The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here, we demonstrate that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9 Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR analysis. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate H3K27 in vitro and in vivo. Laser micro-irradiation data reveal that PHF1 is transiently recruited to DNA double-strand breaks (DSBs), and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at DSB sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as an early DNA damage response cofactor.

h Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-␥)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-␥ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-␥ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

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Chromatin-based regulation of herpesviral transcriptional programs is increasingly appreciated as a mechanism for modulating infection outcomes. Transcriptionally permissive euchromatin predominates during lytic infection, whereas heterochromatin domains refractory to transcription are enriched at lytic genes during latency. Reversibly silenced facultative heterochromatin domains are often enriched for histone H3 trimethylated on lysine 27 (H3K27me3), a modification catalyzed by Polycomb repressive complex 2 (PRC2). The requirement for PRC2 in suppressing the human cytomegalovirus (HCMV) lytic transcriptional program during latency has not been thoroughly evaluated. Therefore, we disrupted PRC2 activity in the highly tractable THP1 and NT2D1 quiescent-infection models by treating cells with small-molecule inhibitors of PRC2 activity. Compared to control cells, disruption of PRC2 in HCMV-infected THP1 or NT2D1 cells resulted in significant increases in viral transcript levels and the detection of viral protein. Using chromatin immunoprecipitation, we demonstrated that enrichment of H3K27me3, deposited by PRC2, correlates inversely with lytic transcriptional output, suggesting that PRC2 catalytic activity at viral chromatin directly represses lytic transcription. Together, our data suggest that PRC2-mediated repression of viral transcription is a key step in the establishment and maintenance of HCMV latency.

The human cytomegalovirus immediate-early 5-kb RNA previously has been shown to be a stable intron that is not required for efficient replication of the virus in cultured fibroblasts. Here we describe a murine cytomegalovirus 7.2-kb ortholog of the human cytomegalovirus 5-kb RNA. The 5 end of the 7.2-kb transcript maps to a consensus splice-donor site that is conserved among all cytomegaloviruses. We constructed mutant viruses lacking the entire 7.2-kb coding domain, the splice-donor site predicted to function in the generation of the intron or a hairpin predicted to stabilize the intron. All three mutant viruses failed to produce the 7.2-kb RNA, supporting our conclusion that it is a stable intron. Each of the mutants replicated with normal kinetics in cultured fibroblasts, but the mutants exhibited a clear defect within infected mice. Although the initial acute phase at 4 days after infection appeared to be normal, none of the mutant viruses progressed to the persistent phase, i.e., little virus was detected in the salivary gland at 14 days after infection. The intron functions as an in vivo virulence factor, facilitating progression from the acute to persistent phase of infection.RNA processing ͉ salivary gland ͉ viral pathogenesis H uman cytomegalovirus (HCMV) is a member of the ␤-herpesvirus family and a widespread human pathogen (1). Primary HCMV infection is generally asymptomatic in healthy individuals, and, after limited acute replication, virus replication persists in epithelial cells of the salivary gland, mammary gland, and kidney. These sites of persistence allow the virus to spread to new hosts via saliva, breast milk, and urine (2).During productive infection, HCMV genes are expressed in an organized cascade of transcription (1). Immediate-early genes are expressed first and encode proteins that establish a cellular environment conducive to viral replication. One immediateearly transcript, known as the 5-kb RNA (3-6), is not known to encode a protein. We have demonstrated that this RNA is an intron that accumulates in the nuclei of infected cells (5).The genomic sequence from which the 5-kb RNA is expressed resides between the UL105 and UL112͞113 loci (Fig. 1A) and has an AT content of Ϸ60%, considerably higher than that of the overall HCMV genome. Six ORFs (UL106-UL111), ranging in length from 93 to 153 aa, have been annotated in the region spanned by the 5-kb RNA, and numerous stop and start codons define additional small ORFs (7). Although the DNA sequences of the 5-kb loci from laboratory-adapted and clinical isolates of HCMV are well conserved, the predicted ORFs vary considerably because of single-nucleotide changes, insertions, and deletions (8, 9). There is no compelling evidence that any one of these ORFs encodes a protein. A polyadenylated 1.1-kb RNA (10) is generated by splicing the 5-kb intron from its primary transcript (5), and it is predicted to encode a 31-aa polypeptide that bears no similarity to other known proteins. A variant of the AD169 strain of HCMV unable to produce the 5-...

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Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5 end of the 5-kb RNA to a consensus splice donor site and localized the 3 end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5 splice donor site that defines the 5 end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV.Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family (reviewed in reference 24). Infection is widespread and generally asymptomatic in healthy individuals. The virus infects a wide variety of cell types such as endothelial cells, fibroblasts, smooth muscle cells, macrophages, and cells of the bone marrow. Following primary exposure, HCMV establishes a lifelong latent infection in the host and latent virus is found in cells of the myeloid lineage (23, 36). Primary or reactivated HCMV infections can cause severe disease in AIDS patients and transplant patients, including retinitis and pneumonia, and are associated with acute organ rejection (28). Congenital HCMV infections are the leading cause of virally induced birth defects, and neonates can suffer life-threatening complications following HCMV infection.During productive infection, HCMV genes are expressed in an organized cascade of immediate-early, early, and late transcription (reviewed in reference 24). The immediate-early gene products serve to establish a cellular environment conducive to viral replication, functioning largely in transcriptional regulation and immune evasion. The expression of early and late genes is dependent upon the activities of the immediate-early gene products. Immediate-early transcripts originate from several loci in the HCMV genome. The most abundant transcripts are derived from the UL123 and UL122 genes, encoding the family of IE1 and IE2 proteins, respectively. The UL36-38, UL115-119, UL69, TRS1, IRS1, and US3 genes encode the remaining immediate-early proteins. Together, the immediateearly genes of HCMV encode a variety of proteins with diverse regulatory activities, serving essential and dispensable functions in cultured cells during HCMV replication.An additional immediate-early t...

Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryILIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-a-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sulficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain