Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.Human cytomegalovirus (HCMV) infects many different cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, smooth muscle cells, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (reviewed in references 5, 30, 38, and 45). In the laboratory, HCMV is normally propagated in primary human fibroblasts because most other cell types yield low titers of virus. Commonly studied laboratory strains, such as AD169, were propagated extensively in fibroblasts, and this was accompanied by deletions or mutations in a cluster of 22 genes known as ULb′ (6). These mutations were correlated with the inability to infect other cell types, including endothelial and epithelial cells and monocyte-macrophages. Targeted mutagenesis of three of the ULb′ genes, UL128, UL130, and UL131, abolished infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (13, 15). Restoration of the UL128-131 genes in laboratory strains of HCMV strains restored the capacity to infect endothelial and epithelial cells and other cells (15, 52).The UL128, UL130, and UL131 proteins assemble onto the extracellular domain of HCMV gH/gL (1, 42, 53). For all herpesviruses, gH/gL complexes mediate entry into cells (12, 33, 39), suggesting that gH/gL/UL128-131 might participate in the entry mechanism. Indeed, we demonstrated that gH/gL/UL128-131 mediates entry into epithelial and endothelial cells by using the fusogenic agent polyethylene glycol to force entry of HCMV UL128-131 mutants into these cell types (41). This was consistent with reports that UL128-, UL130-, and UL131-specific antibodies blocked the capacity of HCMV to infect epithelial and endothelial cells but not fibroblasts (1, 53). Furthermore, expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV infection, consistent with saturable gH/gL/UL128-131 receptors (40). Expression of all five proteins was necessary so that the gH/gL/UL128-131 complexes were exported from the endoplasmic reticulum (ER) and could function (40-42, 53). Together, these data suggested that gH/gL/UL128-131 mediates entry into epithelial/endothelial cells but is not required for entry into fibroblasts. By extension, it was reasonable to propose that other forms of gH/gL might facilitate the entry into fibroblasts.The laboratory HCMV strain AD169 is known to express a second gH/gL complex containing glycoprotein O (gO) (21-23, 53). In cells infected with a recombinant AD169 in which the UL131 mutation was repaired, gH/gL/gO complexes were separate from gH/gL/UL128-131 complexes, i.e., gO was not detected following immunoprecipitation (IP) with UL128- and UL130-specifc antibodies, and gO-specific antibodies did not precipitate UL128 and UL130 (53). AD169 and Towne gO⁻ mutants produce small plaques on fibroblast monolayers and low titers of virus, supporting an important, although not essential, role for gH/gL/gO in virus replication in fibroblasts (11, 19). AD169 does not infect endothelial and epithelial cells, so AD169 gO⁻ mutants were not tested on these cells. Jiang et al. described a gO-null mutant derived from an endotheliotropic HCMV strain, TB40/E (27). The TB40/E gO-null mutant spread normally on endothelial cells, suggesting that gO or gH/gL/gO is less important for infection and spread in these cells. Given that the role of gH/gL in entry is highly conserved among the herpesviruses, it seemed likely that gH/gL/gO might be involved in entry into fibroblasts. Consistent with this notion, Paterson et al. showed that anti-gO antibodies decreased fusion from without caused by infection of cells with HCMV AD169 (37). These observations supported our working model in which gH/gL/UL128-131 mediates entry into epithelial and endothelial cells, while gH/gL/gO mediates entry into fibroblasts. There is also the possibility that gH/gL (lacking gO and UL128-131) might be incorporated into the virion envelope, although there is presently no direct evidence for this. Any gH/gL detected biochemically might result from dissociation of gO or UL128-131 during sample preparation and analysis. gH/gL expressed without other HCMV proteins was retained in the ER (42), arguing against incorporation into the virion.Other herpesviruses, e.g., Epstein-Barr virus, human herpesvirus 6 (HHV-6), and HHV-7, use different forms of gH/gL to enter different cell types via different pathways (25, 34, 43). Similarly, HCMV entry into fibroblasts occurs by fusion at the plasma membrane at a neutral pH and does not require gH/gL/UL128-131 (7), whereas entry into epithelial and endothelial cells involves endocytosis and low pH-dependent fusion and requires gH/gL/UL128-131 (41).All of the biochemical analyses of gO in terms of binding to gH/gL and intracellular transport have involved fibroblast-adapted strain AD169 (21-23, 31, 53). These studies indicated that gO is a 110- to 125-kDa glycoprotein encoded by the UL74 gene (22). Glycosidase digestion experiments demonstrated that the gO polypeptide chain is ∼62 to 65 kDa (21-23, 53). Pulse-chase studies showed that gH/gL assembles in the ER as a disulfide-linked heterodimer (28) that subsequently binds to, and establishes disulfides with, gO (22, 23). The 220-kDa immature gH/gL/gO trimer is initially sensitive to endoglycosidase H (endo H), which removes immature N-linked oligosaccharides from glycoproteins present in the ER (22, 23). Transport of gH/gL/gO to the Golgi apparatus is associated with processing of N-linked oligosaccharides to mature forms that resist endo H. Also associated with transport to the Golgi apparatus is the addition of O-linked oligosaccharides and phosphorylation, increasing the molecular weight of gO (after reduction) to 125 to 130 kDa and that of the gH/gL/gO complex to 240 to 260 kDa (22, 23, 29). It is the mature glycoprotein complex, previously known as gCIII, that is trafficked to HCMV assembly compartments for incorporation into the virion envelope (22, 23, 29).In addressing the function of gO, it is important to recognize that AD169 has adapted to replication in fibroblasts, losing expression of UL131 and failing to assemble gH/gL/UL128-131 complexes (6) (15). There seems to be strong pressure to mutate UL128-131, because clinical strain Merlin acquired a UL128 mutation within 5 passages on fibroblasts (2). It is also reasonable to suggest that fibroblast adaptation includes changes in gO. The gO genes (UL74) of several laboratory and clinical strains and clinical isolates are highly variable (up to 25% of amino acids) (10, 35, 37, 47). However, it is important to note that AD169-derived UL131-repair virus can infect epithelial and endothelial cells (52). Thus, if AD169 gO is important for infection of these cells, then gO must be functionally normal in this regard. These differences in laboratory versus clinical HCMV prompted us to characterize the gO molecule expressed by the HCMV strain TR. HCMV TR is a clinical isolate that was stabilized in the form of a bacterial artificial chromosome (BAC) after very limited passage in fibroblasts (35, 41). HCMV TR expresses gH/gL/UL128-131 (42) and infects epithelial and endothelial cells (41) and monocyte-macrophages well (D. Streblow and J. Nelson, unpublished results).Here, we report our biochemical and cell trafficking analyses of the TR gO protein. We were surprised to find that TR gO was not present in extracellular virus particles. In contrast, gO was detected in extracellular AD169 particles, consistent with previous findings (22). TR gO expressed either in HCMV-infected cells or by using nonreplicating Ad vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of TR gO with gH/gL promoted transport of gH/gL beyond the ER, and gO was slowly lost from gH/gL complexes but not secreted from cells and not observed in extracellular virus particles. Thus, TR gO acts as a chaperone. Consistent with this, in the accompanying paper by Wille et al. (54), a TR gO-null mutant was described that secreted extracellular particles containing markedly reduced quantities of gH and gL. The gO⁻ mutant failed to enter fibroblasts and also epithelial and endothelial cells. Together, these results suggest that it is gH/gL, not gH/gL/gO, which is incorporated into HCMV TR virions. It appears that gH/gL is required for entry into fibroblasts, and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.     

