Second, all screens were carried out in duplicate to determine whether the results were reproducible. Third, prior to any matings in our experiments, all of the binding domain-fusion proteins have been tested for autoactivation and those positive ones were subsequently removed from this study. Fourth, we have employed a high stringency selection in the YTH analysis by utilizing 4 different reporters to ensure the validity of the data. We also observed similar growth of the diploid yeast cells representing the positive interactions. Any protein that resulted in histidine-positive growth with the empty vector controls was classified as a false positive. Fifth, many of the identified interactions were further examined by expressing these proteins in human cells. Whether they associate with each other in human cells was investigated by co-IP experiments. To our best knowledge, 58 of these 79 interactions found in our YTH screens have not been reported previously while 17 have been found in HCMV and 4 in other herpesviruses. Furthermore, all the previously identified 21 interactions and 24 of the new 58 interactions were positive in co-IP experiments, suggesting the presence of these interactions in human cells. The identified interactions can be classified into four different categories based on the locations of these viral proteins in the virion. However, the identity of the tegument densities has not been clearly determined. So far only one interaction between HCMV tegument proteins and capsid proteins has been reported. Gibson and coworkers have shown that UL32 can bind to intranuclear HCMV capsids via its amino-third domain. Diperodon Previous studies using YTH screens as well as our results here failed to detect interactions of UL32 with any HCMV capsid proteins. This may possibly be because the region of the capsid protein that interacts with UL32 is only folded properly when the protein is present in the capsid, and therefore, the interactions between this protein and UL32 can not be detected by YTH screens as these proteins are expressed in the absence of capsid formation. Our YTH and co-IP analyses provide the first direct evidence of potential interactions between tegument protein UL24 and capsid proteins UL46 and UL85. The UL24 protein has been detected in the tegument. Gene-deletion analyses indicate that UL24 is not essential for viral growth in human foreskin fibroblasts but is important for viral replication in microvascular endothelial cells. However, its exact function is currently unknown. It is possible that UL24 may function as a ”hub” to connect viral capsids to other tegument proteins that it interacts with, and facilitate tegument formation by recruiting other tegument proteins to the capsid. We note that our results failed to detect a number of interactions between HCMV virion proteins that have been previously identified by YTH analysis, including the seven interactions reported recently. This may be due to the difference in the yeast strains, activation/DNA binding domain vectors, and the selection medium/reporters that were used for the studies. Little overlap of interactions identified by different research groups has been observed in the YTH studies of other systems including herpesviruses such as EBV and KSHV. There are also a number of previously reported interactions between viral envelope proteins that were not detected in our Labetalol hydrochloride current study. This may not be surprising as most of the viral envelope proteins may not be folded properly in our YTH screen system, and therefore, may not possess the correct conformations for binding and interactions. Moreover, YTH systems that test for protein interactions in the nucleus are prone to false negatives due to the expression of proteins that are normally not found in the nuclear environment. These issues can be resolved by using affinity purification assays to detect interactions in vivo. However, co-IP experiments can only detect relatively stable, high affinity interactions while the YTH methods reveal transient, weak interactions.