Expression of the gH/gL/UL128-131 complex on HCMV strains links the activation of the integrin/Src-mediated signaling pathway to efficient viral internalization into target monocytes

Our results demonstrated that the virus functions as a specific ligand that, through the engagement of integrins and activation of downstream signal transduction pathways, is able to modulate monocyte biology. However, the specific ligand on the viral lipid membrane responsible for inducing the integrin/Src-signaling pathway has not been revealed. In an attempt to answer this question, we began to focus on the HCMV gH complexes, as they were shown to be the predominant HCMV glycoprotein complexes reported to interact with integrins on fibroblasts [28], and because the gH/gL/UL128-131 complex was documented to be required for endothelial/epithelial and monocyte tropism [30], [31], [34], [35], [45]. We first investigated if there was a correlation between the presence of the gH/gL/UL128-131 complex on the viral envelope and the ability of several HCMV strains to induce the key integrin/Src-signaling pathway. In our studies, we used HCMV strains containing the gH/gL/UL128-131 complex [our low fibroblast-passaged Towne (Towne/E p.40), a moderately fibroblast-passaged Towne/E (Towne/E p. 51) and TB40/E [39]], as well as viral strains lacking this complex [a highly fibroblast-passaged Towne (Towne/F p. 57), AD169 and TB40/F (a high passage TB40/E [39])]. Using a monoclonal antibody recognizing pUL130 (utilized as a surrogate marker for the UL128-131 complex), we found in virus lysates that the TB40/E strain possessed the highest level of pUL130 and that the continuous propagation of the Towne/E strain in fibroblasts caused a stepwise reduction in the expression of pUL130, such that the protein was no longer detectable in our Towne strain by passage 57 (Towne/F p.57; Figure 1A, lower panel). pUL130 was also not detected in fibroblast-adapted strains (AD169 and TB40/F). These results are in accord with previous reports demonstrating genetic changes in the UL128-131 region in clinical isolates propagated in fibroblasts [36], [39]. As an internal control, we examined levels of the HCMV tegument protein, pp65 (pUL83), in viral lysates (Figure 1A, upper panel). Similar amounts of pp65 were detected in each of the viral strains, suggesting similar particle to PFU ratios were used during infection of cells with the different viral strains. Monitoring of infected fibroblasts with the different virus strains supports our suggestion of a similar number of infectious particles per strain.

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Figure 1. Presence of the gH/gL/UL128-131 complex links HCMV′s ability to activate and efficiently enter into target monocytes.

(A) Approximately 2×10⁶ virions of Towne (p.40, p.51 and p.57), AD169, TB40/E and TB40/F were spun down through a sucrose cushion, lysed and western blot analyses were performed using antibodies recognizing the HCMV proteins, pp65 and pUL130. (B) Monocytes were isolated and cultured in low serum for 24 h at 37°C/5% CO₂. Monocytes were then mock- or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src and p70 S6 kinase. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (C) Monocytes were mock infected or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169) infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and semi-quantitative PCR was performed using primers complementary to genomic HCMV DNA and cellular GAPDH, as an internal control. PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. (D, E, F, G, H, I and J) Monocytes were mock- or HCMV (TB40/E or AD169)-infected (M.O.I. of 5) for 1 h at 4°C, then 2 mM of DTSSP [3,3′-dithiobis (sulfosuccinimidylpropionate] was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing β1, β3 integrins, HCMV gH or isotype control IgG were added overnight at 4°C to cellular lysates and then protein A/G Sepharose was added for 4 h at 4°C. Protein A/G Sepharose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in sample buffer. Western blot analyses were performed using antibodies recognizing β1 and β3 integrins, as well as the HCMV gH and pUL130. Lysates from HCMV-infected monocytes were also analyzed for equal levels of β1, β3 integrins and actin in samples undergoing immunoprecipitation. All experiments were repeated at least three times and representative results are shown. Note: The arrows point to the band of interest. The asterisks mark non-specific bands. (K) The schematic diagram describes our cumulative data from the immunoprecipitation analysis; illustrating the interaction between the gH/gL/UL128-131 complex of TB40/E strain or gH/gL/(gO) complex of AD169 strain with cellular integrins.

https://doi.org/10.1371/journal.ppat.1003463.g001

Next, we tested our panel of HCMV strains for their ability to induce integrin-mediated signaling. Src tyrosine kinases are established regulators of cellular signaling mediated by integrins [46]–[49], however their activity can also be initiated by other cellular receptors [50]–[53]. Similarly to our previous results [27], here we show that infection of monocytes with Towne/E p.40 caused an increase in the phosphorylation of Src at Tyr416 (∼2 fold increase), when compared to mock-infected monocytes (Figure 1B). Infection with TB40/E had a stronger effect on Src activation (∼3 fold increase over mock); than that observed following infection with Towne/E p.40 (Figure 1B), which correlated with a higher amount of pUL130 in the TB40/E viral particle vs. that seen in the Towne/E p.40 viral particle (Figure 1A). The highly passaged HCMV strains lacking a functional gH/gL/UL128-131 complex, however, were unable to initiate Src phosphorylation (TB40/F) or were inhibitory to Src activation (Towne p.57 and AD169) upon infection of monocytes (Figure 1B). In addition, the initial activation of Src by Towne/E p.40 and TB40/E resulted in the activation of a downstream signaling cascade, measured by phosphorylation of p70 S6 kinase (p70 S6K) at Thr389 (4.6- and 6.2-fold increase, respectively, over those levels seen in mock-infected cells; Figure 1B).

Because the activation of the integrin/Src-signaling pathway was demonstrated to be critical for efficient HCMV entry into monocytes [27], we next investigated the ability of HCMV strains expressing different levels of the gH/gL/UL128-131 complex to efficiently enter monocytes. We only observed efficient entry of Towne/E p.40 and TB40/E into monocytes, as indicated by the presence of internalized viral genomic DNA (Figure 1C). In contrast, the signal from the internalized HCMV genomic DNA was significantly lower in cells infected with Towne/F p.57, AD169 and TB40/F (Figure 1C). Equal loading of samples was ensured by examining the level of GAPDH expression (Figure 1C). Together, our results suggest a link between the presence of the functional gH/gL/UL128-131 complex on the viral envelope and the ability of HCMV to induce the integrin/Src-mediated signaling pathway required for efficient viral entry into target monocytes. In addition, because we used viral strains differentiated only by passage length (which in turn relates to cell tropism), our data provides support for the specific role that UL128-131 plays in these specific strains (TB40/E vs. TB40/F and Towne/E vs. Towne/F); at present we are not aware of other mutations that may be present in these different passaged strains. These data also support our previous observation of the critical role for the integrin/Src-signaling in efficient HCMV internalization into monocytes [27].

The aforementioned results suggest that the gH/gL/UL128-131 complex may physically engage cellular integrins on the surface of monocytes to initiate receptor-mediated signaling. Furthermore, we speculated that the gH/gL/UL128-131 complex not only bound integrins, but that it might engage only select integrins differentiating the biological effect caused by the gH/gL complex without the UL128-131 trimer from that of the gH/gL complex with the UL128-131 trimer. Thus, we next immunoprecipitated from HCMV-infected monocytes β1 or β3 integrins, receptors previously found to interact with the HCMV virion on these cells [27], as well as on other cell types [28], [54], [55], and investigated if the UL128-131 complex was capable of engaging these integrins. To pull down sufficient amounts of interacting proteins for their visualization using western blot analysis, we utilized the DTSSP crosslinker to stabilize the interactions. We chose this crosslinker as it allows the dissociation of crosslinked complexes by 5% β-mercaptoethanol and a separation of the individual proteins in those complexes using a standard SDS-PAGE analysis [56]. We found that pull down of β1 and β3 integrins from TB40/E- and AD169-infected monocytes resulted in the finding that the gH protein only interacts with β1 integrins (Figures 1D and 1E). As the lack of gH protein in β3 integrin-immunoprecipitate does not necessary mean the lack of interaction between these proteins, the reverse immunoprecipitation was performed. By immunoprecipitating the gH protein from the lysate of TB40/E-infected cells, we confirmed that gH of TB40/E interacts exclusively with β1 integrin (Figure 1F). However, the immunoprecipitation of the gH protein from the lysate of AD169-infected monocytes suggested that this HCMV glycoprotein can interact with both β1 and β3 integrins (Figure 1G). More importantly, we found that pUL130 present in the gH/gL/UL128-131 complex of TB40/E strain engages both β1 and β3 integrins on the surface of monocytes (Figure 1D). As predicted pUL130 was not detected in immunoprecipitates from monocytes infected with AD169 strain (Figure 1E). As a control for a non-specific binding, we performed immunoprecipitation analyses on lysates from infected monocytes using an IgG isotype matched antibody. We did not detect any signal in our immunoprecipitated samples; however the specific antibodies (to gH and pUL130) recognized viral proteins in the input viral lysate (Figure 1H). Additionally, an immunoprecipitation assay performed on mock-infected cells using an antibody recognizing HCMV gH did not result in a pulldown of the β1 and β3 cellular integrins (Figure 1I). In contrast, antibodies recognizing the β1 and β3 integrins did detect the appropriate integrins in the input lysate (Figure 1I). Western blot analysis also determined that there were equal amounts of cellular proteins in cell lysates used in our co-immunoprecipitation experiments (Figure 1J). The data together suggest that different components of the gH/gL/UL128-131 complex interact on the surface of monocytes with the distinct integrins that we previously showed were important for enhanced motility of and efficient entry into target monocytes [27]. This ability of viruses expressing the UL128-131 complex to bind to the β1 integrin (via gH) and to both β1 and β3 integrins (via the UL128-131 complex) provides new data as to why HCMV requires both integrins for entry into monocytes [27] (Figure 1K). The results also suggest why only a single β-integrin is likely required for HCMV entry into fibroblasts [28], [55]. Our findings may provide an explanation of why β1 integrins are key regulators of monocyte function as described by Yurochko et al. [57]. Based on our new results, we speculate that the interaction of the UL128-131 with β1 and β3 integrins may allow for a unique, synchronous engagement and activation of both β1 and β3 integrins by HCMV, which through the creation of the appropriate type and level of integrin-mediated signaling allows for efficient viral entry and the early functional changes in infected monocytes to occur.

The HCMV gH/gL/UL128-131 complex is critical for the functional activation of the integrin/Src-mediated signaling pathway and for efficient viral internalization into target monocytes

Long-term passaging of HCMV in fibroblasts alters the virus's genetic composition and those changes are not only limited to the UL128-131 region of the genome [34], [37], [58]–[60]. Consequently, in order to determine if the gH/gL/UL128-131 complex is directly responsible for the initiation of integrin signaling, leading to efficient HCMV internalization into monocytes, we decided to utilize two well-characterized bacterial artificial chromosome (BAC)-based, AD169-derived clones: BADwt, which contains a frame shift mutation in UL131A, and BADrUL131, which possesses a repaired UL131 locus and thus has a functional gH/gL/UL128-131 complex [31]. We confirmed that pUL130 was only detected in lysates of BADrUL131 virus, and not in lysates from BADwt (Figure S1A). As a control, we used the AD169 strain that, as showed in Figure 1A, does not express the gH/gL/UL129-131 complex and the TB40/E strain that possesses the pentameric complex on its envelope (Figure S1A).

We next investigated the ability of these viruses to induce the integrin/Src-signaling pathway. By using western blot analyses, we determined that BADrUL131 was able to increase the level of phosphorylated Src above the levels seen in mock- and BADwt-infected monocytes (Figure 2A). Figure 2A shows a representative western blot experiment with a densitometry analysis, demonstrating 1.6-fold increase of Src activation in BADrUl131-infected monocytes compared to that seen in mock-infected cells. A cumulative densitometry analysis of Src activation in infected monocytes that incorporates results from three repeats of the experiment showed significant changes in the level of phosphorylated Src in BADrUL131-infected cells, when compared to mock- and BADwt-infected cells (Figure S1B). Furthermore, the initial activation of Src in BADrUL131-infected monocytes translated into the activation of downstream signaling, resulting in increased levels of phosphorylated paxillin (2.4-fold increase), Erk (2-fold increase) and SAPK/JNK (3.6-fold increase), compared to those seen in mock-infected cells (Figure 2A). We also noticed that there was a lower level of phosphorylated forms of Erk and SAPK/JNK in BADwt-infected cells, compared to mock-infected monocytes (Figure 2A), suggesting that BADwt infection may have a slight inhibitory effect on the integrin/Src-signaling pathway in target monocytes. We did not observe any significant differences in levels of total Src, paxillin, or Erk. These results not only substantiate the importance of the gH/gL/UL128-131 complex in the activation of integrin-mediated signaling and validate our previous studies demonstrating the ability of HCMV to engage cellular integrins and to induce the integrin/Src/paxillin signaling pathway in infected monocytes [27], but they further provide insight into how HCMV stimulates integrin receptors on target monocytes.

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Figure 2. The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes.

(A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO₂. Monocytes were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p<0.05 (indicated by asterisks) was used for the measurement of statistical significance between samples. (C and D) Monocytes were mock- or HCMV (BADwt or BADrUL131)-infected [M.O.I. of 5 (C) or 1 (D)] and incubated at 37°C in 5% CO₂ for 5 days. (C) Cells were fixed, cytospun, underwent fluorescence in situ hybridization and were analyzed using a confocal microscopy (vDNA – yellow/green & DAPI nuclear stain – pink) or (D) were harvested and semiquantitative PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Note: in the (C) panel, inset pictures are an enlarged version of the representative cell shown in the picture. (E) Monocytes were HCMV (BADwt or BADrUL131)-infected (M.O.I. of 1) and incubated at 37°C in 5% CO₂ for 3 weeks. RNA was harvested from cells, reverse transcribed and semiquantitative PCR was performed using primers complementary to HCMV IE1-72 mRNA and 18S rRNA, as an internal control. Reverse transcriptase negative (RT-) sample was also tested to demonstrate lack of residual, genomic DNA in samples. All experiments were repeated at least three times and representative results are shown.

https://doi.org/10.1371/journal.ppat.1003463.g002

The data presented so far strongly support a clear correlation between the presence of a functional HCMV gH/gL/UL128-131 complex on the viral envelope and the ability of the virus to trigger integrin/Src-signaling in target monocytes. Therefore, we next examined the ability of BADwt and BADrUL131 to enter target monocytes using HCMV entry assay [26], [27]. Based on the level of internalized vDNA in infected monocytes, we found that BADrUL131 was more efficiently (∼3-fold) internalized into monocytes, when compared to BADwt (Figure 2B). The differences in entry seen between BADwt and BADrUL131 were not due to the ability of these viruses to differentially bind monocytes; we did not observe any significant changes in the binding properties of BADwt and BADrUL131 to monocytes (Figure S1C). We also analyzed the ability of the endotheliotropic TB40/E vs. the non-endotheliotropic TB40/F strains to enter monocytes. TB40/F lost its ability to infect endothelial cells, likely due to a frameshift mutation in UL128 gene region [37], [39], [59], as a result of its prolong passaging in fibroblasts. Our results showed that TB40/E was more efficiently (∼3 fold increase) internalized into monocytes than TB40/F. The efficiency of TB40/E internalization into monocytes was similar to that observed following infection with BADrUL131 (Figure 2B). As a control, infected cells were also kept at 4°C without a temperature shift to 37°C. Using this assay, we determined that basal levels of HCMV genomic DNA in cells kept at 4°C was comparable to the levels of BADwt and TB40/F internalization into monocytes at 37°C (Figure S2A). We speculate that even though we saw very low level of viral internalization at 4°C, this entry does not result in a productive infection. Additionally, the proteinase K-treatment might not have removed all non-internalized viral particles from the surface of infected cells, which would also have an effect on the levels of viral genomic DNA in cells maintained at 4°C.

To investigate the ability of BADwt vs. BADrUL131 to establish a productive infection, we performed fluorescence in situ hybridization (FISH) analysis on HCMV-infected monocytes to monitor the localization/presence of vDNA at 5 dpi. The signal from the fluorescence probe recognizing HCMV vDNA was only found in monocytes infected with BADrUL131, not in monocytes infected with BADwt (Figure 2C; see the inset pictures for a magnified view of the representative cells). A majority of the cells analyzed showed evidence of vDNA staining at 5 dpi (∼80%). As a control, we verified that BADwt and BADrUL131-infected fibroblasts were both positive for vDNA using this probe (DNS). As FISH only allows for an examination of a small number of cells, we also wanted to examine the infected cell population as a whole, thus we additionally conducted semi-quantitative PCR and RT-PCR analyses looking at HCMV genomic DNA and HCMV IE mRNA expression at 5 dpi and 3 weeks pi, respectively. The results obtained from these experiments support and extend the FISH data; we saw amplification of the HCMV UL123 genomic sequence only from DNA isolated from BADrUL131-infected cells at 5 dpi (Figure 2D). Similarly, HCMV IE mRNA was only found expressed at 3 weeks pi in monocytes/macrophages infected with BADrUL131 (Figure 2E), suggesting that only monocyte-derived macrophages initially infected as monocytes with BADrUL131 were able to express vRNA and, thus were the only cells productively infected. Taken together, our data indicate that the presence of the gH/gL/UL128-131 complex on the HCMV envelope is important for the activation of virus-induced, integrin/Src-mediated signaling pathway in target monocytes and for the efficient viral internalization into these cells that ultimately results in productive viral infection.

The integrin/Src/paxillin-mediated signaling pathway is critical for BADrUL131 internalization

We have documented that the integrin/Src/paxillin signaling axis must be functionally activated for HCMV to enter blood monocytes [27], and we now demonstrate that the gH/gL/UL128-131 complex is the key trigger for this activation of integrin-mediated signaling and for efficient internalization (Figures 1 and 2). However, because the signaling networks in HCMV-infected monocytes are complex in their nature and involve crosstalk between different receptors [18], [22], [25]–[27], [61]–[63], we wanted to clarify if the Src-mediated signaling pathway interacted molecularly with the EGFR-mediated pathway. Both pathways were shown to be important for efficient HCMV internalization into monocytes; however, both pathways were also found to have a distinct role in regulating the biology of HCMV-infected monocytes [26], [27]. Our new results showed that pretreatment of monocytes with PP2 (specific Src tyrosine kinase inhibitor) and/or AG1478 (specific EGFR tyrosine kinase inhibitor) did not affect the efficiency of BADwt internalization (Figure 3A). However, when monocytes were pretreated with PP2 or AG1478 prior to BADrUL131 infection, viral internalization was inhibited by approximately 56% and 25%, respectively, compared to DMSO-pretreated, BADrUL131-infected cells (Figure 3A). The addition of both PP2 and AG1478 prior to BADrUL131 infection blocked viral internalization by more than 70% compared to DMSO-pretreated, BADrUL131-infected monocytes, however, the cumulative effect of both drugs was not significantly different from the effect of PP2 alone (Figure 3A).

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Figure 3. Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes.

(A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO₂. (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p<0.05 (indicated by asterisks) was used for the measurement of statistical significance between samples. All experiments were repeated at least three times.

https://doi.org/10.1371/journal.ppat.1003463.g003

To test for the importance of integrin engagement by the gH/gL/UL128-131 complex in the efficient HCMV entry into monocytes, we pretreated cells with function blocking antibodies to β1 or β3 integrins prior to infection with BADwt and BADrUL131. As shown by the level of internalized vDNA, we determined that this pretreatment did not have any effect on BADwt entry into target monocytes; however the blocking of β1 or β3 integrins inhibited the ability of BADrUL131 to enter these cells by ∼75% or ∼40%, respectively, as determined by densitometry analysis (Figure 3B). The effect of function blocking antibodies on BADrUL131 internalization correlated with their inhibitory impact on the activation of the integrin/Src/paxillin signaling pathway in monocytes infected with HCMV expressing the pentameric complex (Figure S2B and [27]). We did not detect any effect of these blocking antibodies on the integrin/Src/paxillin signaling axis in cells infected with BADwt (Figure S2C). Together, these data provide additional support for the idea that the functional activation of integrin/Src/paxillin signaling pathway, triggered by the HCMV gH/gL/UL128-131 complex, has a causative effect on efficient HCMV entry into target monocytes. It also suggests that even though BADrUL131 predominantly utilizes the integrin-mediated signaling for its internalization into monocytes, the EGFR-mediated signaling also plays at least a supporting role in this process.

We reported earlier that HCMV infection leads to increased expression of paxillin in target monocytes via integrin/Src-signaling [27]. We also demonstrated that by knocking down paxillin expression, we were able to significantly decrease HCMV entry into monocytes [27]. To assess the importance of paxillin regulation in BADwt vs. BADrUL131 internalization into monocytes, we used siRNA to knock down paxillin expression as previously described [27] and as before we accomplished a paxillin knockdown efficiency of ∼80–90% (Figure S2D). Using our entry assay, we found that the lack of paxillin expression did not influence BADwt entry into monocytes, while in contrast, BADrUL131 internalization into paxillin-deficient monocytes was inhibited ∼45% (Figure 3C). To attempt to answer if the lack of paxillin activation might be responsible for the inability of BADwt to efficiently enter monocytes, a rescue experiment using α-thrombin, documented to increase phosphorylation of Src and paxillin [64], [65], was also performed. We found that treatment of monocytes with α-thrombin was able to increase the levels of the phosphorylated forms of paxillin and Src with the peak of this activation at 15 min. post the α-thrombin treatment (Figure S2E and data not shown). Because our results demonstrated that the kinetics of HCMV- and α-thrombin-mediated activation were similar (Figure S2E and [27]), we wanted to mimic the pace of paxillin phosphorylation triggered by the gH/gL/UL128-131 complex by exposing BADwt-infected monocytes to α-thrombin just before shifting temperature from 4°C to 37°C in our entry assay. We found that the efficiency of BADwt internalization into monocytes with a normal paxillin expression was significantly (2-fold) enhanced by α-thrombin treatment (Figure 3D). Because α-thrombin can stimulate other signal transduction pathways [66]–[69], we wanted to ensure that α-thrombin-mediated effect on BADwt internalization was paxillin-dependent. Thus, we examined the effect of α-thrombin on BADwt internalization into monocytes deficient of paxillin expression. Monocytes were transduced with scrambled siRNA or siRNA specific for paxillin mRNA, as previously described [27]. The positive effect of α-thrombin on the entry of BADwt into control siRNA-transduced monocytes was diminished in monocytes lacking paxillin expression (Figure 3D), thus suggesting that the inducing effect of α-thrombin on BADwt internalization was paxillin-dependent. In summary, these data support the critical role for the activated integrin/Src/paxillin-signaling pathway, induced through the interaction of integrins with the HCMV gH/gL/UL128-131 complex, in efficient HCMV internalization into monocytes.

Fibroblasts infected by BADwt and BADrUL131 show an opposite activation pattern when compared to infected monocytes

Fibroblasts are a very well studied in vitro model system of HCMV infection. Although we have previously shown that HCMV receptor/ligand engagement activates fibroblasts [25], we have also documented that there are key differences between the nature and duration of the signaling during HCMV infection of monocytes vs. fibroblasts. For example, HCMV infection of monocytes results in the induction of cellular differentiation, long-term cellular survival, and PI(3)K-independent HCMV entry into monocytes [17], [18], [26], [61], [70], which is not observed in fibroblasts. Additionally, HCMV clinical isolates were reported to infect fibroblasts less efficiently than highly passaged laboratory adapted strains with the opposite being true for the infection of endothelial cells [71], [72], suggesting to us that the presence of the gH/gL/UL128-131 complex differentially affects fibroblasts vs. monocytes. Thus, we next investigated if there were differences in receptor-mediated signaling in BADwt- vs. BADrUL131-infected fibroblasts. Using western blot analyses, we demonstrated that in fibroblasts, BADwt triggered an increase in the level of phosphorylated Src (1.9-fold increase) as compared to mock-infected fibroblasts (Figure 4A). This initial activation of Src induced downstream signaling, resulting in increased levels of activated paxillin (1.8-fold increase) and Erk (1.6-fold increase) as compared to mock-infected cells (Figure 4A). In contrast, the levels of activated Src and paxillin were not changed in BADrUL131-infected fibroblasts, compared to mock-infected cells (Figure 4A). Interestingly, the level of phosphorylated Erk in fibroblasts infected with BADrUL131 was lower than that seen in mock-infected cells (Figure 4A), suggesting that BADrUL131 may even have an inhibitory influence on molecules downstream of integrin/Src/paxillin-signaling in fibroblasts. As showed by our entry assay, the differences in stimulating the receptor-mediated signaling in fibroblasts by BADwt vs. BADrUL131 did not translate into differences in the ability of these two viruses to be internalized (Figure 4B), supporting previously published data [34].

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Figure 4. Presence of the gH/gL/UL128-131 complex has no effect on the integrin/Src/paxillin signaling pathway in and HCMV internalization into fibroblasts.

(A) Fibroblasts were cultured in low serum for 24 h at 37°C/5% CO₂. Fibroblasts were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 20 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, and Erk. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then left at 4°C or temperature shifted to 37°C for 1 h. (C) Fibroblasts were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. (B and C) Then, fibroblasts were washed and treated with Proteinase K solution for 1 h. Cells were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p<0.05 (indicated by asterisks) was used for the measurement of statistical significance between samples. All experiments were repeated at least three times and representative results are shown.

https://doi.org/10.1371/journal.ppat.1003463.g004

Because of the importance of paxillin regulation for HCMV entry into monocytes, we assessed the role paxillin plays in BADwt vs. BADrUL131 internalization into fibroblasts. siRNA technology was used to knock down paxillin expression and, similarly to monocytes (Figure S2D and [27]), we were able to decrease paxillin expression in siRNA-transfected fibroblasts by 80 to 90% (Figure S2F). We found that the expression of paxillin did not influence either BADwt or BADrUL131 entry into fibroblasts (Figure 4C), suggesting that although BADwt triggers paxillin phosphorylation during infection of fibroblasts (Figure 4A), paxillin regulation is not required for efficient viral internalization into this cell type. To assure that the differences seen in the ability of BADwt and BADrUL131 to stimulate the integrin/Src-signaling in fibroblasts, as well as the similar abilities of these viruses to enter fibroblasts, was not attributed to differences in binding of BADwt vs. BADrUL131 to fibroblasts, we performed a binding assay. We did not observe significant changes in the binding of the BADwt and BADrUL131 to fibroblasts (Figure S1D).

Regulation of the actin cytoskeleton is important for efficient entry of HCMV into monocytes

Results presented earlier in this manuscript demonstrated that the functional regulation of paxillin in target monocytes is central for efficient internalization of HCMV virions possessing the gH/gL/UL128-131 complex. We have also recently documented that paxillin expression at the level of mRNA and protein is elevated upon HCMV infection of monocytes, and its regulation is critical for HCMV-mediated pathological motility of target monocytes [27]. Paxillin is a scaffolding and signal transduction protein that plays an important role in regulating the interaction between multiple proteins involved in cell motility and adhesion [73]. Increased paxillin (Tyr118) phosphorylation has been shown to enhance actin polymerization and cytoskeleton rearrangement [74], [75]. Thus, we hypothesized that paxillin is a central regulator of HCMV-mediated changes in infected monocytes and through its role in actin remodeling governs a “hyper” motility of and HCMV entry into monocytes. The role of paxillin-mediated actin rearrangement in cellular motility has been extensively studied [76]–[78], while the importance of paxillin in rapid actin filament rearrangement during viral entry remains unknown. The modulation of the actin cytoskeleton is utilized by several viruses to enhance their infectivity (i.e. Epstein Barr Virus, Vaccinia virus [79]) and entry (i.e. Human Immunodeficiency Virus, Adenovirus [80], [81]), suggesting that HCMV may also require actin rearrangement as a part of its entry process.

Therefore, we asked if HCMV modulates actin rearrangement in infected monocytes. G-actin is a monomeric form of actin, which can polymerize in an ATP-dependent manner to create conformationally changed F-actin-based filaments [82]. Based on western blot analysis, we found that at 15 min. pi, the ratio of F-actin to total actin fell by approximately 50%, suggesting a decrease in polymerized F-actin in HCMV-infected monocytes when compared to mock-infected cells (Figure S3A). Our results also showed that there was a higher level of F-actin at 60 min. pi in HCMV-infected monocytes when compared to mock-infected cells (∼50% higher; Figure S3A), which corresponds to the increased motility of these cells at later times post infection [17], [18], [26], [27], [61]. As a control for our experiments, monocytes were also treated with jasplakinolide (an inducer of actin polymerization) and with latrunculin A (an inhibitor of actin polymerization) [83], [84]. We found that at 15 min. post treatment, jasplakinolide increased the ratio of F-actin to total actin by approximately 40%, and latrunculin A decreased this ratio by 35% when compared to untreated monocytes (Figure S3B).

The presented data suggest that HCMV induces actin rearrangement very early post infection, correlating with the early time frame of viral internalization ([85] and our unpublished results). Because we showed that HCMV was characterized by a lower efficiency of internalization in paxillin-deficient monocytes [27], we next wanted to investigate if the deficiency in paxillin expression affected actin rearrangement. By comparing levels of F-actin to total actin in siRNA-treated monocytes, we demonstrated that cells transfected with paxillin siRNA for 48 h were characterized by a 40–50% decrease in the ratio of F-actin to total actin when compared to this ratio in mock- and control siRNA-treated monocytes (Figure S3C). These results support the idea that paxillin, via its regulation of the actin cytoskeleton in monocytes, could be involved in viral entry and that its loss leads to the suppression of normal actin turnover and a decrease in actin polymerization [86]–[89].

To examine the direct role of actin rearrangement in the HCMV internalization process into monocytes, cells were pretreated with jasplakinolide or latrunculin A 1 h prior to infection with BADwt, BADrUL131 or TB40/E. From an examination of internalized vDNA, we found that latrunculin A significantly inhibited BADrUL131 (∼60% decrease) and TB40/E (∼53% decrease) entry into monocytes compared to DMSO-treated cells (Figure 5A). When monocytes were pretreated with jasplakinolide, BADrUL131 internalization into monocytes was decreased by ∼70% and TB40/E entry was decreased by more than 80% (Figure 5A). Both pharmacological compounds also had an impact on the internalization process of BADwt; however, the effects were not as substantial as that seen following infection with BADrUL131 and TB40/E. Latrunculin A decreased BADwt internalization efficiency by ∼45%, and jasplakinolide was able to decrease this process by 34%; however, this result was not statistically significant (Figure 5A).

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Figure 5. Regulation of the actin cytoskeleton and dynamin is essential for efficient internalization of clinical-like HCMV isolates into monocytes, but not into fibroblasts.

(A) Monocytes were pretreated with DMSO, 0.5 µM jasplakinolide, or 2.5 µM latrunculin A. (B) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (C) Monocytes were preatreated with 50 µM dynasore. (D) Monocytes were transfected with siRNA complementary to dynamin or a control siRNA for 48 h. (E and F) Fibroblasts were pretreated with DMSO, 0.5 µM jasplakinolide, 2.5 µM latrunculin A, or 50 µM dynasore. (A, B, C, D, E, and F) Cells were then infected with BADwt, BADrUL131 or TB40/E (M.O.I. of 0.1) for 1 h at 4°C washed and then temperature shifted to 37°C for 1 h. Monocytes were then treated with 0.5 µM jasplakinolide at 37°C for an additional 1 h (only B). Cells were then washed and treated with Proteinase K solution for 1 h. Cells were harvested and PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA. For qPCR data, results are plotted as a mean ±SEM. Student's T-tests were performed and p<0.05 (indicated by asterisks) was used for the measurement of statistical significance between samples. For semiquantitative PCR, PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. The experiments were performed at least three times.

