Moreover, the orientation and interaction of HCMV proteins in the virion have not been extensively studied. Much of what is currently known about the interactions among herpesvirus capsid and tegument proteins come from various studies involving protein assays and in particular, YTH analyses. Large-scale YTH analyses have also been applied to interactome studies of many organisms such as Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Plasmodium falciparum, andHelicobacter pylori. In complex organisms where their genome sizes are Albaspidin-AA relatively large, it is difficult to assess the importance of each individual protein to the systems. Therefore, it is important to map the global interactome to assess the true significance of each protein. Global genetic YTH analysis was also used to study the interactions between proteins encoded by vaccinia virus and five herpesviruses, which include herpes simplex virus 1, Varicella-zoster virus, EpsteinBarr virus, Gomisin-D murine cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. Furthermore, the potential interactions among 5 capsid proteins and 28 tegument proteins of HCMV have recently been investigated using the YTH approach. These results have provided significant insights into the interactions among proteins encoded by herpesviruses. In this study, we have carried out a comprehensive YTH analysis to identify potential interactions among 56 HCMV virion proteins, which include 5 capsid proteins, 33 tegument proteins, and 18 envelope proteins. We have identified 79 pairs of potential interactions that are involved in viral capsid proteins, tegument proteins, and envelope proteins. Of the 79 interactions, 58 have not been previously identified to the best of our knowledge, while 21 of them have been reported. Forty-five of these 79 putative interactions were also identified in human cells by co-immunoprecipitation experiments. Our results indicate the presence of several HCMV proteins that serve as ”hubs” for interactions with numerous protein partners, thereby may function as an organizing center for connecting viral proteins in the mature virion and for recruiting other virion proteins. The interactions identified in this study provide a framework to study potential interactions between HCMV proteins and to investigate the functional roles of protein-protein interactions in HCMV virion assembly. We then used the locally written Unix-based scripts or automation of GCG package to analyze the obtained TowneBAC sequence and determine the coding sequences for ORFs that are 100 codons or longer. Each ORF was compared with the set of ORFs that had been predicted or found in all the HCMV strains for which sequences have been determined. This analysis suggested that the TowneBAC sequence encodes at least 150 ORFs with 100 amino acids or longer, and that all these ORFs align with those found in other HCMV strains. We initially selected an optimal PCR primer pair for each ORF. The primer pairs used for amplification of the viral sequences were constructed as follows. The forward primer contained the sequence immediately after the predicted translation initiation codon and 20�C25 additional nucleotides of coding sequence. The reverse primer contained the reverse complement of both the predicted translation termination codon and the preceding 20�C25 nucleotides at the end of the ORF. In addition, these primers also contain 15�C20 nucleotide common sequences that contain sites for restriction enzymes for cloning of the PCR products into the YTH screen vectors and the mammalian expression vectors. Each ORF encoding HCMV virion proteins was amplified individually by PCR. The amplified PCR products covered the entire ORFs minus the translation initiation codon, and were cloned into both the yeast two-hybrid screen ”prey” pGADT7 and ”bait” pGBKT7 vectors. We generated a collection of 118 constructs that 
contained the sequences of the 56 HCMV ORFs, including those coding for exons 1 and 2 of UL89, the amino and carboxyl domains of UL48, and exons 1 and 2 of UL112.