Cloning of HCMV ORFs for yeast two-hybrid analysis

We have previously determined the genomic sequence of a HCMV Towne strain that was cloned into a bacterial artificial chromosome (BAC) vector [32] and was maintained as a single copy BAC-based plasmid in E.coli [7]. When introduced into human cells, the viral DNA sequence of this construct, Towne_BAC, produces infectious progeny and retains wild type growth characteristic in vitro. These results suggested that the genome sequence of Towne_BAC encodes a collection of functional viral genes responsible for viral replication and infection [7], [33].

We then used the locally written Unix-based scripts or automation of GCG package to analyze the obtained Towne_BAC 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 [5], [6], [7], [33], [34], [35]. This analysis suggested that the Towne_BAC 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 [7] (data not shown).

To study potential interactions among HCMV-encoded proteins in viral infectious particles, we cloned and expressed, in the yeast-two hybrid system, 56 HCMV ORFs that encode proteins that have been identified as virion components by mass spectrometry [12], [13]. Those cloned and expressed ORFs include 5 capsid proteins (UL46, UL48.5, UL80, UL85, and UL86), 33 tegument proteins (UL24, UL25, UL26, UL32, UL43, UL47, UL48 cloned individually as N′ and C′-terminal truncated fragments, UL82, UL83, UL94, UL99, US22, US23, US24, UL44, UL45, UL54, UL57, UL69, UL72, UL84, UL89 exon 1 (UL89.1)/UL89 exon 2 (UL89.2), UL97, UL122, UL35, UL51, UL71, UL79, UL88, UL96, UL103, UL104, and UL112 exon 1 (UL112.1)/exon 2 (UL112.2)), and 18 envelope proteins (RL10, UL5, UL22A (also called UL21.5), UL33, UL38, UL41A, UL50, UL55, UL73, UL74, UL75, UL77, UL93, UL100, UL115, UL119, UL132, US27).

We initially selected an optimal PCR primer pair for each ORF (Supporting Information Table S1). The primer pairs used for amplification of the viral sequences were constructed as follows. The forward primer contained (from 5′ to 3′) the sequence immediately after the predicted translation initiation codon and 20–25 additional nucleotides of coding sequence. The reverse primer contained the reverse complement of both the predicted translation termination codon and the preceding 20–25 nucleotides at the end of the ORF (Supporting Information Table S1). In addition, these primers also contain 15–20 nucleotide common sequences that contain sites for restriction enzymes for cloning of the PCR products into the YTH screen vectors (pGADT7 and pGBKT7) and the mammalian expression vectors (pCMV-HA and pCMV-Myc) (Supporting Information Table S1).

Each ORF encoding HCMV virion proteins was amplified individually by PCR. The amplified PCR products covered the entire ORFs minus the translation initiation codon (Figure 1), 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 (Figure 1, Supporting Information Table S2). The viral ORF sequences in these constructs were confirmed using DNA sequencing analysis (data not shown).

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Figure 1. HCMV ORF sequences generated by PCR and cloned in yeast and mammalian expression vectors.

(A) The PCR products for the HCMV ORFs separated on 1% agarose gels. (B) The predicted sizes of the PCR products for the HCMV ORFs based on the PCR primers used.

https://doi.org/10.1371/journal.pone.0017796.g001

Identification of potential interactions among HCMV-encoded virion proteins using YTH analysis

Our strategy was to test each pair-wise combination of HCMV-encoded virion proteins in the YTH assay using a 48-well protein array format. The array consisted of a set of yeast transformants, each expressing a HCMV ORF as a hybrid protein with the Gal4 DNA binding domain (BD). This array was mated to yeast transformants of the opposite mating type carrying one of the HCMV ORFs as a hybrid protein with the Gal4 activation domain (AD). The resulting diploids were cultured on the medium selective for the two-hybrid reporter genes. In this approach, a single protein could be tested for binary interaction with the protein encoded by every cloned HCMV ORF in the array. By generating a different set of activation and DNA binding domain hybrids with HCMV virion proteins, we were able to carry out the mating and selection experiments to assay all of the ∼3500 combinations (Figure 2, Supporting Information Table S2).

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Figure 2. Protein-protein interactions between HCMV ORFs as identified by YTH analysis.

In (A–B), a directional interaction is shown with the interaction existing only in BDUL86-ADUL48.5 (B) but not in BDUL48.5-ADUL86 (A). Interactions detected only in one direction are common in YTH assays and are most likely due to steric hindrance of either bait or prey fusion proteins [21], [22], [23]. In (C–D), an example of reciprocal interactions is shown with BDUL24-ADUL69 and the reverse combination. In (E–F), BDUL-Lam and BDUL-p53 are the human lamin C and murine p53 proteins fused to the BD domain of GAL4, respectively, while ADUL-T is the SV40 large T-antigen fused to the AD domain of GAL4. Murine p53 and SV40 T-antigen interact and serve as a positive control (F) while human lamin C and SV40 T-antigen do not interact and serve as a negative control (E). Diploids were plated on SD/-Leu/-Trp/-Ade/-His/X-α-Gal agar plates to test for protein-protein interactions. Positive interactions had blue yeast colony growth (B, C, D, and F), whereas no interactions were shown by the absence of growth (A and E). We observed similar growth of the diploid yeast cells representing the positive interactions.

https://doi.org/10.1371/journal.pone.0017796.g002

To generate the yeast transformants for YTH analysis, DNAs from each of the 59 generated “bait” pGBKT7-derived constructs were isolated from E.coli, and transformed into yeast strain AH109, resulting in a collection of different yeast transformants that expressed a set of 59 DNA-binding domain (BD) hybrid proteins (Supporting Information Table S2). Similarly, DNAs from the generated “prey” pGADT7-derived constructs were isolated and transformed into yeast strain Y187 to generate yeast transformants expressing a set of 59 activation domain (AD) hybrid proteins.

Prior to performing mating experiments, all 59 pGBKT7-fusion proteins in AH109 strains were tested for autoactivation. Seven AH109 strains, which contained ORFs UL48.5, UL26, UL48N, UL48C, UL94, US23, and UL51, were determined to be autoactivators in the absence of any pGADT7-cloned ORFs (Table 1), and subsequently eliminated from any further mating experiments. Each of the remaining 52 AH109 strains was allowed to mate with each of the 59 constructed Y187 strains in duplicate, and the diploid cells were plated in duplicate on selective medium. Besides selecting for the presence of the two plasmids based on leucine and tryptophan selection, we employed a high stringency selection of positive protein interactions by using 3 more reporter genes to eliminate potential false positives: histidine, adenine, and Mel1, which encodes alpha-galactosidase for blue/white screening. The growth of colonies of the diploid yeast cells with a blue staining within 3 weeks after plating was scored as a positive pair-wise interaction between the two proteins tested while other results (e.g. no growth of yeast colonies) were scored as a negative interaction (Figure 2, Supporting Information Table S2). There was no significant difference in the growth of the diploid yeast cells representing all the positive interactions (data not shown).

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Table 1. Protein-protein interactions of HCMV virion proteins that have either been previously reported or suggested, and were positive in our YTH and co-IP analysis.

https://doi.org/10.1371/journal.pone.0017796.t001

Two-hybrid screens may generate a significant number of false positives that represent random generation of histidine-positive colonies. This is possibly due to rearrangement and deletion of the DNA-binding domain plasmids, recombinational events between the DNA-binding and activation domain plasmids, or genomic rearrangement of the host strain. To exclude these possibilities, three sets of experiments were carried out. First, plasmid DNAs from the yeast transformants were recovered and analyzed. Our results indicated that these constructs were present in all transformants examined and exhibited similar restriction enzyme profiles as those in E. coli. Second, all screens were carried out in duplicate to determine whether the results were reproducible. Third, any protein that resulted in histidine-positive growth with the empty vector controls (i.e. pGADT7 and pGBKT7) was classified as a false positive.

The >3000 combination screen of the mating of 52 HCMV Gal4 DNA-binding and 59 HCMV Gal4 activation domain fusion proteins revealed 79 positive interactions among 37 virion proteins (Supporting Information Table S2 and Figure 3). We identified 4 interactions between capsid proteins, 2 interactions between capsid proteins and tegument proteins, 58 interactions between tegument proteins, 12 interactions between tegument proteins and envelope proteins, and 3 interactions between envelope proteins (Table 1 and 2). The 37 participating proteins include all 5 capsid proteins (UL46, UL48.5, UL80, UL85, and UL86), 27 tegument proteins (UL24, UL25, UL26, UL32, UL43, UL47, UL48N/UL48C, UL82, UL83, UL94, UL99, US22, US23, US24, UL44, UL45, UL54, UL69, UL72, UL89.2, UL122, UL35, UL51, UL71, UL88, UL103, UL112.1), and 5 envelope proteins (UL22A, UL38, UL50, UL77, UL132). Of these 79 identified interactions, 58 are novel (Table 2) while the other 21 have previously been reported (Table 1).

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Figure 3. Interaction map of potential interactions between HCMV virion proteins as revealed by YTH analysis.

The shaded hexagon represents the capsid and the shaded sphere represents the viral particle. The nodes (in yellow) within the capsid represent capsid proteins. The circular nodes in blue represent tegument proteins. The rectangular nodes in orange represent viral envelope proteins. The green lines represent interactions identified in one direction only and the red lines represent reciprocal interactions. The solid lines represent the interactions that were positive in both the YTH analysis and co-IP experiments, while the dashed lines represent the putative interactions that were positive only in the YTH analysis. The ORFs in which deletion resulted in no viral growth, wildtype-like growth, and significant growth defect in human foreskin fibroblasts are marked as “E” (essential), “NE” (non-essential), and “D” [7].

https://doi.org/10.1371/journal.pone.0017796.g003

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Table 2. Putative protein-protein interactions of HCMV virion proteins that have not been previously reported and were determined by our YTH and co-IP analysis.

https://doi.org/10.1371/journal.pone.0017796.t002

Twelve proteins, which include 2 capsid proteins (UL80 and UL85), 9 tegument proteins (UL24, UL25, UL82, UL69, UL89, UL122, UL71, UL103, and UL112), and 1 envelope protein (UL77), were found to be self-interacting. Previous analyses have shown the self-interaction of UL80, UL85, UL25, UL69, and UL112 [10], [15], [20], [36]. These results suggest that all 12 proteins may form dimers or self-assemble into polymers. These notions are consistent with previous observations that UL85 is the minor capsid protein that can self-assemble into pre-caspid particles and that UL80 self-interacts and forms a dimer (Table 1) [10], [15].

Of the 79 interactions identified, 11 of them were reciprocal interactions that were tested and confirmed in both orientations in the YTH assay (Supporting Information Table S2, Figures 2–3), strongly supporting the validity of these interactions. Excluding the 12 self-interactions and 11 reciprocal interactions, the remaining 56 interactions were detected in only one direction. Interactions detected only in one direction are common in YTH analysis and most likely are due to steric hindrance of either bait or prey fusion proteins [21], [22]. Among these 79 interactions, 18 were found to be involved with UL24 while 11 and 10 were with UL25 and UL89.2, respectively (Figure 3, Table 1 and 2). These results raise the possibility that these proteins may function as a hub or organizing center for connecting viral proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. UL24, the only tegument protein identified in this study to interact with a capsid protein (e.g. UL46), may be especially important in this process.

Expression of HCMV virion proteins and investigation of their interactions in human cells

A co-immunoprecipitation (co-IP) assay was employed to validate the yeast two-hybrid approach and to determine whether the identified interactions occur in human cells. Figure 4 shows the results of the co-IP experiments that examined six of the interactions identified in the YTH analysis, UL24-UL25, UL69-UL69, UL82-UL94, UL83-UL25, UL99-UL94 and its reciprocal, UL94-UL99 (lanes 1–6). Two pair interactions, UL99-UL83 and UL25-UL94, which showed no interactions in our YTH screen, were also included as control experiments (lanes 7 and 8). In these experiments, the HCMV ORFs were cloned into mammalian expression vectors pCMV-Myc and pCMV-HA, in which each ORF was expressed as either a fusion protein with a carboxyl terminal c-myc or HA epitope tag, respectively. HeLa and U373MG cells were transfected with the mammalian expression constructs and were harvested at 72 hours. To detect the expression of these proteins, transfected cells were lysed and proteins were separated electrophoretically on SDS-containing gels, transferred electrically to membranes, and reacted with anti-HA and anti-c-myc antibodies. UL24, UL25, UL69, UL82, UL83, UL94 and UL99 were detected as proteins of about 40, 80, 90, 70, 70, 40, and 25 kD, respectively (data not shown, Figure 4), consistent with their coding sequences of 358, 656, 743, 559, 561, 345, and 190 amino acids, as predicted from the HCMV Towne_BAC sequence [7]. The protein species, which migrated at approximately 82 kDa and was detected by the anti-HA antibody in the input protein samples of the cell lysate (Figure 4A) but not in the protein samples immunoprecipitated with either the anti-HA or anti-myc antibodies (Figure 4C and E), may represent a cellular protein that non-specifically reacts with the anti-HA antibody.

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Figure 4. Co-immunoprecipitation (co-IP) of transiently expressed viral proteins.

HeLa cells were co-transfected with a combination of two plasmids expressing HA- and myc-tagged proteins. Panels (A) and (B) are inputs that show the expression of HA- and myc-tagged proteins detected by Western blot analysis with anti-HA and anti-myc antibodies, respectively. Lanes 1 and 9 are from the same cell lysate, likewise, lanes 2 and 10, and so on. Lanes 7, 8, 15, and 16 are negative controls based on our YTH data that revealed no interactions. Panels (C) and (D) represent co-IP experiments (IP-anti-HA) with anti-HA agarose antibodies and visualized by Western blot analysis using anti-HA and anti-myc antibodies, respectively. Panels (E) and (F) are co-IP experiments (IP-anti-myc) with anti-myc agarose antibodies (IP-anti-Myc) and visualized by Western blot analysis using anti-HA and anti-myc antibodies, respectively. The protein species, which migrated at approximately 82 kDa and was detected by the anti-HA antibody in the input protein samples of the cell lysate (A) but not in the protein samples immunoprecipitated with either the anti-HA or anti-myc antibodies (C and E), may represent a cellular protein that non-specifically reacts with the anti-HA antibody.

https://doi.org/10.1371/journal.pone.0017796.g004

The six interactions revealed by our YTH analysis appeared to be identified in the co-IP experiments (Figure 4, lanes 1–6). As an example, to determine whether UL25 and UL94 interact with UL83 and UL99 respectively, protein lysates from the transfected cells were either first immunoprecipitated with anti-HA or anti-c-myc, and then immunoblotted with antibodies against the c-myc and HA epitope tags. The HA-tagged UL25 was co-precipitated with the myc-tagged UL83 (Figure 4C–F, lane 5 and 13), while the HA-tagged UL99 was found to be associated with the myc-tagged UL94 and vice versa (lane 3–4 and 11–12). In contrast, we observed, in control experiments, neither significant binding between the HA-tagged UL99 and the myc-tagged UL83 (lane 7) nor between the HA-tagged UL25 and the myc-tagged UL94 (lane 8). These results confirm the specificity of the co-IP assay and suggest that the UL25-UL83 and UL99-UL94 interactions may occur in human cells. These results are consistent with recent report of interactions between UL69-UL69, UL82-UL94, and UL99-UL94 revealed by YTH and co-IP analysis [20]. Similar co-IP experiments were carried out to study each of the 79 putative interactions identified in the YTH screens in human cells. Forty five of these interactions were positive in our experiments while 34 were negative (Table 1 and 2).

(A) Interactions of viral capsid proteins

Extensive studies have been carried out to investigate the interactions between capsid proteins [10], [11]. Our YTH screens coupled with co-IP experiments revealed four interactions, all of which have been previously identified [15], [16], [17], [18], [19], [20], [42], validating our approach for identifying protein-protein interactions.

(B) Interactions between viral capsid and tegument proteins

Both the YTH and co-IP analyses revealed novel potential interactions of tegument protein UL24 to capsid protein 46 and UL85 (Table 2), which have not been reported previously. It is believed that tegument proteins specifically interact with capsid proteins to initiate the formation of the tegument and to form mature infectious virions. By cryo-EM, an icosahedrally ordered tegument layer was visualized in HCMV particles when compared to precursor capsids prior to DNA encapsidation [8], [9]. Furthermore, distinct tegument densities were observed to interact with all of the structural components of the nucleocapsid. Possible functions of these capsid-proximal organized tegument proteins may be involved in serving as a nucleus for further tegument assembly. 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 [43]. 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 [43].

Our YTH and co-IP analyses provide the first direct evidence of potential interactions between tegument protein UL24 and capsid proteins UL46 and UL85 (Table 2 and Supporting Information Table S2, Figure 3). The UL24 protein has been detected in the tegument [12], [44]. 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 [7]. 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.

(C) Interactions among tegument proteins

It is generally believed that tegument proteins interact with each other as well as with human proteins to maintain the structure of the tegument and play an important role in recruiting proteins during tegument formation. Nineteen specific interactions among HCMV tegument proteins have been reported and include the self-interaction of UL25, UL44, UL45, UL69 and UL112, and the interaction of UL25-UL26, UL32-UL35, UL32-UL82, UL35-UL82, UL43-UL83, UL45-UL25, UL45-UL69, UL82-UL94, UL88-UL48, UL99-UL94, UL32-UL45, UL48-UL45, UL69-UL88, UL94-US22 [20], [22], [36], [39], [45].

Of the 79 interactions identified by our YTH analysis, 58 (>73%) are between tegument proteins (Table 1 and 2). Our results confirm the previously identified 13 interactions (i.e. self-interaction of UL25, UL69, and UL112; and interaction of UL25-UL26, UL32-UL35, UL32-UL82, UL35-UL82, UL43-UL83, UL45-UL25, UL45-UL69, UL82-UL94, UL88-UL48, and UL99-UL94) (Table 1). Furthermore, these results suggest the presence of 45 possible interactions between tegument proteins, 21 of which were also positive in co-IP experiments (Table 2). The salient features of these interactions are as follows:

1. Both YTH and co-IP experiments identified 7, 9, and 5 interactions that were involved with UL24, UL25, and UL89.2, respectively. Thus, UL24, UL25, and UL89 may function as a hub or organizing center for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly.
2. Previous studies have suggested that proteins of similar functions may interact with each other to achieve their function synergistically and co-operatively [21], [22], [31]. For example, UL35 and UL82 have been shown to interact with each other, and form a complex that functions to activate viral transcription cooperatively and synergistically [45]. Identifying the binding partners of an unknown protein may shed light into its potential biological function. Excluding those interactions that are involved in UL24, UL25, and UL89.2, which may function to recruit tegument proteins, the majority (24) of the remaining 25 interactions identified in YTH experiments are either between essential genes or between genes that are important for viral replication in human foreskin fibroblasts (Table 1 and 2) [7]. For example, both the YTH and co-IP analysis identified the interactions of UL71, an essential protein, with UL51 and UL89, which are essential for viral DNA genome cleavage and packaging [1], [7], [46]. These results suggest that UL71 may play its essential role in supporting viral DNA encapsidation. Further studies will be carried out to investigate the roles of UL71 and its interactions with UL51 and UL89 in viral replication.

(D) Interactions between viral tegument proteins and envelope proteins and among envelope proteins

Little is currently known about the interactions between viral tegument and envelope proteins. Our YTH analysis suggests 12 potential interactions between tegument proteins and envelope proteins. In particular, the UL24-UL77 and UL132-UL48 interactions were identified by both the YTH and co-IP analyses. Furthermore, novel interactions between envelope proteins, UL22A-UL132 and UL77-UL50, were also identified by both the YTH and co-IP experiments. Both UL77 and UL50 are essential for viral replication [7], [46]. However, the exact function of UL77 is currently unknown while UL50, along with a non-virion protein UL53, functions to dismantle the inner nuclear membrane and facilitate the budding of the nucleocapsid into the nuclear membrane during the initial step of acquiring the viral envelope [47], [48]. It will be interesting to investigate whether UL77 interacts with UL50 and exerts similar function in supporting the budding of the nucleocapsid through the nuclear membrane. UL132 encodes a glycoprotein that may play an important structural function during viral replication in vitro [49]. ORF UL22A, which is also called UL21.5, is believed to encode a RANTES decoy receptor glycoprotein found in the virion [12], [50]. It will be interesting to determine whether UL22A forms a complex with UL132 to exert their functions in supporting viral growth during HCMV infection.

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 (i.e. self-interaction of UL44 and UL45; interaction of UL32-UL45, UL48-UL45, UL69-UL88, UL94-US22, UL86-UL46) reported recently [20]. 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 [21], [22], [23], [24]. There are also a number of previously reported interactions between viral envelope proteins that were not detected in our 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. This is because the dissociation constant (K_d) for a typical YTH assay is 10–100 µM compared to ∼10 mM for co-IP experiments [51]. Fifty-seven percent of the interactions revealed by our YTH analysis was confirmed in co-IP experiments, consistent with previous observations that about 50–60% of the YTH interactions are positive in immunoaffinity pulldown experiments in the studies of other viruses [21], [22], [23], [24], [31]. While we can not completely rule out the possibility that those interactions which are negative in co-IP experiments may not be present in mammalian cells, it is conceivable that these interactions are so transient and weak that they are only detected by the YTH approach but not by the immunoaffinity pulldown assay.

The HCMV virion represents one of the most complex viral particles found in nature. It contains more than 55 HCMV proteins of at least 100 amino acids, and in addition, at least 10 viral-encoded small peptides/proteins of less than 100 amino acids and over 70 human cellular proteins [12], [13]. Of the 56 ORFs we studied, 19 were either not found to interact between themselves or with any other of the 37 HCMV proteins. We can not completely exclude the possibility that there were interactions among themselves or with the other 37 ORFs in human cells that could not be detected by our YTH assays. It is also conceivable that these proteins may interact with the viral encoded small peptides, human proteins, and other constituents of the virion particles (e.g. lipids). Further studies to identify the partners of these proteins and study their potential interactions with the partners will provide insight into the mechanism of HCMV virion assembly and formation, and facilitate the development of novel compounds and new strategies for the treatment and prevention of HCMV infection.

Construction of plasmids

HCMV Towne_BAC (accession no. AY315197) [7] was used as the template for PCR amplification of HCMV ORFs . For genes that encode spliced transcripts, cDNAs from Towne_BAC-infected cells were used as the templates. Table S1 (Supporting Information) lists the primers used for cloning HCMV sequences in the pGBKT7 bait and pGADT7 prey plasmids for expression in yeast and in pCMV-Myc and pCMV-HA for expressing in human cells (Clontech, Mountain View, CA). Each primer sequence contains an outer and inner restriction enzyme site for cloning the HCMV sequences at the multiple cloning site (MCS) of the yeast and mammalian expression plasmids, respectively (Table S1) (Supporting Information). PCR amplification was performed using iProof high-fidelity DNA polymerase (Bio-Rad, Inc., Hercules, CA). The resultant constructs were confirmed by restriction digest profile and sequencing.

Yeast two-hybrid (YTH) analysis

The DNAs of the viral ORFs that were cloned into both pGBKT7 and pGADT7 were transformed into AH109 (MATa) and Y187 (MATα) yeast strains, respectively (Matchmaker 3 System, Clontech). AH109 strains harboring pGBKT7 plasmids were maintained in minimal SD media with tryptophan dropout supplement (SD/-Trp) while Y187 strains harboring pGADT7 plasmids were maintained in minimal SD media with leucine dropout supplement (SD/-Leu).

Prior to performing matings, individual AH109 strain was plated on SD/-Ade/-His/-Trp agar with 40 µg/ml X-α-Gal, and tested for autoactivation. AH109 strains containing the sequences of seven ORFs (UL48.5, UL26, UL48N, UL48C, UL94, US23, and UL51) were determined to be autoactivators in the absence of any pGADT7-cloned ORFs, and subsequently eliminated from further mating experiments.

Yeast mating was carried out by inoculating fresh colonies (<2 weeks old) of both AH109 and Y187 strains into 1.5 ml microcentrifuge tubes containing 0.5 ml of YPDA media and incubated at 30°C with shaking at 200 rpm for 24 hours. There were a total of 3068 possible combinations (52 AH109×59 Y187) and each mating combination was performed in duplicates. To select for diploids that have successfully been mated, yeast mating cultures were centrifuged at 14,000 rpm for 15 seconds, resuspended in 0.5 ml Tris-EDTA buffer, and plated in one well of 48-well plates containing 1 ml of SD/-Leu/-Trp agar and incubated at 30°C for 3–5 days. Diploid colonies from each successful mating were then picked from each well, resuspended with TE buffer, and plated in one well of 48-well plates containing 1 ml of SD/-Ade/-His/-Leu/-Trp (quadruple drop-out (QDO)) agar with 40 µg/ml of X-α-Gal.

Three weeks after plating the mated diploid yeasts, the QDO plates were scored for positive protein-protein interactions. QDO/X-α-Gal plates represent a high selection stringency for eliminating possible false positives. AH109 strains contain three reporters-ADE2, HIS3, and MEL1 (encodes α-galactosidase) under unique GAL4 upstream activating sequences (UASs) and TATA boxes. The mating between AH109 transformed with a plasmid expressing the GAL4 binding domain (BD)-p53 fusion protein and Y187 transformed with a plasmid expressing the GAL4 activation domain (AD)-T (SV40 T antigen) fusion protein was used as the positive control. The mating between AH109 transformed with a plasmid expressing the BD-Lamin fusion protein and Y187 transformed with a plasmid expressing AD-T was used as the negative control. Growth of the diploid yeast cells representing the positive interactions was further analyzed.

When scoring the duplicates, if both had no yeast growth after 3 weeks incubation, the fusion proteins were considered non-interacting partners. When the duplicates both had blue yeast colonies or if only one of the wells had blue yeast colonies, that mating combination was repeated until the results from two of three independent experiments are consistent.

Co-immunoprecipitation (co-IP) analysis

HeLa and U373MG cells, which were obtained from American Type Culture Collection (ATCC) (Manassas, VA), were co-transfected with the DNAs of the generated expression constructs that were derived from pCMV-myc and pCMV-HA and contained the HCMV ORF sequences, with the aid of Lipofectamine 2000 (Invitrogen, Carlsbad, CA). In some experiments, U373MG cells expressing a T7 RNA polymerase, which supported HCMV lytic replication, were co-transfected with the constructs that were used for the YTH analysis. The expression of T7 polymerase in these cells induced the expression of the HCMV ORFs cloned in the pGBKT7 and pGADT7 vectors. Cell lysates were harvested at 3 days post-transfection. Co-immunoprecipitation experiments were performed using the ProFound Mammalian HA Tag IP/Co-IP and c-myc Tag IP/Co-IP kits following the manufacturer's protocol (Pierce, Rockford, IL).

Western blot analysis

The denatured polypeptides from cell lysates were separated on SDS-polyacrylamide gels cross-linked with N,N″-methylenebisacrylamide, transferred electrically to nitrocellulose membranes [14], [52]. Detection of tagged proteins was performed by incubation with mouse anti-myc or rabbit anti-HA antibodies (Clontech, Mountain View, CA) and reacted in an enzyme-linked immunoassay with either anti-mouse IgG or anti-rabbit IgG conjugated with horse radish peroxidase (Vector Laboratories, Burlingame, CA) [53]. The membranes were subsequently stained with a chemiluminescent substrate with the aid of a Western chemiluminescent substrate kit.