The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.

The herpes simplex virus type 1 (HSV-1) UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component or CCSC. Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid binding domain of UL25 located on the surface of the mature DNA-containing capsid.

The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes.The herpes simplex virus 1 (HSV-1) virion consists of a 152-kbp double-stranded DNA (dsDNA) genome that is enclosed in an icosahedral capsid, which is itself surrounded by an amorphous protein layer called the tegument, and a lipid envelope containing viral glycoproteins. Viral DNA replication generates concatemers of head-to-tail genomes in the nucleus of the infected cell (26,27). DNA replication is concurrent with the expression of HSV late genes, including the capsid structural proteins VP5 (UL19), VP19c (UL38), VP23 (UL18), and VP26 (UL35) (reviewed in reference 24). Using the scaffold proteins encoded by the HSV-1 UL26 and UL26.5 genes, the capsid structural proteins self-assemble into a spherical capsid precursor, or procapsid (19,31,36). Packaging of viral genomes into procapsids requires several tightly coupled events: (i) proteolysis and expulsion of the scaffold proteins, (ii) recognition and cleavage of viral genome termini by the terminase complex, (iii) docking of the terminase-DNA complex on the capsid portal, (iv) insertion of DNA into the procapsid, and (v) sealing of the portal. The product of DNA packaging is an angular capsid that contains tightly packed viral DNA (2, 10). The mature capsid, also known as a C capsid, then exits the nucleus and is incorporated into virions.In the absence of successful packaging, two capsid forms accumulate in the nuclei of infected cells. B capsids contain remnants of the cleaved scaffold proteins VP21, VP22a, and VP24, and A capsids are empty (10). In the current model of HSV DNA packaging, B and A capsids form when DNA packaging is termina...

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.DNA packaging is a critical step in the lytic replicative cycle of herpes simplex virus type 1 (HSV-1). During the packaging reaction, replicated DNA is resolved from branched concatemers to single-unit-length genomes that are inserted into preformed capsids (10, 31). Three types of capsids are found in the nuclei of cells infected with HSV-1: A (empty), B (intermediate), and C (full) (reviewed in reference 14). The three are distinguishable morphologically in electron micrographs, and they can be separated from each other preparatively by sucrose density gradient ultracentrifugation. They differ in the material present inside the capsid cavity. C capsids contain the virus DNA, and A and B capsids lack DNA. The B capsid cavity is filled primarily with VP22a, the cleaved form of the scaffolding protein, while in A capsids, the cavity lacks both DNA and protein. While B capsids may form after spontaneous maturation of the procapsid, empty A capsids result from abortive DNA packaging (39). Seven HSV-1 genes have been identified that are required for DNA encapsidation (1, 2, 21-23, 27, 30, 34). Null mutants of the UL6, UL15, UL17, UL28, UL32, and UL33 genes produce only B capsids, and these mutations block cleavage of viral genomes from the replicated concatemer. In contrast, disruption of the UL25 gene results in the accumulation in the nucleus of both A and B capsids in addition to unpackaged, genome-length DNA (23, 33). Thus, UL2...

Ejection of DNA from the capsid is an early step in infection by all herpesviruses. Ejection or DNA uncoating occurs after a parental capsid has entered the host cell cytoplasm, migrated to the nucleus and bound to a nuclear pore. DNA exits the capsid through the portal vertex and proceeds by way of the nuclear pore complex into the nucleoplasm where it is transcribed and replicated. Here we describe use of an in vitro uncoating system to determine which genome end exits first from the herpes simplex virus (HSV-1) capsid. Purified DNA-containing capsids were bound to a solid surface and warmed under conditions in which some, but not all, of the DNA was ejected. Restriction endonuclease digestion was then used to identify the genomic origin of the ejected DNA. The results support the view that the S segment end exits the capsid first. Preferential release at the S end demonstrates that herpesvirus DNA uncoating conforms to the paradigm in dsDNA bacteriophage where the last end packaged is the first to be ejected. Release of HSV-1 DNA beginning at the S end causes the first gene to enter the host cell nucleus to be α4, a transcription factor required for expression of early genes.