https://doi.org/10.1371/journal.ppat.1003463.g005

The results presented so far suggest that paxillin, through its regulation of the actin cytoskeleton, affects HCMV internalization into monocytes. To address paxillin's possible direct influence of HCMV entry into monocytes via actin rearrangement, we examined if we could rescue the low entry efficiency of BADrUL131 into paxillin-deficient monocytes ([27] and Figure 3C) by inducing actin polymerization in these cells. Shelhass et al. [90] has demonstrated that human papillomavirus (HPV) localizes to long, tubular invaginations of plasma membrane in cells treated with cytochalasin D (an inhibitor of actin cytoskeleton dynamics). These structures were shown to hold several viral particles and are thought to emerge due to the inability of endocytic vesicles to pinch off. We hypothesized that HCMV infection of paxillin-deficient monocytes may mimic aspects of HPV entry and thus we could induce the completion of the HCMV entry process into paxillin-deficient monocytes by inducing actin polymerization. Monocytes were treated with siRNA recognizing paxillin mRNA or control siRNA for 48 h and then infected at 4°C with BADrUL131. After an hour-long-incubation at 37°C; cells were treated with an inducer of actin polymerization (jasplakinolide) for an additional hour. Again, we found that BADrUL131 was not able to efficiently enter paxillin-deficient monocytes (Figure 5B). Importantly, jasplakinolide increased (∼4×) the internalization of BADrUL131 in paxillin-deficient monocytes compared to that seen in monocytes treated only with paxillin siRNA (Figure 5B). The level of BADrUL131 internalization in jasplakinolide-treated cells was higher than that seen in control siRNA-treated cells (Figure 5B), which may suggest that there is a concentration of HCMV particles inside plasma membrane structures when paxillin expression is limited, that in turn fully enter when actin polymerization is induced. Noteworthy, we saw an inhibitory effect of jasplakinolide-treatment on BADrUL131 internalization into control siRNA-transfected monocytes (Figure 5B), which may suggest that not all viral particles entered monocytes within the first hour at 37°C. Together, these data strongly support the notion that HCMV utilizes a paxillin-regulated actin rearrangement as a mechanism for its efficient entry into monocytes. It also indicates that there is an interrelationship between the presence of the gH/gL/UL128-131 complex on the HCMV envelope and the dependence of HCMV entry on actin regulation. Moreover, these studies provide new clues about the molecular mechanisms for how HCMV mediates enhanced motility of and efficient viral entry into target monocytes.

The efficient internalization of HCMV in target monocytes is dynamin-dependent

The involvement of actin rearrangement in the internalization process of HCMV into monocytes indicates that the virus might use one of the endocytic pathways to enter these cells [91]. To address this possibility, we tested a panel of pharmacological inhibitors targeting different modes of endocytosis. Monocytes were pretreated with nystatin (disrupts caveolar structure and function [92]), genistein (disrupts caveolae-mediated endocytosis [93]), Rac1 inhibitor (Rho GTPase inhibitor [94]) and dynasore (dynamin inhibitor [95]) prior to BADrUL131 infection. We found that among these inhibitors, only dynasore significantly (∼35%) blocked HCMV internalization into monocytes (Figure 5C and Figure S3D). Additionally, we analyzed the ability of BADrUL131 to enter dynamin-deficient monocytes. Cells were transduced with siRNA recognizing dynamin mRNA or a control siRNA for 48 h. Even though, we only obtained ∼30% dynamin knockdown in monocytes, as determined by examining the level of dynamin mRNA (Figure S3E), BADrUL131 entry into these monocytes, as showed by levels of internalized vDNA using a semiquantitative PCR analysis, was inhibited by 60% (Figure 5D), which provided an even stronger support for the importance of dynamin regulation in efficient HCMV entry into monocytes. We found that dynasore did not have any effect on BADwt entry into monocytes (Figure 5C), suggesting again that only virus expressing the gH/gL/UL128-131 complex is able to utilize a dynamin-dependent entry mode into monocytes, which in turn leads to productive infection of HCMV in monocyte-derived macrophages (Figure 2C, 2D, and 2E). Additionally, we demonstrated that dynasore, jasplakinolide and latrunculin A were not able to inhibit HCMV entry into fibroblasts, as the efficiency of BADwt and BADrUL131 internalization was not altered by drug-pretreatment in these cells (Figure 5E and 5F). Interestingly, we also observed that the role of integrin/Src/paxillin-signaling, the actin cytoskeleton and dynamin regulation in efficient HCMV entry into monocytes matched their role in HCMV internalization into epithelial cells, as seen by levels of internalized BADrUL131 and TB40/E DNA in epithelial cells pretreated with PP2, AG1478, dynasore, jasplakinolide, and latrunculin A (Figure S3F). Taken together, our data indicate that HCMV, expressing the gH/gL/UL128-131 complex, uses a dynamin- and actin-dependent endocytic-like route to enter into target monocytes and that the internalization process of HCMV is strikingly different to that utilized by the virus in fibroblasts, however it closely resembles a type of HCMV entry utilized in epithelial cells.

Ethics statement

We explicitly state that the Louisiana State University Health Sciences Center-Shreveport (LSUHSC-S) Institutional Review Board (IRB) approved our study (the approved study number is H99-064) and that all HIPAA and LSUHSC-S IRB guidelines were followed for the use of human subjects in our study. In addition, the National Institutes of Health has approved our study using human subjects as part of our funded application (AI050677). We also explicitly state that informed written consent was provided by study participants (using an approved consent form, from the approved protocol H99-064); that is, an approved consent form was signed by the participants and collected for all subjects utilized in our study. No vulnerable populations were utilized in our study (minors, pregnant women, prisoners, etc.); thus only the participants themselves signed the consent approved forms and no legal guardians were required to provide consent to participate in the study. As per HIPAA and LSUHSC-S IRB guidelines, we follow all protocols on the welfare of human subjects and on the privacy of all collected information. The LSUHSC-S IRB is under the guidance of the Human Research Protection Program (HRPP) and is headed by the chancellor of the University, Dr. Robert Barish, M.D. (the website for our HRPP is as follows [http://www.lsuhscshreveport.edu/HRPP/HRPPHome.aspx]). The board is as follows: Institutional Official (Robert A. Barish, MD, MBA); HRPP (Robert E. Walter, MD, MPH; Aliese Seawright, MS, C.I.P; Kathleen Bloomingdale, BS; and Janice Soderstrom, RN, BSN); QA/QI (Sherry Mosura, RN, BSN, CCRC; Yvette Viverette, RN, CCRC); and, IRB (Christina Copeland, MPH; Shweta Khedlekar, MS; Ann Johnson; Crystal McGee).

HCMV culture and infection

AD169-derived bacterial artificial chromosome (BAC) viruses BADwt and BAD rUL131 (containing a repaired UL131A mutation, such that the gH/gL/UL128-131 complex is identical to that of HCMV TR strain), were previously constructed and characterized [31]. In addition to these BAC-based viruses, several additional HCMV strains were used: a green fluorescent protein (GFP)-labeled TB40/E-UL32 (TB40/E) [39]; a high fibroblast-passaged TB40 (TB40/F) that has lost the ability to infect endothelial cells [39]; our Towne/E (with the UL128-131 complex intact; Towne p.40); a intermediate fibroblast-passaged Towne (Towne p.51); and, a high fibroblast-passaged Towne (without a functional UL128-131 complex; Towne p.57) [123]. The TB40/E and TB40/F strains [39] are related (TB40/F is a high passaged TB40/E) as is our Towne/E and Towne/F strains [39]. TB40/E and Towne/E retain endothelial and epithelial cell, as well as monocyte tropism, while TB40/F and Towne/F have lost the ability to infect these clinically relevant cells types and are restricted to fibroblast infection. As shown by the data in Figure 1, those viruses retaining endothelial and epithelial cell, and monocyte tropism possess an intact UL128—131 complex, while those restricted to fibroblast infection have lost this complex. It is currently unclear if additional mutations have been incorporated into the more highly passaged viruses. The viruses were cultured in human embryonic lung (HEL) fibroblasts with 4% heat-inactivated fetal bovine serum (FBS) and purified on a sucrose gradient and resuspended in RPMI 1640 (Cellgro, Mediatech Inc., Herndon, VA). Monocytes were infected with purified virus at the multiplicity of infection (M.O.I.) of 0.1 for entry assays or M.O.I. of 5 for the various functional assays, unless otherwise stated. Mock infection was carried out by adding an equivalent volume of RPMI 1640 (Cellgro) to monocytes.

Human peripheral blood monocyte isolation

Double-density gradient centrifugation was used to purify human peripheral blood monocytes [57]. Whole blood was collected from donors by venipuncture. Mononuclear cells were then collected by centrifugation through a Ficoll Histopaque 1077 (Sigma-Aldrich, Inc., St. Louis, MO) gradient. Next, the collected cells were washed in 1× Phospho-Buffered Saline (PBS; Cellgro) to remove platelets. Monocytes were then isolated by centrifugation through a Percoll (Pharmacia Biotech, Inc., Piscataway, NJ) gradient and suspended in RPMI 1640 (Cellgro) supplemented with 1% human serum (Sigma-Aldrich Inc.).

Cell treatment and culture

After isolation, monocytes were cultured under non-adherent conditions (unless specified otherwise) in RPMI 1640 (Cellgro) supplemented with 1% human serum (Sigma-Aldrich Inc.) at 37°C with 5% CO₂ overnight, prior to any treatment, if not stated otherwise. Primary human embryonic lung (HEL) fibroblasts (passage 13–20) were cultured in Eagle's minimal essential media (MEM, Cellgro) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gemini, Woodland, CA), penicillin (100 IU/ml; Cellgro), and streptomycin (100 µg/ml; Cellgro) and Plasmocin (25 µg/ml; InvivoGen, San Diego, CA). Primary human mammary epithelial cells (HMEC) were culture in EBM-2 Basal Medium (Cambrex Inc., East Rutherford, NJ). The following standard treatment groups were employed in our study: dimethyl sulfoxide (DMSO; Sigma-Aldrich Inc.) as a solvent control; 1 µM PP2 (Src tyrosine kinase inhibitor; EMB Biosciences, Inc.; La Jolla, CA); 1 µM AG1478 (EGFR tyrosine kinase inhibitor; EMB Biosciences, Inc.); 0.5 µM jasplakinolide (inducer of actin polymerization; Enzo Life Sciences International, Inc.; Plymouth Meeting, PA); 2.5 µM latrunculin A (inhibitor of actin polymerization; Enzo Life Sciences International, Inc.); 50 µg/ml nystatin (disrupts caveolar structure and function; Enzo Life Sciences International, Inc.); 200 µM genistein (disrupts caveolae-mediated endocytosis; Enzo Life Sciences International, Inc.), 100 µM Rac1 inhibitor (Rho GTPase inhibitor; Enzo Life Sciences International, Inc.) and 50 µM dynasore (dynamin inhibitor; Enzo Life Sciences International, Inc.) were added 1 h prior to HCMV infection at 37°C with 5% CO₂. In one of the viral entry assays, 0.5 µM jasplakinolide was added after the shift in temperature to 37°C. In one of the viral entry assays, 5 U/ml of α-thrombin (Enzyme Research Laboratories, Inc., South Bend, IN) was added prior to the shift in temperature to 37°C. Cells were also treated with 5 µg/ml of function-blocking anti-β1 integrin (Millipore, Bedford, MA) and/or anti-β3 integrin (Millipore) antibodies for 1 h at 4°C prior to HCMV infection, as well as with an IgG isotype control (Santa Cruz Biotechnology, Inc.)

Western blot analysis

Monocytes or fibroblasts were pretreated with pharmacological agents and then infected. To examine the kinetics of receptor-mediated signaling pathways, cells were harvested using lysis buffer [50 mM Tris-HCl at pH 7.5 (Fisher Scientific, Fair Lawn, NJ), 5 mM ethylenediaminetetraacetic acid (EDTA; BioRad Laboratories, Hercules, CA), 100 mM sodium chloride (Fisher Scientific), 1% Triton X-100 (Fisher Scientific), 0.1% sodium dodecyl sulfate (SDS; MP Biomedicals, Inc., Solon, OH), and 10% glycerol (MP Biomedicals, Inc.)] at the time points indicated. Samples were mixed with Laemmli's SDS-Sample Buffer (Boston BioProducts, Boston, MA) containing 2-mercaptoethanol (Fisher Scientific). Equal protein amounts of the different samples were separated by continuous polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to ImmunoBlot polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories). Western blot analyses were performed using primary antibodies recognizing the phosphorylated and non-phosphorylated forms of Src [phospho-Src (Tyr416) antibody (Cell Signaling Technology, Inc., Danvers, MA) and pan-Src (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)], of paxillin [phospho-paxillin (Tyr18) antibody and pan-paxillin antibody (Cell Signaling Technology, Inc.)], of SAPK/JNK [phospho-SAPK/JNK (Thr183/Tyr185) antibody and pan-SAPK/JNK antibody (Cell Signaling Technology, Inc.)], of Erk [phospho-Erk (204) antibody and pan-Erk1 antibody (Santa Cruz Biotechnology, Inc.)], of p70 S6K [phospho-p70 S6K (Thr389) and pan-p70 S6K antibody (Cell Signaling Technology, Inc.)], as well as antibodies recognizing cellular F-actin (Thermo Fisher Scientific; Rockford, IL), and recognizing HCMV proteins: pUL130 (gift from Dr. Thomas Shenk) and pp65 (Virusys Corporation; Taneytown, MD). Probing for actin (Santa Cruz Biotechnology, Inc.) was used as a loading control. Donkey anti-rabbit (GE Healthcare Life Sciences, Piscataway, NJ), donkey anti-mouse (GE Healthcare Biosciences) and donkey anti-goat (Santa Cruz Biotechnology, Inc.) conjugated with horseradish peroxidase (HRP) were used as secondary antibodies. Western blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences).

Immunoprecipitation

Monocytes were infected with HCMV at 4°C for 1 h. Subsequently, 2 mM of DTSSP [3,3′-dithiobis(sulfosuccinimidylpropionate)] (Thermo Fisher Scientific; Rockford, IL) was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing gH (Virostat Inc., Portland, ME), pUL130, β1 (Millipore), β3 integrins (Millipore) or an IgG isotype control (Santa Cruz Biochenology, Inc.) were added to lysate overnight at 4°C and then Protein A/G PLUS Agarose was added for 4 h at 4°C. Protein A/G PLUS Agarose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in Laemmli Sample Buffer (BioRad Laboratories) containing β-mercaptoethanol (Fisher Scientific). Proteins were separated on SDS-PAGE gels, and transferred to ImmunoBlot PVDF membranes. Antibodies recognizing the HCMV gH (Virostat Inc.), pUL130, β1 or β3 integrins (Santa Cruz Biotechnology, Inc.) were used in the western blot assays.

Quantitative and semiquantitative reverse transcriptase-PCR

Monocytes or fibroblasts were pretreated and infected. Total cellular DNA was harvested with E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek, Inc.). The quantitative PCR amplification and detection were performed as previously described [27]. Semiquantitative PCR was also used to assess the entry efficiency of different HCMV strains and the efficiency of dynamin knockdown. Product amplification was carried out using MyCycler thermocycler (BioRad Laboratories), with the following PCR mix: 1× iTaq buffer (BioRad Laboratories) containing 1.25 U of iTaq DNA polymerase (Invitrogen Corp.) and a 50 µM concentration of each deoxynucleotide (Invitrogen Corp.). Primers specific for HCMV IE1-72 (sense, 5′-AGTGACCGAGGATTGCAACG-3′; antisense, 5′-CCTTGATTCTATGCCGCACC-3′), GAPDH (sense, 5′-GAAGGTGAAGGTGGAGT-3′; antisense, 5′-GAAGATGGTGATGGGATTTC-3′), 18S rRNA (sense, 5′-CGAGCCGCCTGGATACC-3_; antisense, 5′-CAGTTCCGAAAACCAACAAAATAG-3′) and dynamin (Santa Cruz Biotechnolgy, Inc.) were used to amplify target sequences. All primers, exempt primers for dynamin, were obtained from Integrated DNA Technologies, Inc. (Coralville, IA).

siRNA transfection

Monocytes or fibroblasts were resuspended in Human Monocyte Nucleofector Solution or Basic Nucleofector Solution - Primary Fibroblasts, respectively (Lonza Group Ltd, Basel, Switzerland) containing 300 nM of paxillin siRNA (Dharmacon, Inc., Lafayette, CO; sequence: 5′-GUGUGGAGCCUUCUUUGGUUU-3′), 300 nM of dynamin siRNA (Santa Cruz Biotechnology, Inc.; sequence: 5′-CCAUCAUGCACCUCAUGAUTT), 300 nM control siRNA (Santa Cruz Biotechnology, Inc.) or RNase-free water (Invitrogen Corp.) and then transfected using an AMAXA Nucleofector (Lonza Group Ltd). siRNA-transfected monocytes or fibroblasts were mixed with pre-equilibrated Human Monocyte Nucleofector Medium [supplemented with 10% human serum (Sigma-Aldrich Inc.)] or Basic Nucleofector Medium – Primary Fibroblasts [supplemented with 4% fetal bovine serum (Sigma-Aldrich Inc.)], respectively (Lonza Group Ltd) and incubated for 48 h at 37°C in 5% CO₂.

HCMV entry assay

Performed as previously described [26], [27]. Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C. Monocytes or fibroblasts were then harvested, total DNA was isolated and quantitative real-time PCR or semiquantitative PCR were performed using primers complementary to genomic HCMV DNA (the UL123/IE1-72 Exon 1 region) and cellular 18S rRNA. Results were plotted as a mean ±SEM. Student's T-tests were performed and p value of <0.05 was used for the measurement of statistical significance between samples. To monitor the accuracy of the assay, representative samples of infected monocytes were kept at 4°C without a temperature shift and processed as described above.

Measurement of a functional HCMV entry

Isolated monocytes were infected with BADwt or BADrUL131 (MOI = 1) at 37°C for 1 hour then plated on fibronectin-coated cell culture dishes in RPMI 1640 supplemented with 10% human serum. Cells were incubated at 37°C in 5% CO₂ for 5 days and 3 weeks post infection and media was changed every 5 days. At 5 days post infection, total DNA was extracted. At 3 weeks post infection, total RNA was extracted. Semiquantitative PCR and RT-PCR were performed using primers complementary to the viral UL123/IE1-72 Exon 1 region and cellular 18S rRNA.

Fluorescence in situ hybridization

Alexa Fluor 488-labelled DNA probe was synthesized according to a protocol in FISH Tag DNA Green Kit (Invitrogen) using BAC DNA encoding the whole HCMV genome. Monocytes were HCMV infected (M.O.I. of 5) and kept at 37°C in 5% CO₂ in RPMI 1640 supplemented with 10% human serum for 5 days. Cells were washed twice with 1× PBS and then fixed for 15 min. with 4% paraformaldehyde (Fisher Scientific). Cells were cytospun onto slides, dried and fixed with methanol (VWR, International, Radnor, PA):acetic acid (VWR International) (3∶1) for 15 min at room temperature (RT). Then cells were dehydrated at RT by subsequent washes with 70%, 85%, and 100% ethanol (Alcohol and Chemicals Co., Shelbyville, KY). Next, DNA inside cells was denatured by incubating cells with 70% formamide (Fisher Scientific) in 2× SSC (Boston BioProducts) at 70°C for 2 min. The dehydratation step followed at −20°C with subsequent washes with 70%, 80%, and 95% ethanol. Denatured DNA probe was then applied to cell spots and hybridization continued overnight at 37°C. After this time, cells were washed with 50% formamide in 2× SSC at 37°C, washed in 1× PBS and the blocking solution [1× PBS/5% normal goat serum (Santa Cruz Biotechnology, Inc.)/1∶80 of Fc Blocking Reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany)/0.3% Triton X-100 (Fisher Scientific)] was applied for 1 h at RT. Next, cells were incubated overnight with 20 µg/ml of anti-Alexa 488 rabbit IgG (Invitrogen) resuspended in the blocking solution. After this time, cells were washed with 1× PBS, the blocking solution was applied for 1 h. Cells then were incubated for 1 h with goat anti-rabbit Alexa 647 IgG (Invitrogen) resuspended in the blocking buffer. Next, cells were washed and mounted using SlowFade Gold with DAPI antifade reagent (Invitrogen). Images were taken using Leica TCS SP5 confocal microscope.