Epstein-Barr virus protein BKRF4 restricts nucleosome assembly to suppress host antiviral responses.

Inhibition of host DNA damage response (DDR) is a common mechanism used by viruses to manipulate host cellular machinery and orchestrate viral life cycles. Epstein-Barr virus tegument protein BKRF4 associates with cellular chromatin to suppress host DDR signaling, but the underlying mechanism remains elusive. Here, we identify a BKRF4 histone binding domain (residues 15-102, termed BKRF4-HBD) that can accumulate at the DNA damage sites to disrupt 53BP1 foci formation. The high-resolution structure of the BKRF4-HBD in complex with a human H2A-H2B dimer shows that BKRF4-HBD interacts with the H2A-H2B dimer via the N-terminal region (NTR), the DWP motif (residues 80-86 containing D81, W84, P86), and the C-terminal region (CTR). The "triple-anchor" binding mode confers BKRF4-HBD the ability to associate with the partially unfolded nucleosomes, promoting the nucleosome disassembly. Importantly, disrupting the BKRF4-H2A-H2B interaction impairs the binding between BKRF4-HBD and nucleosome in vitro and inhibits the recruitment of BKRF4-HBD to DNA breaks in vivo. Together, our study reveals the structural basis of BKRF4 bindings to the partially unfolded nucleosome and elucidates an unconventional mechanism of host DDR signal attenuation.

HIV-1 Preintegration Complex Preferentially Integrates the Viral DNA into Nucleosomes Containing Trimethylated Histone 3-Lysine 36 Modification and Flanking Linker DNA.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Structural basis for retroviral integration into nucleosomes.

Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.

Human H4 tail stimulates HIV-1 integration through binding to the carboxy-terminal domain of integrase.

The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

Intasome architecture and chromatin density modulate retroviral integration into nucleosome.

BACKGROUND: Retroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors. RESULTS: Using in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts. CONCLUSIONS: Thus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density.

Human cytomegalovirus major immediate early 1 protein targets host chromosomes by docking to the acidic pocket on the nucleosome surface.

The 72-kDa immediate early 1 (IE1) protein encoded by human cytomegalovirus (hCMV) is a nuclearly localized promiscuous regulator of viral and cellular transcription. IE1 has long been known to associate with host mitotic chromatin, yet the mechanisms underlying this interaction have not been specified. In this study, we identify the cellular chromosome receptor for IE1. We demonstrate that the viral protein targets human nucleosomes by directly binding to core histones in a nucleic acid-independent manner. IE1 exhibits two separable histone-interacting regions with differential binding specificities for H2A-H2B and H3-H4. The H2A-H2B binding region was mapped to an evolutionarily conserved 10-amino-acid motif within the chromatin-tethering domain (CTD) of IE1. Results from experimental approaches combined with molecular modeling indicate that the IE1 CTD adopts a ß-hairpin structure, docking with the acidic pocket formed by H2A-H2B on the nucleosome surface. IE1 binds to the acidic pocket in a way similar to that of the latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus. Consequently, the IE1 and LANA CTDs compete for binding to nucleosome cores and chromatin. Our work elucidates in detail how a key viral regulator is anchored to human chromosomes and identifies the nucleosomal acidic pocket as a joint target of proteins from distantly related viruses. Based on the striking similarities between the IE1 and LANA CTDs and the fact that nucleosome targeting by IE1 is dispensable for productive replication even in "clinical" strains of hCMV, we speculate that the two viral proteins may serve analogous functions during latency of their respective viruses.

Characterization of nucleosome positioning in hepadnaviral covalently closed circular DNA minichromosomes.

Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.

RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus.

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions.

Functional coupling between HIV-1 integrase and the SWI/SNF chromatin remodeling complex for efficient in vitro integration into stable nucleosomes.

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier.

During lytic infections, herpes simplex virus type 1 DNA is in complexes with the properties of unstable nucleosomes.

The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.

Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications.

Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes.

Baculoviruses and nucleosome management.

Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management.

The regulation of HIV-1 gene expression: the emerging role of chromatin.

Host and viral factors that regulate the expression of the human immunodeficiency virus type 1 (HIV-1) 5' long terminal repeat (LTR) promoter have been studied since the recognition that HIV is the cause of the acquired immunodeficiency syndrome (AIDS). However, complex modifications of nucleosomes within chromatin has been recently recognized as an important mechanism of gene regulation. Nucleosome remodelling can alter the accessibility of DNA to specific activators or repressors, general transcription factors, and RNA polymerase. Emerging data now suggests that dynamic regulation of chromatin structure in the vicinity of the LTR promoter adds an additional level of complexity to the regulation of HIV expression. A better understanding of the role of chromatin in the regulation of HIV expression could lead to much-needed therapies against proviral genomes that are being actively transcribed, and those that are quiescent and persistent.

High-density nucleosome occupancy map of human chromosome 9p21-22 reveals chromatin organization of the type I interferon gene cluster.

Genome-wide investigations have dramatically increased our understanding of nucleosome positioning and the role of chromatin in gene regulation, yet some genomic regions have been poorly represented in human nucleosome maps. One such region is represented by human chromosome 9p21-22, which contains the type I interferon gene cluster that includes 16 interferon alpha genes and the single interferon beta, interferon epsilon, and interferon omega genes. A high-density nucleosome mapping strategy was used to generate locus-wide maps of the nucleosome organization of this biomedically important locus at a steady state and during a time course of infection with Sendai virus, an inducer of interferon gene expression. Detailed statistical and computational analysis illustrates that nucleosomes in this locus exhibit preferences for particular dinucleotide and oligomer DNA sequence motifs in vivo, which are similar to those reported for lower eukaryotic nucleosome-DNA interactions. These data were used to visualize the region's chromatin architecture and reveal features that are common to the organization of all the type I interferon genes, indicating a common nucleosome-mediated gene regulatory paradigm. Additionally, this study clarifies aspects of the dynamic changes that occur with the nucleosome occupying the transcriptional start site of the interferon beta gene after virus infection.

Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell.

Hepatitis B virus (HBV) nucleoprotein complexes were isolated from nuclei of the human hepatoblastoma cell line HepG2.2.15. Under conditions of physiological ionic strength, the complexes sedimented at a rate corresponding to about 82 S. They contained viral DNA, histone, and nonhistone proteins. For DNA a circular, covalently closed structure was shown both by CsCl gradient centrifugation and electron microscopy. Spread preparations revealed the typical "beads-on-a-string" appearance of nucleosomally organized DNA. The average number of nucleosomes was 18, resulting in a biochemical repeat unit of HBV chromatin of approximately 180 base pairs of DNA. This value was confirmed by experiments analyzing the structure of the HBV chromatin by the use of micrococcal nuclease. Electron microscopy demonstrated that exposure to high ionic strength conditions resulted in removal of nucleosomes from the complexes, but also revealed proteinaceous structures remaining bound to viral DNA molecules. The nature of these residual proteins is discussed. Since native nucleoprotein complexes could be precipitated with HBV-core antibodies, core protein appeared to be one of the nonhistone proteins.

Apoptosis is induced by hantaviruses in cultured cells.

Hantaviruses replicate in primary and cultured animal cells with little or no cytopathic effect. We report here that the cultured Vero E6 cells infected by the Hantaan or by the Prospect Hill viruses exhibited characteristic features of apoptosis, including condensation and segmentation of nuclei and internucleosomal cleavage of nuclear DNA. Apoptosis was not seen in the cells adsorbed by UV-inactivated virus, indicating that the viral replication is required for the induction of apoptosis. Furthermore, level of the proto-oncogenic Bcl-2 protein was significantly reduced, whereas its mRNA level remained unchanged in Hantaan virus-infected cells, suggesting possible involvement and posttranscriptional regulation of this antiapoptotic protein in the process.

DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes.

The choice of retroviral integration sites is strongly influenced by chromatin: integration in vitro occurs more efficiently into nucleosomal DNA than into naked DNA, and a characteristic pattern of preferred insertion sites with a 10 bp periodicity is observed at the outer face of the nucleosomal DNA. At least three features of nucleosomal DNA could be responsible for the creation of these favored sites: the presence of histones, attachment of the DNA to a protein surface, and DNA bending. To test each of these possibilities, we studied integration in vitro with human immunodeficiency virus and murine leukemia virus integrases into four model targets that mimic features of nucleosomal DNA: (i) catabolite activator protein-DNA complexes; (ii) lac repressor-operator complexes; (iii) lac repressor-induced loops; and (iv) intrinsically bent A-tract DNA. We found that bending of the target DNA can create favored integration sites at the outer face of the helix, irrespective of whether the bent DNA is attached to a protein surface. Our findings offer an explanation for the preferred usage of nucleosomes as integration targets. In addition, they suggest that bending of the target DNA might be an intrinsic feature of the integration reaction.

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor.

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.