Human Cytomegalovirus Vaccine Based on the Envelope gH/gL Pentamer Complex

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.     

Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.     

The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as “vehicles” for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.     

Isolation of Human Monoclonal Antibodies That Potently Neutralize Human Cytomegalovirus Infection by Targeting Different Epitopes on the gH/gL/UL128-131A Complex

Human cytomegalovirus (HCMV) is a widely circulating pathogen that causes severe disease in immunocompromised patients and infected fetuses. By immortalizing memory B cells from HCMV-immune donors, we isolated a panel of human monoclonal antibodies that neutralized at extremely low concentrations (90% inhibitory concentration [IC₉₀] values ranging from 5 to 200 pM) HCMV infection of endothelial, epithelial, and myeloid cells. With the single exception of an antibody that bound to a conserved epitope in the UL128 gene product, all other antibodies bound to conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex. Antibodies against gB, gH, or gM/gN were also isolated and, albeit less potent, were able to neutralize infection of both endothelial-epithelial cells and fibroblasts. This study describes unusually potent neutralizing antibodies against HCMV that might be used for passive immunotherapy and identifies, through the use of such antibodies, novel antigenic targets in HCMV for the design of immunogens capable of eliciting previously unknown neutralizing antibody responses.Human cytomegalovirus (HCMV) is a member of the herpesvirus family which is widely distributed in the human population and can cause severe disease in immunocompromised patients and upon infection of the fetus. HCMV infection causes clinical disease in 75% of patients in the first year after transplantation (58), while primary maternal infection is a major cause of congenital birth defects including hearing loss and mental retardation (5, 33, 45). Because of the danger posed by this virus, development of an effective vaccine is considered of highest priority (51).HCMV infection requires initial interaction with the cell surface through binding to heparan sulfate proteoglycans (8) and possibly other surface receptors (12, 23, 64, 65). The virus displays a broad host cell range (24, 53), being able to infect several cell types such as endothelial cells, epithelial cells (including retinal cells), smooth muscle cells, fibroblasts, leukocytes, and dendritic cells (21, 37, 44, 54). Endothelial cell tropism has been regarded as a potential virulence factor that might influence the clinical course of infection (16, 55), whereas infection of leukocytes has been considered a mechanism of viral spread (17, 43, 44). Extensive propagation of HCMV laboratory strains in fibroblasts results in deletions or mutations of genes in the UL131A-128 locus (1, 18, 21, 36, 62, 63), which are associated with the loss of the ability to infect endothelial cells, epithelial cells, and leukocytes (15, 43, 55, 61). Consistent with this notion, mouse monoclonal antibodies (MAbs) to UL128 or UL130 block infection of epithelial and endothelial cells but not of fibroblasts (63). Recently, it has been shown that UL128, UL130, and UL131A assemble with gH and gL to form a five-protein complex (thereafter designated gH/gL/UL128-131A) that is an alternative to the previously described gCIII complex made of gH, gL, and gO (22, 28, 48, 63).In immunocompetent individuals T-cell and antibody responses efficiently control HCMV infection and reduce pathological consequences of maternal-fetal transmission (13, 67), although this is usually not sufficient to eradicate the virus. Albeit with controversial results, HCMV immunoglobulins (Igs) have been administered to transplant patients in association with immunosuppressive treatments for prophylaxis of HCMV disease (56, 57), and a recent report suggests that they may be effective in controlling congenital infection and preventing disease in newborns (32). These products are plasma derivatives with relatively low potency in vitro (46) and have to be administered by intravenous infusion at very high doses in order to deliver sufficient amounts of neutralizing antibodies (4, 9, 32, 56, 57, 66).The whole spectrum of antigens targeted by HCMV-neutralizing antibodies remains poorly characterized. Using specific immunoabsorption to recombinant antigens and neutralization assays using fibroblasts as model target cells, it was estimated that 40 to 70% of the serum neutralizing activity is directed against gB (6). Other studies described human neutralizing antibodies specific for gB, gH, or gM/gN viral glycoproteins (6, 14, 26, 29, 34, 41, 52, 60). Remarkably, we have recently shown that human sera exhibit a more-than-100-fold-higher potency in neutralizing infection of endothelial cells than infection of fibroblasts (20). Similarly, CMV hyperimmunoglobulins have on average 48-fold-higher neutralizing activities against epithelial cell entry than against fibroblast entry (10). However, epitopes that are targeted by the antibodies that comprise epithelial or endothelial cell-specific neutralizing activity of human immune sera remain unknown.In this study we report the isolation of a large panel of human monoclonal antibodies with extraordinarily high potency in neutralizing HCMV infection of endothelial and epithelial cells and myeloid cells. With the exception of a single antibody that recognized a conserved epitope of UL128, all other antibodies recognized conformational epitopes that required expression of two or more proteins of the gH/gL/UL128-131A complex.     

Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

Histone deacetylation plays a pivotal role in regulating human cytomegalovirus gene expression. In this report, we have identified candidate HDAC1-interacting proteins in the context of infection by using a method for rapid immunoisolation of an epitope-tagged protein coupled with mass spectrometry. Putative interactors included multiple human cytomegalovirus-coded proteins. In particular, the interaction of pUL38 and pUL29/28 with HDAC1 was confirmed by reciprocal immunoprecipitations. HDAC1 is present in numerous protein complexes, including the HDAC1-containing nucleosome remodeling and deacetylase protein complex, NuRD. pUL38 and pUL29/28 associated with the MTA2 component of NuRD, and shRNA-mediated knockdown of the RBBP4 and CHD4 constituents of NuRD inhibited HCMV immediate-early RNA and viral DNA accumulation; together this argues that multiple components of the NuRD complex are needed for efficient HCMV replication. Consistent with a positive acting role for the NuRD elements during viral replication, the growth of pUL29/28- or pUL38-deficient viruses could not be rescued by treating infected cells with the deacetylase inhibitor, trichostatin A. Transient expression of pUL29/28 enhanced activity of the HCMV major immediate-early promoter in a reporter assay, regardless of pUL38 expression. Importantly, induction of the major immediate-early reporter activity by pUL29/28 required functional NuRD components, consistent with the inhibition of immediate-early RNA accumulation within infected cells after knockdown of RBBP4 and CHD4. We propose that pUL29/28 modifies the NuRD complex to stimulate the accumulation of immediate-early RNAs.     

Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions

Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of (3)H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with (14)C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus. (14)C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before (14)C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug.     

Characterization of the human cytomegalovirus gH/gL/UL128-131 complex that mediates entry into epithelial and endothelial cells

The entry of human cytomegalovirus (HCMV) into biologically relevant epithelial and endothelial cells involves endocytosis followed by low-pH-dependent fusion. This entry pathway is facilitated by the HCMV UL128, UL130, and UL131 proteins, which form one or more complexes with the virion envelope glycoprotein gH/gL. gH/gL/UL128-131 complexes appear to be distinct from the gH/gL/gO complex, which likely facilitates entry into fibroblasts. In order to better understand the assembly and protein-protein interactions of gH/gL/UL128-131 complexes, we generated HCMV mutants lacking UL128-131 proteins and nonreplicating adenovirus vectors expressing gH, gL, UL128, UL130, and UL131. Our results demonstrate that UL128, UL130, and UL131 can each independently assemble onto gH/gL scaffolds. However, the binding of individual UL128-131 proteins onto gH/gL can significantly affect the binding of other proteins; for example, UL128 increased the binding of both UL130 and UL131 to gH/gL. Direct interactions between gH/UL130, UL130/UL131, gL/UL128, and UL128/UL130 were also observed. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface was dramatically increased when all of UL128, UL130, and UL131 were coexpressed with gH/gL (with or without gO expression). Incorporation of gH/gL complexes into the virion envelope requires transport beyond the ER. Thus, we concluded that UL128, UL130, and UL131 must all bind simultaneously onto gH/gL for the production of complexes that can function in entry into epithelial and endothelial cells.     

The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)₂ motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.     

A Human Cytomegalovirus gO-Null Mutant Fails To Incorporate gH/gL into the Virion Envelope and Is Unable To Enter Fibroblasts and Epithelial and Endothelial Cells

Human cytomegalovirus (HCMV) depends upon a five-protein complex, gH/gL/UL128-131, to enter epithelial and endothelial cells. A separate HCMV gH/gL-containing complex, gH/gL/gO, has been described. Our prevailing model is that gH/gL/UL128-131 is required for entry into biologically important epithelial and endothelial cells and that gH/gL/gO is required for infection of fibroblasts. Genes encoding UL128-131 are rapidly mutated during laboratory propagation of HCMV on fibroblasts, apparently related to selective pressure for the fibroblast entry pathway. Arguing against this model in the accompanying paper by B. J. Ryckman et al. (J. Virol., 84:2597-2609, 2010), we describe evidence that clinical HCMV strain TR expresses a gO molecule that acts to promote endoplasmic reticulum (ER) export of gH/gL and that gO is not stably incorporated into the virus envelope. This was different from results involving fibroblast-adapted HCMV strain AD169, which incorporates gO into the virion envelope. Here, we constructed a TR gO-null mutant, TRΔgO, that replicated to low titers, spread poorly among fibroblasts, but produced normal quantities of extracellular virus particles. TRΔgO particles released from fibroblasts failed to infect fibroblasts and epithelial and endothelial cells, but the chemical fusogen polyethylene glycol (PEG) could partially overcome defects in infection. Therefore, TRΔgO is defective for entry into all three cell types. Defects in entry were explained by observations showing that TRΔgO incorporated about 5% of the quantities of gH/gL in extracellular virus particles compared with that in wild-type virions. Although TRΔgO particles could not enter cells, cell-to-cell spread involving epithelial and endothelial cells was increased relative to TR, apparently resulting from increased quantities of gH/gL/UL128-131 in virions. Together, our data suggest that TR gO acts as a chaperone to promote ER export and the incorporation of gH/gL complexes into the HCMV envelope. Moreover, these data suggest that it is gH/gL, and not gH/gL/gO, that is present in virions and is required for infection of fibroblasts and epithelial and endothelial cells. Our observations that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial/endothelial cells differ from models for other beta- and gammaherpesviruses that use one of two different gH/gL complexes to enter different cells.Human cytomegalovirus (HCMV) infects a broad spectrum of cell types in vivo, including epithelial and endothelial cells, fibroblasts, monocyte-macrophages, dendritic cells, hepatocytes, neurons, glial cells, and leukocytes (6, 28, 36). Infection of this diverse spectrum of cell types contributes to the multiplicity of CMV-associated disease. HCMV infection of hepatocytes and epithelial cells in the gut and lungs following transplant immunosuppression is directly associated with CMV disease (3, 44). HCMV can be transported in the blood by monocyte-macrophages, and virus produced in these cells can infect endothelial cells, leading to virus spread into solid tissues such as the brain, liver, and lungs, etc. (16). Despite the broad spectrum of cells infected in vivo, propagation of HCMV in the laboratory is largely limited to normal human fibroblasts because other cells produce little virus. HCMV rapidly adapts to laboratory propagation in fibroblasts, losing the capacity to infect other cell types, i.e., epithelial and endothelial cells and monocyte-macrophages (9, 16, 18, 43). This adaptation to fibroblasts involves mutations in the unique long b′ (ULb′) region of the HCMV genome, which includes 22 genes (9). Targeted mutation of three of the ULb′ genes, UL128, UL130, and UL131, abolished HCMV infection of endothelial cells, transmission to leukocytes, and infection of dendritic cells (17, 18). Restoration of UL128-131 genes in HCMV laboratory strain AD169 (which cannot infect epithelial and endothelial cells) produced viruses capable of infecting these cells (18, 48). There is also evidence that the UL128-131 proteins are deleterious to HCMV replication in fibroblasts, resulting in rapid loss or mutation of one or more of the UL128-131 genes during passage in fibroblasts (2).A major step forward in understanding how the UL128-131 genes promote HCMV infection of epithelial and endothelial cells involved observations that the UL128-131 proteins assemble onto the extracellular domain of the membrane-anchored HCMV glycoprotein heterodimer gH/gL (1, 49). Antibodies to UL128, UL130, and UL131 each neutralized HCMV for infection of endothelial or epithelial cells (1, 49). All herpesviruses express gH/gL homologues and, where this has been tested, all depend upon gH/gL for replication and, more specifically, for entry into cells (14, 15, 31, 38). Indeed, we showed that the gH/gL/UL128-131 complex mediated entry into epithelial and endothelial cells (40). All five members of the gH/gL/UL128-131 complex were required for proper assembly and export from the endoplasmic reticulum (ER) and for function (39, 41). In addition, the expression of gH/gL/UL128-131, but not gH/gL or gB, in epithelial cells interfered with HCMV entry into these cells (39). This interference suggested that there are saturable gH/gL/UL128-131 receptors present on epithelial cells, molecules that HCMV uses for entry. There was no interference in fibroblasts expressing gH/gL/UL128-131, although some interference was observed with gH/gL (39). As noted above, gH/gL/UL128-131 plays no obvious role in entry into fibroblasts and, in fact, appears to be deleterious in this respect (2, 18, 40).HCMV also expresses a second gH/gL complex, as follows: gH/gL/gO (20, 21, 22, 30, 48). Fibroblast-adapted HCMV strain AD169 expresses a gO protein that is a 110- to 125-kDa glycoprotein (21). Pulse-chase studies suggest that gH/gL assembles first in the ER before binding and forming disulfide links with gO (21, 22). The 220-kDa immature gH/gL/gO complex is transported from the ER to the Golgi apparatus and increases in size to ∼280 to 300 kDa before incorporation into the virion envelope (21). gH/gL/gO complexes are apparently distinct from gH/gL/UL128-131 complexes because gO-specific antibodies do not detect complexes containing either UL128 or UL130 and UL128-specific antibodies do not precipitate gO (49). Towne and AD169 gO-null mutant laboratory strains can produce small plaques on fibroblasts, leading to the conclusion that gO is not essential. However, the AD169 and Towne mutants produced ∼1,000-fold less infectious virus than wild-type HCMV (14, 19), which might also be interpreted to mean that gO is very important or even essential for replication. Thus, the prevailing model has been that wild-type HCMV particles contain the following two gH/gL complexes: gH/gL/gO, which promotes infection of fibroblasts, and gH/gL/UL128-131, which promotes entry into epithelial and endothelial cells. Supporting this model, there are two different entry mechanisms, as follows: HCMV enters fibroblasts by fusion at the plasma membrane at neutral pH (12), whereas entry into epithelial and endothelial cells involves endocytosis and a low pH-dependent fusion with endosomes (40). This model of HCMV entry parallels models for Epstein-Barr virus (EBV) entry that use gH/gL to enter epithelial cells and gH/gL/gp42 to enter B cells (24). Similarly, HHV-6 uses gH/gL/gO and gH/gL/gQ, which bind to different receptors (33).Many of the studies of gH/gL/gO have involved the fibroblast-adapted HCMV strain AD169, which fails to express UL131 and assemble gH/gL/UL128-131 or AD169 recombinants in which UL131 expression was restored (20, 21, 22, 48, 49). It seemed possible that the adaptation of AD169 to long-term passage in fibroblasts might also involve alterations in gO. HCMV gO is unusually variable (15 to 25% amino acid differences) among different HCMV strains compared with other viral genes (13, 34, 35, 37, 46). In recent studies, Jiang et al. (26) described a gO-null mutant derived from the HCMV strain TB40/E, a strain that can infect endothelial cells following extensive passage on these cells. The TB40/E gO-null mutant spread poorly on fibroblasts compared with wild-type TB40/E, and there was little infectious virus detected in fibroblast culture supernatants. However, the few TB40/E gO-null mutant particles produced by fibroblasts that could initiate infection of endothelial cells were able to spread to form normal-sized plaques on endothelial cells. These results further supported the model for which gH/gL/gO is required for infection of fibroblasts but not for epithelial/endothelial cells. Those authors also concluded that gO is important for the assembly of enveloped particles in fibroblasts, based on observations of few infectious virus particles in supernatants and cytoplasmic accumulation of unenveloped capsids (26).Our studies of gH/gL/UL128-131 have involved the clinical HCMV strain TR (39, 40, 41, 47). HCMV TR was originally an ocular isolate from an AIDS patient (45) and was passaged only a few times on fibroblasts before being genetically frozen in the form of a bacterial artificial chromosome (BAC) (34, 40). HCMV TR infects epithelial and endothelial cells (40) and monocyte-macrophages (D. Streblow and J. Nelson, unpublished results) well. In the accompanying paper (42), we characterized the biochemistry and intracellular trafficking of TR gO. TR gO expressed either in TR-infected cells or by using adenovirus vectors (expressed without other HCMV proteins) was largely retained in the ER. Coexpression of gO with gH/gL promoted transport of gH/gL beyond the ER. Importantly, TR gO was not found in extracellular virions. In contrast, AD169 gO was present in extracellular virus particles, as described previously (20, 21). We concluded that TR gO is a chaperone that promotes ER export of the gH/gL complex, but gO dissociates prior to incorporation into the virus envelope. Moreover, these differences highlight major differences between gO molecules expressed by fibroblast-adapted strain AD169 and low-passage TR.To extend these results and characterize how TR gO functions, whether in virus entry or virus assembly/egress, we constructed a TR gO-null mutant. TRΔgO exhibited major defects in entering fibroblasts, as evidenced by increased virus infection following treatment with the chemical fusogen polyethylene glycol (PEG). Unexpectedly, the mutant also failed to enter epithelial and endothelial cells, and again, PEG partially restored entry. Relatively normal numbers of TRΔgO particles were produced and released into cell culture supernatants, although even with PEG treatment, most of these virus particles remained defective in initiating immediate-early HCMV protein synthesis. Western blot analyses of TRΔgO extracellular particles demonstrated very low levels of gH/gL incorporated into virions, which likely explains the reduced entry of TRΔgO. However, the small amounts of gH/gL complexes that were present in TRΔgO virions were associated with increased quantities of UL130, and these TRΔgO particles spread better than wild-type HCMV on epithelial cell monolayers. Together with the results shown in the accompanying paper (42), we concluded that HCMV TR gO functions as a chaperone to promote ER export of gH/gL to HCMV assembly compartments and the incorporation of gH/gL into the virion envelope. The highly reduced quantities of gH/gL in virions are apparently responsible for the inability of HCMV to enter fibroblasts and epithelial and endothelial cells. These results suggest a modified version of our model, in which gH/gL, not gH/gL/gO, mediates entry into fibroblasts and both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.     

Fetal Human Cytomegalovirus Transmission Correlates with Delayed Maternal Antibodies to gH/gL/pUL128-130-131 Complex during Primary Infection

Primary human cytomegalovirus (HCMV) infections during pregnancy are associated with a high risk of virus transmission to the fetus. To identify correlates of intrauterine HCMV transmission, serial serum samples from HCMV transmitter and non-transmitter pregnant women with primary HCMV infection were analyzed for the presence of neutralizing antibodies against different glycoproteins and glycoprotein complexes, which are known to mediate entry into distinct types of host cells. Neutralizing activity was detected in the sera early after primary infection; absorption with a soluble pentameric complex formed by gH/gL/pUL128-131, but not with gH/gL dimer or with gB, abolished the capacity of sera to neutralize infection of epithelial cells. Importantly, an early, high antibody response to pentamer antigenic sites was associated with a significantly reduced risk of HCMV transmission to the fetus. This association is consistent with the high in vitro inhibition of HCMV infection of epithelial/endothelial cells as well as cell-to-cell spreading and virus transfer to leukocytes by anti-pentamer antibodies. Taken together, these findings indicate that the HCMV pentamer complex is a major target of the antibody-mediated maternal immunity.