Human Cytomegalovirus Exploits ESCRT Machinery in the Process of Virion Maturation ^▿

Cells.

Primary HFs were cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen Corp., Carlsbad, CA) supplemented with 10% FetalClone III (HyClone, Logan, UT) at 37°C with 5% CO₂. HFs between passages 5 and 15 were used for transfections and infections. Medium was changed every other day in cell culture. HFs that stably express IE1 protein (ihfie1.3) (21) were used for viral plaque assays of the IE1-deficient Towne virus (CR208) (54).

Viruses, DNA constructs, and complementation assays.

HCMV parental Towne strain (51) and IE1-deficient CR208 mutant (54) have been described previously. Plasmids expressing WT and DN forms of Vps4A (pBJ-Vps4A and pBJ-Vps4A_E228Q, respectively) (60) were provided by Arianna Calistri, University of Padua, Padua, Italy. Plasmid expressing CHMP1A_WT protein (pCDNA3-myc-CHMP1A) (55) was obtained from Sylvie Urbe, University of Liverpool, Liverpool, United Kingdom, and the plasmids expressing Tsg101_DN (pCR3.1-GFP-Tsg101_1-157) (37), ALIX_DN (pCR3.1-YFP-ALIX_176-869) (38), and WWP1_DN (pCR3.1-YFP-WWP1_341-547) (36) were obtained from Juan Martin-Serrano, King's College, London, England. Plasmid expressing CHMP1A_DN protein (pEYFPN1-CHMP1A) was kindly provided by Colin Crump, Cambridge University, United Kingdom. Glycoprotein M (gM) and gN amplified from HCMV-TowneBAC were cloned into pDsRed-Monomer-N1 vector (Clontech Laboratories, Inc., Mountain View, CA) to generate DsRed-tagged gM (pON2995) and gN (pON2996).

CR208 complementation assays were performed as described earlier (54). Briefly, primary HFs in six-well tissue culture plates were cotransfected 24 h postseeding with pCMV-IE1₄₉₁-GFP plasmid (pON2994) (54) and either vector control or one of the plasmids expressing WT or a DN ESCRT protein. Cells were analyzed by epifluorescence microscopy for expression and evaluation of IE1₄₉₁-GFP (localized to the nucleus) and ESCRT proteins (expressed predominantly in cytoplasm) in all wells at 24 h posttransfection (hpt). Immunofluorescence was used to optimize plasmid concentrations providing equivalent number of cells coexpressing these proteins. This optimum concentration of plasmids also resulted in maximum inhibition. These cells were infected at 48 hpt with CR208 at a multiplicity of infection (MOI) of 0.002. At such low MOIs, CR208 replicates only in the cells that express IE1 (54). DMEM containing 0.16 mg of pooled human γ-immunoglobulin/ml (to restrict viral spread through medium) was added to the cells after adsorption, and medium was changed every 2 days until day 10 postinfection when the cells were fixed in methanol for 5 min and stained with Giemsa stain (standard Fluka) (Sigma-Aldrich Corp., St. Louis, MO) 1:10 in phosphate-buffered saline (PBS) for 1 h. After staining, plates were washed with distilled water and dried in air, and plaques were counted under optical microscope. Alternatively, antigen (ppUL44) spread was assessed in this assay after fixing cells in 3.7% formaldehyde for 10 min in 1× PBS at day 6 postinfection. Protocols for immunofluorescence microscopy (62) were followed to detect viral antigens. During WT or a complemented virus infection, ppUL44 was expressed in the nucleus with a delayed early kinetics; staining the nucleus diffusely during early infection and localized to nuclear replication compartments later in the infection (21). Noncomplemented CR208 either failed to express ppUL44 or expressed it very poorly, making it easier to identify cells that had expressed IE1 from complementing plasmid before infection. Spread of ppUL44 to cells around the primary infected cell depends on successful maturation and release of virus with spread to adjacent cells where replication ensued. Therefore, restriction of ppUL44 expression to nuclear replication compartments of a single cell was used to indicate the inhibition of viral spread from a primary infected cell in this experiment. A significant decrease in the number of ppUL44 foci (three or more adjacent ppUL44⁺ cells) or plaque formation due to coexpression of a particular DN protein compared to WT-ESCRT protein was interpreted as a viral replication defect. To examine whether infected cell lysates from CR208 complementation assays form plaques on IE1 expressing ihfie1.3 cells, medium was discarded after 3 or 6 days postinfection (dpi), and cells were harvested by brief trypsinization and pipetting in 750 μl of fresh DMEM. These harvested cells were mixed with equal amounts of autoclaved skim milk, sonicated for 10 s on ice, and diluted 10- to 100-fold in serum-free medium for plaque assay on ihfie1.3 cells (21). Medium (DMEM containing 10% serum and 0.16 mg of pooled human γ-immunoglobulin/ml) was changed after 5 days and, at 10 days postinoculation, the cells were washed twice with 1× PBS, fixed in methanol for 5 min, and stained with Giemsa stain as described above. After staining, plates were washed with distilled water and dried in air, and the plaques were counted under a dissecting microscope.

Mutagenesis and secondary spread assays.

HCMV UL32 open reading frame (encoding tegument phosphoprotein pp150) cloned in pLNCX vector (pON2780) (2) was used as a template for QuikChange mutagenesis (Qiagen, Inc., Valencia, CA). Proline and tyrosine in PTAP domain of pp150 were mutated simultaneously to glutamate and phenylalanine, respectively. Inserted mutations were confirmed by diagnosis of new silent restriction sites that were introduced and by sequencing in this region to confirm insertion of desired mutations without undesirable changes. Secondary spread assays were performed as described previously (2, 62). Briefly, primary HFs in six-well tissue culture dishes were cotransfected 24 h after seeding with either Towne-BAC-GFP (13) or ΔUL32-BAC-GFP (2, 62) with pp71 expression plasmid (pON2788) (41) and either WT or mutant UL32 LNCX constructs. Medium (DMEM containing 10% serum and 0.16 mg of pooled human γ-immunoglobulin/ml) was changed every other day until day 10 posttransfection when the cells were fixed in 3.7% formaldehyde-PBS for 10 min prior to evaluation by epifluorescence microscopy. Viral spread was defined as three or more adjacent eGFP-positive cells.

Antibodies.

A mouse monoclonal antibody to ppUL44 (ICP36, cell clone CH16) was purchased from Virusys Corporation, Sykesville, MD. Monoclonal anti-c-myc antibody (9E10; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used to detect myc-tagged CHMP1A and monoclonal anti-FLAG antibody (F3165; Sigma-Aldrich Corporation, St. Louis, MO) was used to detect FLAG-tagged VPS proteins. Hoechst 33258 (AnaSpec Corporation, San Jose, CA) staining identified the nuclei. Secondary antibodies Alexa-594 anti-mouse immunoglobulin G (IgG; H+L) or Alexa-488 anti-mouse IgG (H+L) were purchased from Molecular Probes/Invitrogen, Inc., (Carlsbad, CA).

Microscopy.

Samples were prepared by using established protocols for immunofluorescence assay and confocal fluorescence microscopy. Briefly, HFs were grown on coverslip inserts in 24-well tissue culture dishes. After transfections and infections, at the endpoint, the cells were fixed in 3.7% formaldehyde for 10 min and incubated in 50 mM NH₄Cl in 1× PBS for 10 min to reduce the autofluorescence. This followed washing in 1× PBS, incubation in 0.5% Triton X-100 for 20 min to permeabilize the cells, and finally washing and incubation with primary and secondary antibodies at a 1:1,000 dilution in 0.1% bovine serum albumin in 1× PBS. Coverslips were retrieved from the wells and were mounted on glass slides with a drop of mounting medium (Gel/Mount; Biomeda, Foster City, CA) and dried overnight before imaging. Images were acquired on a Carl-Zeiss LSM 510 META confocal fluorescence microscope or Zeiss Axio Imager A1 epifluorescence microscope.

Employment of ESCRT machinery by HCMV in a Vps4- and CHMP1-dependent manner.

