Bromodomain and extraterminal inhibitors block the Epstein-Barr virus lytic cycle at two distinct steps.

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Epstein-Barr virus latency type and spontaneous reactivation predict lytic induction levels.

The human Epstein-Barr virus (EBV) evades the immune system by entering a transcriptionally latent phase in B cells. EBV in tumor cells expresses distinct patterns of genes referred to as latency types. Viruses in tumor cells also display varying levels of lytic transcription resulting from spontaneous reactivation out of latency. We measured this dynamic range of lytic transcription with RNA deep sequencing and observed no correlation with EBV latency types among genetically different viruses, but type I cell lines reveal more spontaneous reactivation than isogenic type III cultures. We further determined that latency type and spontaneous reactivation levels predict the relative amount of induced reactivation generated by cytotoxic chemotherapy drugs. Our work has potential implications for personalizing medicine against EBV-transformed malignancies. Identifying latency type or measuring spontaneous reactivation may provide predictive power in treatment contexts where viral production should be either avoided or coerced.

Histone chaperones link histone nuclear import and chromatin assembly.

Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

Histone chaperones link histone nuclear import and chromatin assembly.

Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

Interaction with the histone chaperone Vps75 promotes nuclear localization and HAT activity of Rtt109 in vivo.

Modification of histones is critical for the regulation of all chromatin-templated processes. Yeast Rtt109 is a histone acetyltransferase (HAT) that acetylates H3 lysines 9, 27 and 56. Rtt109 associates with and is stabilized by Nap1 family histone chaperone Vps75. Our data suggest Vps75 and Nap1 have some overlapping functions despite their different cellular localization and histone binding specificity. We determined that Vps75 contains a classical nuclear localization signal and is imported by Kap60-Kap95. Rtt109 nuclear localization depends on Vps75, and nuclear localization of the Vps75-Rtt109 complex is not critical for Rtt109-dependent functions, suggesting Rtt109 may be able to acetylate nascent histones before nuclear import. To date, the effects of VPS75 deletion on Rtt109 function had not been separated from the resulting Rtt109 degradation; thus, we used an Rtt109 mutant lacking the Vps75-interaction domain that is stable without Vps75. Our data show that in addition to promoting Rtt109 stability, Vps75 binding is necessary for Rtt109 acetylation of the H3 tail. Direct interaction of Vps75 with H3 likely allows Rtt109 access to the histone tail. Furthermore, our genetic interaction data support the idea of Rtt109-independent functions of Vps75. In summary, our data suggest that Vps75 influences chromatin structure by regulating histone modification and through its histone chaperone functions.

Phosphorylation by casein kinase 2 regulates Nap1 localization and function.

In Saccharomyces cerevisiae, the evolutionarily conserved nucleocytoplasmic shuttling protein Nap1 is a cofactor for the import of histones H2A and H2B, a chromatin assembly factor and a mitotic factor involved in regulation of bud formation. To understand the mechanism by which Nap1 function is regulated, Nap1-interacting factors were isolated and identified by mass spectrometry. We identified several kinases among these proteins, including casein kinase 2 (CK2), and a new bud neck-associated protein, Nba1. Consistent with our identification of the Nap1-interacting kinases, we showed that Nap1 is phosphorylated in vivo at 11 sites and that Nap1 is phosphorylated by CK2 at three substrate serines. Phosphorylation of these serines was not necessary for normal bud formation, but mutation of these serines to either alanine or aspartic acid resulted in cell cycle changes, including a prolonged S phase, suggesting that reversible phosphorylation by CK2 is important for cell cycle regulation. Nap1 can shuttle between the nucleus and cytoplasm, and we also showed that CK2 phosphorylation promotes the import of Nap1 into the nucleus. In conclusion, our data show that Nap1 phosphorylation by CK2 appears to regulate Nap1 localization and is required for normal progression through S phase.

Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae.

Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes. EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events. In exponentially growing S. cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1. After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction. Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions. The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions.