The viral tegument is a layer of proteins between the herpesvirus capsid and its outer envelope. According to phylogenetic studies, only a third of these proteins are conserved amongst the three subfamilies (Alpha-, Beta- and Gammaherpesvirinae) of the family Herpesviridae. Although some of these tegument proteins have been studied in more detail, the structure and function of the majority of them are still poorly characterized. VP22 from Herpes simplex virus 1 (subfamily Alphaherpesvirinae) is a highly interacting tegument protein that has been associated with tegument assembly. We have determined the crystal structure of the conserved core domain of VP22, which reveals an elongated dimer with several potential protein–protein interaction regions and a peptide-binding site. The structure provides us with the structural basics to understand the numerous functional mutagenesis studies of VP22 found in the literature. It also establishes an unexpected structural homology to the tegument protein ORF52 from Murid herpesvirus 68 (subfamily Gammaherpesvirinae). Homologues for both VP22 and ORF52 have been identified in their respective subfamilies. Although there is no obvious sequence overlap in the two subfamilies, this structural conservation provides compelling structural evidence for shared ancestry and functional conservation.

Kaposi’s sarcoma-associated herpesvirus encodes four viral homologues to cellular interferon regulatory factors (IRFs), where the most studied is vIRF-1. Even though vIRF-1 shows sequence homology to the N-terminal DNA-binding domain (DBD) of human IRFs, a specific role for this domain in vIRF-1’s function has remained uncertain. To provide insights into the function of the vIRF-1 DBD, we have determined the crystal structure of it in complex with DNA and in its apo-form. Using a thermal stability shift assay (TSSA), we show that the vIRF-1 DBD binds DNA, whereas full-length vIRF-1 does not, suggesting a cis-acting regulatory mechanism in similarity to human IRFs. The complex structure of vIRF-1 DBD reveals interactions with the DNA backbone and the positioning of two arginines for specific recognition in the major grove. A superimposition with human IRF-3 reveals a similar positioning of the two specificity-determining arginines, and additional TSSAs indicate binding of vIRF-1 to an IRF-3 operator consensus sequence. The results from this study, therefore, provide support that vIRF-1 has evolved to bind DNA and plays a role in DNA binding in the context of transcriptional regulation and might act on some of the many operator sequences controlled by human IRF-3.

Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcomaassociated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49 KSHV ) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49 KSHV protein to a resolution of 2.4 Å. The ORF49 KSHV protein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49 KSHV protein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49 KSHV protein is stabilized in a dose-responsive manner by doublestranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49 KSHV protein binds these oligonucleotides. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49 KSHV ). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49 KSHV protein. This led to the discovery of novel DNA binding properties of the ORF49 KSHV protein. Evolutionary conserved structural elements with the functional homologs of ORF49 KSHV were also established with the structure.

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS) in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors.