A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism Gang Lia,1, Christopher C. Nguyena, Brent J. Ryckmanb, William J. Brittc,d, and Jeremy P. Kamila,1 a Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71103; bDivision of Biological Sciences, Cellular, Molecular and Microbial Biology Program, and Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812; and Departments of cMicrobiology and dPediatrics, University of Alabama at Birmingham, Birmingham, AL 35233

Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/ UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.

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he lipid bilayer membranes of living cells pose an existential challenge to viruses. In enveloped viruses, viral glycoproteins execute a highly regulated fusion event between virion and cellular membranes, thereby delivering the viral genome and other contents of the virion into the host cell. Antibody responses that block entry are considered neutralizing and represent an important host defense against viral pathogens. In many enveloped viruses, one or two viral glycoproteins suffice to carry out binding and membrane fusion events that mediate entry. In herpesviruses, however, at least four envelope glycoproteins are typically involved. The core machinery for herpesvirus entry comprises three highly conserved viral glycoproteins, glycoprotein B (gB), glycoprotein H (gH), and glycoprotein L (gL), along with one or more accessory glycoproteins necessary for binding to cell surface receptors (reviewed in refs. 1, 2). gB is thought to be the proximal mediator of membrane fusion, whereas gH and gL form a complex, termed gH/gL, which has been found to regulate the fusogenic activity of gB (3–6). In a number of beta and gamma herpesviruses, including the human pathogens human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and Epstein–Barr virus (EBV), two different gH/gL complexes are found on the virion envelope and are necessary for the viruses to enter the full range of cell types that they infect in vivo. Of the two gH/gL complexes expressed in HCMV virions, the gH/gL complex with glycoprotein O (gO), gH/gL/gO, suffices for www.pnas.org/cgi/doi/10.1073/pnas.1419875112

entry into fibroblasts, a cell type in which fusion events at the plasma membrane initiate infection (7). Infection of several other types of cells, including monocytes, dendritic cells, endothelial cells, and epithelial cells, requires the pentameric complex of gH/gL and three small glycoproteins—UL128, UL130, and UL131 (UL128-131)—and appears to involve fusion at endosomal membranes (8–16). Strains of HCMV, such as AD169 and Towne, that have undergone extensive serial passage in cultured fibroblasts fail to express the pentameric gH/gL/UL128-131 complex on virions and thus are unable to infect epithelial and endothelial cells (12, 13, 15); however, repair of a frameshift mutation in the UL131 gene of strain AD169 restores expression of gH/gL/UL128-131 (11, 12) and expands its cell tropism. Less extensively passaged HCMV strains that retain expression of gH/gL/UL128-131 can efficiently infect epithelial and endothelial cells (13, 17, 18). Nonetheless, several such strains replicate to ∼1,000-fold lower titers on epithelial cells compared with strain AD169 repaired for UL131 (11). AD169 lacks a ∼15-kb region at the end of the unique long genome region, termed the ULb′ (19). We were intrigued by the rather striking differences in cell tropism between laboratory strain AD169 repaired for expression of the pentameric gH/gL/UL128-131 complex, and strains, such as TB40/E, that have largely intact ULb′ regions and maintain expression of gH/gL/UL128-131. We hypothesized the ULb′ region encodes an additional factor involved in HCMV cell tropism. Our studies addressing this hypothesis led us to identify a new function for UL148, a gene within the ULb′. We found that UL148 encodes an endoplasmic reticulum (ER) resident glycoprotein that influences virion cell tropism by regulating the composition of alternative gH/gL complexes. Significance The entry of a virus into a cell is a fundamental step during infection. In certain herpesviruses, including Epstein–Barr virus, human herpesvirus 6, and human cytomegalovirus (HCMV), a viral glycoprotein complex, gH/gL, plays key roles in entry and is found in two different forms on virions. The relative abundance of the two different types of gH/gL complexes is influenced by the type of cell from which the virus is produced and influences the tropism of the virus for different cell types. We have identified a viral glycoprotein, UL148, that influences the cell tropism of HCMV virions by regulating the relative amounts of these two gH/gL complexes. Our findings have implications for understanding how herpesviruses navigate through host tissues. Author contributions: G.L. and J.P.K. conceived of the study; G.L., B.J.R., W.J.B., and J.P.K. designed research; G.L. and C.C.N. performed research; G.L. and J.P.K. contributed new reagents/analytic tools; G.L. and J.P.K. analyzed data; and J.P.K. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence may be addressed. Email: [email protected] or jkamil@ lsuhsc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1419875112/-/DCSupplemental.

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Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved February 27, 2015 (received for review October 17, 2014)

Titer, log10(IU/mL)

TB_WT TB_148HA TB_∆148

TB_WT TB_148HA TB_∆148

C kDa 250

gH/gL/gO

150 gH/gL/UL128 0

0.5

4

1.5

Fibro. Epi.

1.0

6 dpi

1.2

non-reducing

10 12

**

**

0.9

100

gO

75 37

gH gL

37

0.6

UL130

37

ns

0.3 TB_WT TB_148HA TB_∆148

8

55

0.0

TB

0.0

2

A

6

HA gB reducing

_∆ 14 8

5

H

4

48

3 dpi

TB

2

_1

1.5

1

epi./fibro.

0

Relative infectivity

B

Epithelial cells (ARPE-19)

TB _W T TB _1 48 H A TB _∆ 14 8

Fibroblasts 7 6 5 4 3 2 1

TB _W T

A

Results To determine whether the HCMV UL148 gene encoded a protein that influenced virion cell tropism, we constructed two recombinant viruses based on an infectious bacterial artificial chromosome (BAC) clone of HCMV strain TB40/E (17): TB_148HA and TB_Δ148 (Fig. S1). TB_148HA is a derivative of the wild type TB40/E (TB_WT) that expresses an influenza hemagluttinin epitope (HA) tag at the C terminus of UL148. TB_Δ148 is a derivative of TB_148HA in which a large portion of UL148, comprising most of the 5′ half of the gene, was deleted (Fig. S1). A ∼35-kDa protein immunoreactive to both anti-HA antibodies and to a polyclonal antisera raised against a UL148 synthetic peptide was detected from cells infected with TB_148HA, but not from cells infected with TB_Δ148 (Fig. S1). The protein was expressed with leaky late kinetics and was interpreted as the protein encoded by UL148. During multiplicity of infection (MOI) 1 infection of human foreskin fibroblasts (HFFs), the replication of TB_Δ148 was indistinguishable from that of TB_WT and TB_148HA (Fig. 1A). However, in ARPE-19 human retinal pigment epithelial cells (20), TB_Δ148 replicated to 100-fold higher titers than TB_148HA or TB_WT (Fig. 1A), and caused markedly enhanced cytopathic effects (Fig. S2). When the ability to enter and initiate viral gene expression of TB_Δ148, TB_WT, and TB_148HA was compared side-by-side in HFFs and ARPE-19 cells, TB_Δ148 virions showed approximately equivalent infectivity for HFFs and ARPE-19 cells, whereas virions of WT and TB_148HA showed fivefold higher infectivity for HFFs than for ARPE-19 (Fig. 1B and Figs. S3 and S4). When UL148 was expressed in trans before infection with TB_Δ148, the tropism of progeny virions was restored to that seen for TB_WT, suggesting that the tropism phenotype of TB_Δ148 is related to the absence of the protein encoded by UL148 (Fig. S5). The protein encoded by UL148 is predicted to harbor a signal peptide at the N terminus, which would be cleaved (21), leaving a 265-aa ectodomain anchored by a 23-aa transmembrane helix that terminates in a short (8-aa) cytoplasmic tail (22) (Fig. S1). Nonetheless, how the protein might influence HCMV replication in epithelial cells is unclear. Because alternative gH/gL complexes play important roles in cell tropism of HCMV, we were curious as to whether the tropism phenotype of the TB_Δ148 might involve differences in their expression. Using a gH-specific monoclonal antibody (mAb) to detect gH/gL complexes resolved under nonreducing conditions, we noted that a ∼300-kDa band, which represents the mature gH/gL/gO complex (12), was ∼2.5-fold less abundant in TB_Δ148 virions than in virions of TB_WT or TB_148HA (Fig. 1C and Fig. S6). Although TB40/E 4472 | www.pnas.org/cgi/doi/10.1073/pnas.1419875112

Fig. 1. Disruption of UL148 enhances infection of epithelial cells and alters the ratio of gH/gL complexes in strain TB40/E. (A) Supernatants of cells infected at MOI 1 were collected at indicated time points, and infectivity was determined. IU, infectious units; dpi, days postinfection. (B) Virus preparations were measured by qPCR for genome copies/mL, and TCID 50 was determined in parallel on human fibroblasts and ARPE-19 epithelial cells. TCID 50 results were normalized to viral genome copies and are compared relative to infectivity of WT virus on fibroblasts, which was set to 1.0. Results from three independent experiments are depicted. In both A and B, error bars represent SEM. (C) Lysates of glyceroltartrate gradient-purified virions were electrophoresed under nonreducing conditions, and gH/gL/gO and gH/gL/UL128 complexes were detected using a gH mAb (Upper). Duplicate aliquots of sample were treated with reducing agent and monitored for expression of the indicated proteins (Lower).

virions contain relatively low levels of UL128 (18), a ∼135-kDa band, representing a disulfide-linked complex of gH/gL with UL128 (12), was detectable as well, albeit faintly (Fig. 1C). Whereas levels of gB were consistent across all lanes, TB_Δ148 virions showed decreased levels of gO, gH, and gL compared with TB_WT or TB_148HA (Fig. 1C). To become incorporated into virions, UL130 first must assemble within the ER as subunit of the complete gH/gL/ UL128-131 complex; otherwise, protein constituents of the gH/gL complexes fail to transit to the Golgi (23). The observation of similar UL130 levels across all three lysates thus suggests that virion levels of gH/gL/UL128-131 are not strongly affected by disruption of UL148. Even though we did detect UL148 in TB_148HA virions (Fig. 1C), the bulk of UL148 remained within infected cells, whereas the opposite was true for gH and gB (Fig. S3). Although the significance of the virion-associated UL148 remains unknown, our results suggest that disruption of UL148 leads to decreased levels of gH/gL/gO in virions and enhanced tropism for ARPE-19 epithelial cells. We failed to detect differences in mRNA levels for gH, gL, gO, or UL131 that could explain the tropism differences and effects on gH/gL expression that we observed for TB_Δ148 (Fig. S1). We did find that gH/gL/gO from TB_Δ148-infected cells migrated more rapidly than that from TB_WT- or TB_148HAinfected cells, however (Fig. 2A). gH/gL/gO forms a 220-kDa complex within the ER, but matures to the ∼300-kDa form found on virions only on reaching the Golgi (4, 24, 25). To address whether these mobility differences might indicate differences in glycosylation, we incubated cell lysates of TB_Δ148 and TB_148HA infections with endoglycosidase H (endoH), PNGase F, or buffer alone and assessed for effects on protein mobility. EndoH treatment appeared to enhance overall detection of gO, perhaps because the anti-gO polyclonal antibody that we used was raised against a synthetic peptide, and thus may more efficiently recognize target epitope(s) in the context of partially or fully deglycosylated protein (Fig. 2B). Nonetheless, levels of the slowest migrating, endoH-resistant form of gO were nearly twofold higher on average in TB_148HA-infected HFFs than in TB_Δ148-infected HFFs (Fig. 2B and Fig. S6). Levels of the slowest-migrating form of gH were 1.3-fold higher on average in TB_148HA-infected HFFs compared with TB_Δ148infected HFFs, and the difference was significant (Fig. 2B and Fig. S6). Moreover, the slowest-migrating, endoH-resistant form composed a larger proportion of the gO signal in TB_148HA lysates compared with that in TB_Δ148. EndoH is unable to remove highly branched oligosacharrides found on glycoproteins that traffic to the Golgi, whereas PNGase F removes all N-linked oligosaccharides. Thus, as expected, gO and gH from both lysates Li et al.

Li et al.

37

75 55

3

r AD

13

1_

gO UL130 gH gB

B 6

*

6 fibro. epi.

4 2 0

4 2 0 A

kDa 100

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8 14

H

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HA

c Lu

48

were fully sensitive to PNGase F (Fig. 2B). These results are consistent with the possibility that gH/gL/gO maturation is delayed in the absence of UL148. Moreover, the HA-immunoreactive band, which we interpreted to represent UL148, was fully sensitive to endoH (Fig. 2B), as was a band immunoreactive to anti-UL148 antibodies (Fig. S7). These results suggest that UL148 is mostly retained within the ER during infection, consistent with the presence of a putative RXR ER retention motif (26, 27), RRR, in its predicted cytoplasmic tail (Fig. S1). Furthermore, in confocal microscopy studies of cells infected with TB_148HA, anti-HA antibodies stained a semicontinuous ring-like structure, which colocalized with the ER marker calnexin. The anti-HA staining pattern did not overlap with the compartments stained by antibodies specific for syntaxin-6, a trans-Golgi network (TGN) marker, or by antibodies specific for gH, which instead costained a juxtanuclear structure, previously

A

_1

Fig. 2. UL148 is an ER-resident glycoprotein that influences the maturation of gH and gO. (A) (Upper) Infected fibroblasts were lysed at 72 hpi, and expression of the gH/gL/gO complex was determined under nonreducing conditions. (Lower) Duplicate aliquots were also assayed under reducing conditions for expression of gO, gH, UL130, and β-actin. (B) 72-hpi lysates of fibroblasts infected with TB_148HA or its UL148-null derivative, TB_Δ148, were incubated in the presence of endoH (h) or PNGase F (f), or in buffer lacking enzyme (c), and subsequently assayed by Western blot analysis. (C) Fibroblasts infected with TB_148HA were fixed at 72 hpi and imaged by confocal microscopy after staining with antibodies specific for HA (red) to detect UL148; calnexin (green), an ER marker; syntaxin 6 (green), a TGN marker; and gH, as indicated. DAPI counterstaining of nuclei (blue) is shown in merged images.

uc

Fig. 3. Introduction of UL148 to a laboratory-adapted HCMV strain is sufficient to influence the ratio of gH/gL complexes and cell tropism. (A) Purified virions were compared for the expression of gO, UL130, gH, and gB. (B) TCID50 measurements were normalized to viral genomes per milliliter, and results for each condition are shown relative to those of ADr131_Luc on fibroblasts, which were set to 1.0. Error bars represent SEM. ns, not significant. *P < 0.05, Student t test.

PNAS | April 7, 2015 | vol. 112 | no. 14 | 4473

MICROBIOLOGY

merge

31

gH

r1

HA

AD

merge

epi./fibro.

syntaxin-6

_L

HA

31

merge

r1

calnexin

AD

HA

A

C

H

actin

48

50

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_1

HA

actin

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31

gH

r1

75 37

uc

gO 100 gH 75 37 UL130

100

AD

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100 75 50

identified as the cytoplasmic virion assembly compartment (cVAC), the site in which newly formed virions acquire an infectious envelope (28, 29) (Fig. 2C). Our results are consistent with the notion that the cVAC displaces ER from the side of the nucleus on which it forms, and further suggest that UL148 is an ER-resident glycoprotein (Fig. 2C). We next wished to examine how UL148 expression would affect a laboratory-adapted strain that otherwise lacks UL148. For this, we constructed a new recombinant virus, ADr131_148HA, that harbors an intragenic cassette driving expression of UL148 and, in accordance with the example of previous studies (11, 12), was repaired for the frameshift in UL131 to restore expression of gH/gL/UL128-131 (Fig. S8). Having confirmed that ADr131_148HAinfected cells expressed UL148 (Fig. S8), we compared gH/gL expression and tropism of ADr131_148HA to its parental virus, ADr131_Luc, which harbors a luciferase gene instead of UL148. ADr131_148HA virions showed increased levels of gO and gH and decreased levels of UL130 (Fig. 3A). Accordingly, ADr131_148HA showed an ∼fourfold decrease in tropism for epithelial cells compared with ADr131_Luc (Fig. 3B). Notably, a similar difference in tropism for epithelial cells was observed between EBV virions produced in B cells compared with those produced in epithelial cells (30). However, despite the increased levels of gO, ADr131_148HA virions did not exhibit increased infectivity for fibroblasts (Fig. 3B), and for unknown reasons, ADr131_148HA exhibited a 20-fold replication defect compared with parental ADr131_Luc (Fig. S8). Nonetheless, these results suggest that roles for UL148 could in large part explain the differences in expression of gH/gL complexes and cell tropism between laboratory strain AD169 repaired for UL131 (e.g., BADrUL131) and strains with largely intact ULb′ regions, such as TB40/E, FIX, and TR (Figs. 1 and 3) (11, 12). Because it seemed plausible that the roles of UL148 might involve protein–protein interactions between UL148 and either gH/gL complexes or their constituent proteins, we next conducted coimmunoprecipitation (co-IP) studies. We detected gH, gL, UL130, and UL131, but not gO, UL128, or gB, from antiHA immunoprecipitates of TB_148HA infections (Fig. 4A). We detected no proteins from anti-HA immunoprecipitates from TB_Δ148-infected cell lysates, suggesting that our anti-HA immunoprecipitates are specific for UL148-interacting proteins. Reciprocally, we detected an anti-HA immunoreactive band matching the expected size of UL148 in immunoprecipitates for

Relative infectivity

non-reducing

TB_∆148 c h f

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TB_148HA c h f

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TB _W T TB _1 48 H A TB _∆ 1 gH/gL/gO 48

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gL

75 37

TB

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48 ∆1 TB _

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20 37

48 H

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48 H

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kDa 75 37

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gH gL

UL131

20

UL131

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37

UL130

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UL128

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UL128

100

gO

55 37

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Discussion Despite the clear importance of alternative gH/gL complexes in virion cell tropism of several beta and gamma herpesviruses, the mechanisms that regulate their relative abundance during infection have for the most part remained elusive. Our results argue that HCMV makes use of a virally encoded protein, UL148, to modulate the relative abundance on virions of two alternative gH/gL complexes by influencing their assembly and/or maturation, a finding that suggests a novel mechanism for regulation of virion tropism in a herpesvirus. In strain TB40/E, the presence of UL148 was associated with increased levels of gO, whereas in strain AD169 repaired for expression of gH/gL/ UL128-131, reintroduction of UL148 was associated with both an increase in gO levels and a decrease in UL130 levels (Figs. 1 and 3). These observations suggest that UL148 increases the abundance of the gH/gL/gO complex, and, depending on the genetic background of the virus, that it may do so at the expense of the gH/gL/UL128-131 complex. Although gO null viruses are profoundly defective for entry into all cell types (31–33), the reduced virion gH/gL/gO levels in the UL148 null TB40/E virus were not associated with any overt replication defects during infection of fibroblasts at MOI 1, even though infectivity per genome-containing particle was decreased by approximately twofold (Fig. 1). Moreover, the UL148 null virus showed strikingly enhanced replication, infectivity, and cytopathic effects in epithelial cells, even though virion levels of gH/gL/UL128-131 appeared to be unaffected (Fig. 1 and Fig. S2). These observations suggest not only that HCMV virions may express gH/gL/gO at levels in excess of those needed for entry into fibroblasts, but that the ratio of gH/gL/gO to gH/gL/UL128131 can strongly influence tropism. Indeed, cell type-specific differences in signaling between gH/gL/UL128-131 and gH/gL/gO have been suggested to have consequences for viral fitness (16). We interpret the small amounts of UL148 detected in virions (Fig. 1) with caution. Because the mature envelope of HCMV is derived from post-ER compartments, most bona fide virion envelope glycoproteins can be detected in endoH-resistant forms in infected cells, and they are highly abundant in virions. The vast majority of UL148 was detected in the ER of infected cells in an endoH-sensitive form (Fig. 2 and Figs. S3 and S7), consistent with the presence of an ER retention motif (RXR) in the cytoplasmic tail (26, 27); therefore, it seems unlikely that UL148 would be efficiently incorporated into the mature virion envelope. Taken together, these observations suggest that UL148 exerts its influence on virion cell tropism via its effects on the maturation of gH/gL complexes. EBV arguably provides the most well-understood example of how gH/gL complexes, and hence virion cell tropism, are regulated in a herpesvirus (30, 34). Class II HLA is bound by the gH/gL accessory protein gp42. During replication in B cells, gp42 interacts with class II HLA in the ER and is prevented from reaching assembly sites. Thus, EBV virions produced from B cells are enriched for gH/gL complexes lacking gp42 and exhibit increased tropism for epithelial cells. Because epithelial cells do not express class II HLA, virions produced from epithelial cells contain high amounts of gH/gL/gp42 and show greater tropism for B cells; therefore, it might be proposed that EBV makes use of a cellular protein, HLA class II,

A

TB _

gH (Fig. 4B). The simplest interpretation of our co-IP results is that UL148 interacts with immature gH/gL complexes that contain UL130 and/or UL131, but not gO or UL128. Taken together, our findings identify UL148 as a virally encoded factor that acts within the ER to influence the cell tropism of a herpesvirus by regulating the composition of alternative gH/gL complexes on virions. Furthermore, the data are consistent with a mechanism involving protein–protein interactions occurring in the ER between UL148 and a specific subset of immature gH/gL complexes and/or their constituent proteins.

HA

100

gO

75

gH

Fig. 4. Co-IP studies from infected cells. (A) Anti-HA immunoprecipitates from lysates of TB_Δ148- and TB_148HA-infected fibroblasts were assayed alongside input lysates for detection of gH, gL, UL128, UL130, UL131, gO, gB, and HA epitope. (B) Lysates of TB_148HA-infected fibroblasts were immunoprecipitated using anti-gH mAb or nonspecific control IgG and monitored alongside input lysates for detection of HA epitope, gO, and gH.

to regulate expression of its alternative gH/gL complexes, whereas HCMV makes use of a viral protein, UL148, to do so. The finding that both viruses use factors within the ER to regulate gH/gL complexes illustrates the relevance of the organelle as a foundry for determining the tropism of herpesvirus virions. Because the ER is where newly translated proteins begin their journey through the secretory pathway, factors within this organelle are well positioned to influence the repertoire of glycoproteins available for incorporation into the virion envelope. The finding that UL148 interacts with gH/gL complexes containing UL130 and UL131, but not UL128 or gO, provides clues as to the mechanism by which UL148 might influence the assembly of gH/gL complexes (Fig. 4). A recent study indicated that UL128 and gO form disulfide bonds to the same cysteine residue on gL (35). This finding is consistent with previous data showing that (i) whereas UL128, UL130, and UL131 each can bind independently to gH/gL, the presence of UL128 greatly increases the loading of UL130 and UL131 (23), and (ii) gO and the UL128-131 proteins compete for binding of gH/gL (12, 36). In this context, we propose a model in which UL148 favors increased levels of gH/gL/gO on virions by competing with UL128 for loading onto partially assembled gH/gL complexes containing UL130 and/or UL131 (Fig. 5). Because gO and UL128 each form disulfide bonds to gH/gL, whereas UL130 and UL131 do not (12, 24), UL148 may promote the maturation of gH/gL/gO by occluding the formation of a disulfide bond between gH/gL and UL128. Formation of a disulfide bond between UL128 and gH/gL might irreversibly commit a gH/gL dimer toward assembly into the pentameric gH/gL/UL128-131 complex, whereas binding of UL130 and UL131 to gH/gL may reversibly interfere with loading of gO. Once bound to UL148, complexes of gH/gL with UL130 and/or UL131 might eventually dissociate, providing an opportunity for gO to load. Given that our results suggest that UL148 exerts its influence via protein–protein interactions with gH/gL complexes or their constituent proteins (Fig. 4), it is worth considering how other factors that affect assembly of gH/gL complexes might impact Li et al.

*Sturgill ER, et al. (2014) Natural killer cell evasion by Rhesus cytomegalovirus: Discovery of NKG2D immunoevasins. in The 39th Annual International Herpesvirus Workshop (Kobe, Japan), p 193.

Li et al.

Endoplasmic Reticulum gO

s-s

gH/gL

s-s

Golgi Apparatus

UL128

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gH/gL/UL128-131

gH/gL/UL130/UL148 gH/gL/UL131/UL148 s-s:disulfide bond Fig. 5. Model for the regulation of alternative gH/gL complexes by UL148. During the assembly of gH/gL complexes in the ER, UL148 competes with UL128 for loading onto immature gH/gL complexes that contain UL130 and/or UL131. The resulting gH/gL/UL130/UL148 and gH/gL/UL131/UL148 complexes are retained in the ER. Because gO and UL128 form covalent, disulfide linkages (s-s) with gH/gL, whereas UL131 and UL130 do not, gH/gL/ UL130 and gH/gL/UL131 complexes can dissociate to allow loading of gO. By competing for loading of UL128 onto gH/gL, UL148 selectively promotes the formation of gH/gL/gO complexes, which mature to the Golgi apparatus and ultimately become incorporated into virions.

Materials and Methods Cells and Viruses. HFFs were cultured as described previously (45). ARPE-19 cells (20) were cultivated in the same media conditions used for HFFs. TB40-BAC4 (17), a BAC clone of HCMV strain TB40/E, was a gift from Christian Sinzger (Ulm University Medical Center, Ulm, Germany). All other viruses were derived from either TB40-BAC4 or the strain AD169 derivative pp28_Luc (46). Details of the construction of TB_Δ148, TB_148HA, ADr131_Luc, and AD131_148HA are provided in SI Materials and Methods. Infectious virus was reconstituted from BAC DNA, propagated on HFFs, concentrated by ultracentrifugation through a 20% sorbitol cushion, and measured for infectious units (IU) per milliliter, all as described previously (45, 47). Replication kinetics studies were conducted using infected cell supernatants, as described previously (45, 47). Glycerol-tartrate gradient purification was performed as described elsewhere (48, 49). Tropism Studies. HFFs were infected at MOI 1, and 144-hpi supernatants were collected and measured for infectivity in TCID50 assays conducted in parallel in fibroblasts and ARPE-19 epithelial cells (Fig. S3). TCID50 assays were fixed at 24 hpi and stained using IE1 mAb 1B12 (50) to visualize wells positive for infection, as described previously (45, 47). Duplicate aliquots of supernatants were treated with RQ1 RNase-Free DNase I (Promega), proteinase K digested at 37 °C overnight, and phenol-chloroform extracted. Viral DNA was precipitated and resuspended in 10 mM Tris·HCl and 0.5 mM EDTA, and then subjected to real-time quantitative PCR (qPCR) to quantify viral genomes, as described previously (45, 47). Infectivity, in TCID50 units normalized to viral genome copies, was calculated for each experimental condition. In the case of ADr131_148HA vs. ADr131_Luc (Fig. 3B), glycerol-tartrate gradient-purified virions were used as starting material instead of 144-hpi supernatants. Immunoprecipitiation. Infected HFFs were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholate). Lysates were clarified by centrifugation at 13,800 × g (4 °C, 20 min), and then rotated at 4 °C overnight in the presence of rabbit anti-HA polyclonal antibody (Bethyl Laboratories; A190-108A), mouse anti-gH mAb 14–4b (51, 52), or control IgG (as indicated), together with protein A/G agarose beads (Thermo Scientific; Pierce 20423). Following washing steps, bound proteins were eluted by a 5-min incubation at 90 °C in SDS/PAGE sample buffer (20% glycerol, 4% SDS, 100 mM Tris·Cl pH 6.8, 4 mM EDTA, and 5% beta-mercaptoethanol). Endoglycosidase Analysis. EndoH (EndoH f) and PNGase F enzymes (both from New England Biolabs) were used according to the manufacturer’s recommendations. Confocal Microscopy. Infected HFFs were paraformaldehyde-fixed at 72 hpi. The following primary antibodies were used: rabbit anti-calnexin (2679) and anti-syntaxin 6 (1869; both from Cell Signaling Technologies), mouse

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its function. Polymorphisms in the UL128-131 locus of strains TB40/E and FIX, when incorporated into strain Merlin, were found to strongly influence virion incorporation of UL128 and cell tropism (18). Similarly, polymorphisms in gH/gL or gO might contribute to cell tropism differences by affecting the expression of alternative gH/gL complexes. Therefore, strain-specific polymorphisms in the proteins that participate in virion gH/gL complexes might help explain why reintroduction of UL148 to strain AD169 (repaired for UL131) led to both a decrease in virion levels of gH/gL/UL128-131 and an increase in levels of gH/gL/gO, whereas disruption of UL148 in strain TB40/E appeared to only decrease virion levels of gH/gL/gO (Figs. 1 and 3). Although the UL148 gene is disrupted in laboratory strains such as AD169 and the Towne short variant, it is typically found intact in less extensively passaged strains (19, 37–39). To our knowledge, viruses with functional UL128-131 loci have not been found to harbor spontaneous disruptions in UL148, suggesting that there may be selective pressure, at least during passage in fibroblasts, for the virus to retain UL148 as long as gH/gL/ UL128-131 is expressed, presumably owing to the potential of UL148 to preserve high levels of gH/gL/gO during comaturation of gH/gL/UL128-131. Thus, we speculate that during tissue culture adaptation, disruptions in UL128-131 likely would occur before loss of UL148 expression. Although we found that HCMV deploys UL148 to regulate its virion gH/gL complexes, it would be premature to exclude the possibility that the regulation of cell tropism in HCMV might involve cellular proteins as well. Along these lines, it is intriguing to note that another research group recently found that the rhesus cytomegalovirus (RhCMV) homolog of UL148, Rh159, binds to natural killer (NK) cell activating ligands within the ER and prevents their movement to the cell surface.* Interestingly, Rh159 was identified as an epithelial tropism factor of RhCMV (40), even though the Rh159 mutant in the latter study evaluated was in the 68–1 strain background, which lacks expression of the pentameric gH/gL complex (41, 42) and thus should be at least partially defective for entry into epithelial cells. That herpesviruses would encode ER-resident proteins to prevent surface presentation of immune cell activating ligands is hardly surprising; however, NK cell ligands and alternative HCMV glycoprotein complexes involved in cell tropism each transit through the ER. Because virally encoded ER-resident proteins would be able to influence viral fitness through effects on both cell tropism and immune evasion, it is conceivable that the evolutionary process referred to as “tinkering” (43) might well have saddled a viral NK cell evasion gene with roles in the regulation of virion tropism, or vice versa. Regardless of whether UL148 shares the potential of Rh159 to bind NK cell-activating ligands, its effects on gH/gL complexes might be regulated via interactions with cellular factors. Cell type-specific expression of such factors could provide a mechanism for cell tropism switching observed in HCMV (44), akin to that seen for EBV. Recognizable homologs of UL148 appear to be conserved only among primate cytomegaloviruses. Nonetheless, it seems likely that other beta and gamma herpesviruses also might express ER-resident glycoproteins that influence the composition of alternative gH/gL complexes on their virions in a manner analogous to that of UL148. To infect host tissues involved in latency, persistence, and shedding, HCMV requires the ability to enter an array of distinct cell types. Further investigation into the mechanisms by which UL148 influences HCMV cell tropism may provide new and unexpected insights into how the virus navigates through the human host.

ACKNOWLEDGMENTS. We thank David C. Johnson (Oregon Health Sciences University), Thomas Shenk (Princeton University), Christian Sinzger (Ulm University Medical Center), Don Coen (Harvard Medical School), and Michael

McVoy (Virginia Commonwealth University) for contributing reagents, and Malgorzata Bienkowska-Haba and Georgia Morgan (Louisiana State University Health Sciences Center) for assistance with confocal microscopy. We are also grateful to Christine M. O’Connor (Cleveland Clinic), Lindsey Hutt-Fletcher (Louisiana State University Health Sciences Center-Shreveport), and Klaus Früh and Elizabeth Sturgill (Oregon Health Sciences University) for helpful discussions. This work was supported by the National Institutes of Health (Grants P20GM103433 and P30GM110703) and by awards from the American Heart Association.

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anti-HA (sc7392; Santa Cruz Biotechnology) or rabbit anti-HA (Bethyl), and anti-gH clone AP86 (12). Alexa Fluor-labeled goat polyclonal antibodies were used for secondary detection using a Leica TCS SP5 spectral confocal microscope. Additional details are provided in SI Materials and Methods.

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Li et al.