Polybrene (Sigma-Aldrich, MO, USA) was added to the cell mixture (8 g/mL final concentration), and cells were placed into a 96-well plate. deficiency), KSHV BAC16, and its revertants. The results revealed that both ORF7-KO KSHVs showed significantly reduced viral production but there was no effect on lytic gene expression and viral genome replication. Complementation assays showed virus production from cells harboring ORF7-KO KSHV could be recovered by ORF7 overexpression. Additionally, exogenously expressed ORF7 ZK-261991 partially induced nuclear relocalization of the other terminase components, ORF29 and ORF67.5. ORF7 interacted with both ORF29 and ORF67.5, whereas ORF29 and ORF67.5 failed to interact with each other, suggesting that ORF7 functions as a hub molecule in the KSHV terminase complex for interactions between ORF29 and ORF67.5. These findings indicate that ORF7 plays a key role in viral replication, as a component of terminase. ZK-261991 subfamily [1,2]. KSHV was first discovered in 1994 in a Kaposis sarcoma lesion from a patient with AIDS [1]. It was subsequently found to be closely associated with Kaposis sarcoma, primary effusion lymphoma (PEL), multicentric Castlemans disease, and KSHV-associated inflammatory cytokine syndrome [2,3,4,5]. KSHV establishes a life-long infection in human B-cells or vascular endothelial cells and exists in either a latent or lytic state. During latent infection, the KSHV genome circularizes to form an episome in the nucleus and expresses several latent-associated gene products, which contribute to the promotion of cell proliferation and anti-apoptosis activity [6]. KSHV shifts its life cycle from a latent to a lytic infection by the expression of a replication and transcription activator (RTA/ORF50). During the lytic cycle, most lytic-related genes are expressed in the infected cell, and virions are assembled and then egress from the infected cell. As with other herpesviruses, KSHV virions consist of a linear, double-stranded DNA genome enclosed within an icosahedral capsid shell, tegument proteins, and a lipid bilayer envelope spiked with glycoproteins [7]. The fundamental structure of virus particles and the process of viral capsid formation are thought to be analogous among the human herpesviruses [8]. The process of KSHV encapsidation in an infected cell nucleus remains largely unknown, whereas in herpes simplex virus 1 (HSV-1) it is well understood [9,10]. KSHV genes correspond to homologs in HSV-1; their common names are listed in Table 1. In the case of HSV-1, the first step in capsid formation is the assembly in the nucleus of the procapsid, which consists of an external and an internal capsid shell [9,10]. A major capsid protein and an assembly protein form a heterodimer; these heterodimers then self-assemble to form a procapsid. The external capsid proteins (containing a large amount of major capsid proteins, small capsid proteins, triplex proteins, portal capping proteins, and more) form the spherical shell (i.e., the external capsid shell) of a procapsid. The internal capsid proteins (assembly proteins and considerably lower amounts of previral protease) form the spherical internal backbone (i.e., the internal capsid shell) of a procapsid. The next step in encapsidation is the viral protease-mediated processing of internal scaffold proteins of the procapsid. The viral protease cleaves the C-terminal region of assembly proteins and eliminates the internal capsid proteins (assembly proteins) from the interior cavity of the procapsid [11]. Thereafter, a spherical external ZK-261991 capsid shell causes a conformational change in the capsids morphology, and it becomes an icosahedral capsid [12,13]. Table 1 Capsid and capsid-related molecules of HSV-1 and KSHV. Asterisk (*) indicates PPP3CC one nucleoside deletion site. Lowercase, underlined uppercase indicates mutagenesis site, and uppercase indicates pEP-KanS sequence. Uppercase indicates restriction enzyme site. 2.2. Construction of ORF7-KO KSHV BAC16 Wild-type (WT) KSHV BAC16 (WT-BAC16) was kindly provided by Jae U. Jung. ORF7-KO recombinant KSHV BAC16 (frameshift-induced ORF7-BAC16 (FS-ORF7-BAC16) and stop codon-induced ORF7 (ST-ORF7-BAC16)) were generated from WT-BAC16 using a two-step markerless Red recombination system [25,26,27]. Its revertants, ST Rev-BAC16 and FS Rev-BAC16, were generated from ST-ORF7-BAC16 and FS-ORF7-BAC16, respectively. This mutagenesis of BAC clones was performed according to previously described protocols [25], using the mutagenesis primers shown in Table 2. Insertions and deletions of kanamycin-resistance cassettes (KanR) in each mutant were analyzed by digestion of BglII and agarose gel electrophoresis. ORF7 and mutated sites in each BAC clone (ORF7 coding region (nt6609Cnt8696; accession number: “type”:”entrez-nucleotide”,”attrs”:”text”:”GQ994935″,”term_id”:”261853473″GQ994935)) were confirmed by Sanger sequencing. 2.3. Establishment of Doxycycline-Inducible Recombinant KSHV-Expressing Cells To obtain efficient recombinant.