We inhibited the function of individual ESCRT proteins to evaluate impact on HCMV replication in an assay where an HCMV-Towne IE1 mutant virus (CR208) was complemented in trans (54). HFs were infected with CR208 under conditions where viral replication was restricted to cells that transiently expressed the IE1 protein, as well as a WT or DN ESCRT protein. This assay allowed disruption of ESCRT components specifically in virus-infected cells where replication proceeds. A vector (eGFPC₁) lacking IE1 was used as a negative control in this assay. The transfection efficiency of HFs is low (<10%), such that only the infected subpopulation of cells were studied. Equivalent coexpression of IE1 and ESCRT proteins was confirmed by fluorescent protein detection (IE1, Tsg101, and CHMP1A) or immunofluorescence (Vps4) at 48 hpt (data not shown). IE1 was consistently nuclear, and ESCRT components were localized to the cytoplasm. At 48 hpt, monolayers were exposed to CR208 virus at an MOI of 0.002, conditions where this mutant fails to replicate unless complemented. At 72 h postinfection, HCMV-infected cells contain nuclear antigen ppUL44 in a replication compartment pattern. The number of ppUL44-positive cells was not influenced by expression of WT or DN ESCRT components (data not shown). Cells were fixed at endpoint (6 dpi) and stained for ppUL44 expression to evaluate virus production and spread (Fig. 1). Coexpression of IE1 with eGFPC1, Vps4A_WT, or Tsg101_DN yielded similar numbers of ppUL44-positive foci, whereas Vps4A_DN or CHMP1A_DN expression resulted in a seven- to eightfold reduction in numbers of infected foci (Fig. 1A). At this time, ppUL44 was localized to replication compartments in primary transfected cells independent of viral spread (Fig. 1B) confirming that DN ESCRT components inhibit viral replication at late times (after viral DNA replication and during maturation). A combination of CHMP1A_DN and Vps4A_DN resulted in a marginal further reduction in the levels of viral spread compared to Vps4A_DN alone (at 10 dpi), suggesting these proteins function within the same pathway (Fig. 1C), a finding consistent with their roles in MVB formation (27). ALIX_DN and WWP1_DN did not inhibit CMV replication in a similar complementation assay (Fig. 1D). Thus, HCMV replication was inhibited by disruption of either Vps4A (ATPase) or CHMP1A (ESCRT-III component) function. These proteins typically influence the outcome of ESCRT recruitment via upstream parallel pathways (using Tsg101, ALIX, or HECT-Ub ligases) utilized by other viruses (35).

Tsg101-independent nature of ESCRT engagement by HCMV.

Given our observations that Vps4A and CHMP1A are important for HCMV replication, opposite to those previously reported (16), we also evaluated the role of Tsg101 and PTAP recognition in CMV replication. There are only two HCMV proteins (pUL56 and pp150) with putative PTAP motifs that characterize a subset of L domains. These motifs are conserved in all characterized strains of HCMV. pUL56 is a nonstructural protein involved in packaging of viral DNA in the nucleus (4), whereas pp150 (ppUL32) is a major tegument protein required in cytoplasmic maturation steps (2, 62), making this protein an attractive candidate for interaction with ESCRT components. To further examine any role of Tsg101_DN in viral replication, we mutated the pp150 PTAP motif to EFAP. When this mutant was assessed for its ability to complement viral replication in a standard assay (2, 62), the mutant protein retained activity similar to WT-pp150 (Fig. 2). Thus, pp150 PTAP was completely dispensable for virus replication in HFs, a result that was consistent with the lack of any impact of Tsg101_DN in the present study. HCMV thus appears to exploit ESCRT machinery in a Tsg101-independent manner.

Localization of ESCRT proteins in the vicinity of cytoplasmic viral AC.

It has been reported that HCMV derives its final envelope from endocytic membranes (64) and that viral envelope carries the MVB marker CD63 (16) in a pattern that is reminiscent of the role ESCRT machinery plays in the generation of MVBs in uninfected cells (50). To gain insights into the recruitment of ESCRT machinery by HCMV during infection, we studied the localization of ESCRT proteins in HCMV-infected and mock-infected HFs. HCMV-infected cells develop morphologically distinct nuclear (replication compartment) and cytoplasmic (AC) inclusions, where virus replication and maturation, respectively, take place (43). Here, AC was recognized based on the hallmark morphology of CMV-infected cells with nucleus transforming into a kidney bean shape and the AC pressing against this newly formed depression in the nucleus (11, 62). We determined the localization pattern of ESCRT proteins relative to the AC. Both Vps4A_WT (Fig. 3A and B) and Vps4A_DN (Fig. 3E and F) proteins were distributed in the cytoplasm of mock-infected HFs, with Vps4A_DN also accumulating in aggregates at the periphery of the nucleus. During the late phase (5 dpi) of WT-HCMV infection, both of these proteins were found in cytoplasm, as well as in the AC region (Fig. 3C, D, G, and H). Cells containing either Vps4A_WT or Vps4A_DN expressed both early (ppUL44) (Fig. 1B) and late (MCP, gB, gM-gN, and pp150) viral proteins (data not shown), confirming a late replication defect. This was further supported by localization of ppUL44 in nuclear replication compartments in these cells, an expression pattern associated with late infection. CHMP1A_WT protein (Fig. 3I and J) was expressed diffusely in the cytoplasm, as well as in punctate form in the nucleus of mock-infected HFs, but the CHMP1A_DN (Fig. 3M and N) expressed only in cytoplasm of mock-infected HFs in punctate form. In infected cells, CHMP1A_WT showed a predominantly cytoplasmic expression pattern, adjacent to the AC region in infected HFs (Fig. 3K and L), and a similar expression pattern was observed for CHMP1A_DN (Fig. 3O and P) during late stages of infection. Cells expressing either CHMP1A_WT or CHMP1A_DN proteins expressed early (ppUL44) and late (MCP, gB, gM-gN, and pp150) (Fig. 4F and I and data not shown) viral proteins. Appearance of distinct aberrant endosomal vesicles (class E compartments) has been used as a criterion for impairment of ESCRT function in yeast (3, 53) and mammalian cells (12, 17, 26). Aberrant vesicles in the present study were observed only on expression of DN-ESCRT proteins (Fig. 3E to H and M to P) and not on WT-ESCRT proteins expression (Fig. 3A to D and I to L), confirming inhibition of ESCRT pathway by DN-ESCRT proteins.

In order to determine the localization of CHMP1A_WT and CHMP1A_DN relative to viral markers of maturation, both were evaluated relative to viral glycoproteins (gM-gN). Localization of gM-gN proteins has been correlated with the site of viral envelopment in CMV-infected cells (30). Here, gM-gN proteins were expressed in a ring pattern in the vicinity of AC (Fig. 4A and C) and eGFP control protein expressed in diffused form in the whole cell (Fig. 4B and C). WT CHMP1A localized to the periphery of AC during late infection (5 dpi) and appeared to form a concentric ring inside of the gM-gN region (Fig. 4D to F). Similar expression pattern was observed for DN CHMP1A (Fig. 4G to I). Thus, both WT and DN CHMP1A proteins showed similar expression patterns, although DN CHMP1A blocked CMV replication and WT CHMP1A did not (Fig. 1A). The presence of late viral proteins in cells that express DN ESCRT proteins suggests a block in viral life cycle at late stages of infection. Localization of CHMP1A and Vps4A proteins in the vicinity of AC, where the final steps in virus maturation and envelopment occur, indicates an important role of ESCRT pathway during these processes in HCMV life cycle. AC is formed during late stages of HCMV infection (11) and has a concentration of several viral (pp28, pp150, gM-gN, and gB) and cellular (Golgin-97, EEA-1, mannosidase-II, GM130, p230, and Bip/GRP78) markers (11, 30, 62). The AC is a highly organized structure (11), and a number of HCMV mutants with defects in maturation accumulate in AC (57, 58, 62). It may be hypothesized that viral proteins recruit ESCRT components to these sites and ESCRT machinery plays an important role in CMV maturation steps that are associated with this distinct cellular compartment.

Inhibition of ESCRT components and CMV maturation.

Because maturation and envelopment occurs in AC (43) and these compartments incorporate endosomal markers (11) corresponding to MVBs (16), we examined cells that expressed DN-ESCRT components VPS4_DN or CHMP1A_DN to determine whether virus particles that were detected in these cells were replication competent (mature). For this, we evaluated the ability of infected cell lysates from CR208 complementation experiments to form plaques on complementing ihfie1.3 cells. At day 3 postinfection, more than 100 viral plaques were produced from lysates of cells expressing WT-ESCRT or eGFP control proteins, whereas viral plaques were not detected when the lysates of ESCRT inhibited cells were tested (Fig. 5B). BDCRB [2-bromo-5,6-dichloro-1-(-d-ribofuranosyl) benzimidizole] blocks encapsidation of CMV DNA in the nucleus (65); therefore, BDCRB treated cells served as a negative control in this assay. At day 6 postinfection, lysates from ESCRT inhibited cells produced ∼100-fold fewer viral plaques compared to lysates from cells with WT-ESCRT or control plasmids (Fig. 5C). These experiments demonstrated that inhibition of ESCRT machinery blocked HCMV replication prior to virus release. We believe that these data, together with the localization patterns, suggest a block to secondary envelopment within the AC, a step analogous to HSV maturation, as well as to RNA virus envelopment.