitated by both wild-type and ΔFN mutant.

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).

The interactions of the viral replication components with the HCF-1 amino terminus and Asf1b with the HCF-1 carboxy terminal FN domain (Fig. 4 C) provides the potential that HCF-1 functions to couple Asf1b to the viral replication machinery. Consistent with this, coimmunoprecipitation of Asf1b with FLAG epitope-tagged UL30 and UL52 was significantly enhanced from cells coexpressing HCF-1-V5 (Fig. 5 A, compare lanes 1 with 3 and 5 with 7). Additionally, Asf1b was readily detected in a sequential immunoprecipitation with FLAG-UL52 followed by HCF-1-V5 (Fig. 5 B).

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%.

Asf1b, as noted above, is a histone H3/H4 chaperone. To determine if Asf1b links HCF-1 to histone H3, HCF-1 was immunoprecipitated from extracts of cells transfected with control (GFP) shRNA or Asf1b shRNA and the immunoprecipitates were probed for HCF-1 and histone H3. As shown in Fig. 5 C, HCF-1 efficiently immunoprecipitated histone H3 from control cells but not from cells depleted of Asf1b (compare lane 2 with 5). Taken together, these data indicate that HCF-1 couples the histone chaperone Asf1b/histone complex to viral replication components.

Asf1b Is Critical for Efficient Viral DNA Replication.

In cellular DNA replication, the Asf1 proteins are critical for managing the flow of histones and the progression of the replication fork via coordinated disassembly and reassembly of nucleosomes. In this capacity, recipient Asf1 proteins are associated with the replicative helicase MCM complex for disassembly and transfer of histone H3/4 ahead of the advancing replication fork to the histone donor Asf1/CAF-1/PCNA complex for reassembly on replicated DNA (21, 29 –31). Asf1 mutations or depletion of the proteins results in defects in replication fork progression (23). Similarly, overexpression of histone H3/H4 blocks fork progression via saturation of the recipient Asf1 proteins (21). In contrast, the mechanisms by which chromatin is managed during HSV-1 DNA replication are largely unknown. The high-level colocalization of Asf1b with the viral single-strand binding protein and the coupling of Asf1b to viral replication machinery via HCF-1 would argue that Asf1b would also play an important role in viral DNA synthesis.

To investigate the impact of Asf1b on viral gene expression and the accumulation of viral DNA, cells were transfected with control (GFP) shRNA or Asf1b specific shRNA. As shown in Fig. 6 A, no significant impact of Asf1b depletion was seen on the expression of viral immediate early (IE, ICP4, and ICP27) or early (E, UL29) proteins. However, the expression of a viral late protein (L, gC), a class that is dependent upon viral DNA synthesis, was severely reduced (compare 18 hr timepoint). Additionally, qPCR analysis of viral DNA at late times postinfection showed significant reduction in viral DNA accumulation in Asf1b depleted cells (4.3-fold, Fig. 6 B) whereas no impact was seen on the levels of input genomes (2 hr postinfection). As demonstrated by qRT-PCR analyses, this reduction in DNA accumulation was not due to reduced levels of HSV-1 DNA replication components (Fig. S5). Finally, viral yields from Asf1b depleted cells were reduced (approximately 10-fold) relative to control shRNA treated cells at 18 hr postinfection (Fig. 6 C). The results demonstrate that Asf1b has no significant role in early stages of viral gene expression but plays an important role in viral DNA replication.

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.

Discussion

HCF-1 is an essential cellular transcriptional coactivator. Multiple interactions with transcription factors and other coactivators have been described in the regulation of cellular and viral transcription. In addition, the protein is critical for proper cell cycle progression (32, 33), maintenance of ES cell pluripotency (34), and cellular differentiation (35), likely via its transcriptional functions. However, insights into the mechanism(s) by which this protein mediates activation have only recently come from the observations that HCF-1 is a component of multiple chromatin modification/modulation complexes.

Given the numerous interactions of the protein with transcription factors of various families, it is likely that HCF-1 functions to target specific chromatin modification complexes. This is supported by the HCF-1 dependent regulation of the IE genes of the α-herpesviruses HSV-1 and VZV, where an HCF-1 complex containing both the histone methyltransferase Set1 or MLL1 and the demethylase LSD1 is recruited to the IE promoters by the viral encapsidated activators.

Interestingly, HSV-1 enters the cell devoid of nucleosomes. Histones are rapidly deposited, likely via a cellular response to infection. Whereas the chromatin structure does not appear to be that of canonically spaced nucleosomes, the levels of genome-associated nucleosomes are similar to that observed on cellular DNA (36). The role of chromatin in regulating HSV-1 gene expression is supported by (i) the presence of nucleosomes exhibiting repressive or activating modifications that correlate with repression or activation of viral gene expression (5, 10, 11, 36 –38) and (ii) deposition of the histone variant H3.3 on the viral genome and the requirement for the H3.3 histone chaperone HIRA for efficient viral gene expression (39).

As in viral gene expression, the virus must also utilize mechanisms to manage chromatin during DNA replication. Although the levels of nucleosomes on the genome appear to be reduced relative to cellular DNA at later times in infection (36, 40), the absolute amount of viral DNA associated with histones increases during replication (36). Additionally, it has also been observed that histone H3.1 is incorporated in a replication dependent manner (39), indicating that some level of histone assembly occurs on replicated viral DNA. As HSV-1 encodes much of the enzymology required for replication of its DNA, these observations suggest that the virus must interface with cellular machinery to manipulate chromatin during replication.

Here we demonstrate that both the cellular transcriptional coactivator HCF-1 and the histone H3/H4 chaperone Asf1b localize to viral replication foci and colocalize with the viral single strand binding protein UL29. HCF-1 interacts with the UL9 origin binding helicase, UL52 component of the helicase-primase, and the UL30 DNA polymerase through its amino terminal domain and Asf1b through its carboxyterminal domain. Thus, HCF-1 bridges viral replication machinery and a component of cellular chromatin modulation.

The Asf1 proteins play critical roles in cellular DNA replication and repair processes by (i) modulating the flow of dissembled histones ahead of the advancing MCM replication fork (21), (ii) coordinating histone assembly on replicated and repaired DNA in conjunction with Chromatin Assembly Factor-1 (CAF-1) (29, 31), (iii) promoting histone H3K56 acetylation for CAF-1 dependent chromatin assembly (22, 41, 42), and (iv) signaling termination of DNA repair (43, 44). The significance of these proteins is apparent from studies in which Asf1 depletion or mutation results in inhibition of replication fork progression, decreased replication fork stability, and increased exposure to replication DNA strand breaks (23, 45, 46).

The data presented here and the described roles of Asf1b lead to a proposed model in which HCF-1 is required to mediate Asf1b dependent reorganization of nucleosomes at viral replication forks (Fig. 7). The interactions of HCF-1 with multiple replication components may also reflect a coordinated transfer of HCF-1-Asf1-H3/H4 in this process. Notably, both chromatin remodeling components and DNA damage repair machinery accumulate in viral replication foci (47 –49). In addition to their role in DNA replication, the Asf1 proteins play a significant role in repair processes, in part, by mediating histone modifications that promote chromatin reassembly and by signaling the completion of repair. It is possible, therefore, that HCF-1/Asf1b interactions with HSV replication proteins may also reflect repair processes that are an inherent component of HSV DNA replication. Interestingly, a significant percentage of newly replicated viral DNA appears to be relatively free of nucleosomes (40); a requirement for packaging of viral progeny genomes. Disassembly of nucleosomes promoted by HCF-1-Asf1b, in the absence of substantial reassembly, may play a part in providing a pool of nucleosome free progeny DNA for encapsidation.

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.

Whereas it is clear that additional components and their roles remain to be determined, the data illustrate a novel role for the essential transcriptional coactivator HCF-1. Significantly, the interaction of HCF-1 and Asf1b is likely to reflect a critical role for this coactivator in cellular DNA replication and/or repair.

Materials and Methods

Cells, Virus, and Plasmids.

HeLa, CV-1, and Vero cells were maintained and infected with HSV-1 (strain F) according to standard protocols. Epitope-tagged UL30, UL9, UL19, UL52, UL42, Asf1b, and β-galactosidase were cloned in pcDNA3. HCF-1-V5 has been previously described (50).

Antibodies.

Antibodies used are listed in SI Text.

Yeast Two-Hybrid Analysis.

Yeast two-hybrid analyses were done according to standard protocols in strain AH109. Constructs expressing Gal4-DNA binding domain and activation domain fusion proteins were constructed in pGBKT7 and pGADT7, respectively. The domains of HCF-1 and HSV-1 replication proteins used to produce the fusion constructs are shown in Fig. 2 and Fig. S2.

Coimmunoprecipitations.

2.5 × 10⁶ CV-1 cells were transfected with the appropriate expression plasmids using Lipofectamine 2000 (Invitrogen). Forty-eight h later, lysates were prepared in [50 mM Tris pH7.5, 150 mM NaCl, 0.1%NP-40, 5% glycerol, 1 mM NaF, 10 mM β-glycerophosphate, 0.1 mM Na3VO4, Complete Protease Inhibitor]. Sequential coimmunoprecipitations were done as described above except that lysates were incubated with anti-FLAG M2 affinity resin and washed extensively. Bound proteins were eluted with FLAG peptide (100 ug/ml) and reprecipitated with anti-V5 affinity gel. For endogenous coimmunoprecipitations, antibodies were bound to protein G Dynal beads and incubated with nuclear extracts in NE buffer [20 mM Hepes pH7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT]. Western blots were developed using Super Signal Dura (Pierce), and quantitated using a Kodak 40000 MM Image Station.

Asf1b Depletions.

HeLa cells were transfected with 3 ug control GFP or Asf1b specific shRNAs (Origene, TR30003 and TR317316, respectively) using Fugene 6 (Roche). Viral infections and coimmunoprecipitations were done after 72 hr.

Immunofluorescence Microscopy.

Immunofluorescent staining was done according to standard protocols. For select samples, the cells were treated with [10 mM Pipes pH 6.8, 300 mM Sucrose, 100 mM NaCl, 3 mM Mgcl₂, 1 mM EGTA, 0.5% Triton-100, Complete Protease Inhibitor] prior to fixation in 3.2% paraformaldehyde (21). Stained cells were visualized using a Leica SP5 confocal microscope and LASAF software (V2.182). ROI analyses were done as described in SI Text using Imaris software.

qPCR and qRT-PCR.

Viral DNA samples were quantitated by qPCR, in duplicate, using an ABI PRISM 7900HT. Samples were normalized to actin genomic DNA. Viral DNA was quantitated relative to a purified viral DNA standard. For qRT-PCR, oligo-dT primed cDNA was produced from total RNA of Asf1b depleted or control cells using RNAqueous-4PCR and RETROscript (Ambion). qPCR and qRT-PCR primer sequences are listed in SI Text. Statistical analyses (two-tailed T tests) were done using Prism 5.0b.