A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

ABSTRACTHuman cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling anex vivoinfection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation.

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Influence of human cytomegalovirus glycoprotein O polymorphism on the inhibitory effect of soluble forms of trimer- and pentamer-specific entry receptors

10.1101/778241 ◽

2019 ◽

Author(s):

Nadja Brait ◽

Tanja Stögerer ◽

Julia Kalser ◽

Barbara Adler ◽

Ines Kunz ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Envelope Glycoprotein ◽

Hcmv Infection ◽

Therapeutic Option ◽

Cell Types ◽

Intragenic Recombination ◽

Response Curves ◽

Entry Receptor

AbstractHuman cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO-trimer and gH/gL/UL128L-pentamer, are important for cell-free HCMV entry. While soluble Nrp2-Fc (sNrp2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble PDGFRα-Fc (sPDGFRα-Fc) interacts with gO thereby inhibiting infection of all cell types. Since gO is the most variable subunit we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc.Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, GT5 sequences in TB40-BAC4-luc background. All mutants were tested for fibroblast and epithelial cell infectivity, for virions’ gO and gH content, and for infection inhibition by sPDGFRα-Fc and sNrp2-Fc.Full-length and partial gO GT swapping may strongly alter the virions’ gO and gH levels associated with enhanced epithelial cell infectivity. All gO GT mutants except recombinant gO GT1c/3 displayed a near-complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%) and all on fibroblasts (≥ 99%). While gO GT replacement did not influence sNrp2-Fc inhibition at 1.25 μg/ml on epithelial cells (96%-98%), it rendered mutants with low gO levels moderately accessible to fibroblasts inhibition (20%-40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope >1.0), sNrp2-Fc dose-response curves on epithelial cells displayed slopes of ~1.0 suggesting functional differences between these entry inhibitors.Our findings suggest that targeting of gO-trimer rather than UL128-pentamer might be a promising target to inhibit infectivity independent of the cell type, gO polymorphism, and gO/gH content. However, intragenic gO recombination may lead to moderate resistence to sPDGFRα-Fc inhibition.ImportanceHuman cytomegalovirus (HCMV) is known for its broad cell tropism as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered as a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the virions’ amount of trimer and the highly polymorphic gO sequence. In this study, we show that gO polymorphism rather than gO levels may affect the inhibitory capacity of sPDGFRα. The finding that gO intragenic recombination may lead to moderate evasion from sPDGFRα inhibition is of major value to the development of potential anti-HCMV therapeutic compounds based on sPDGFRα.

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Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors

Journal of Virology ◽

10.1128/jvi.00107-20 ◽

2020 ◽

Vol 94 (14) ◽

Author(s):

Nadja Brait ◽

Tanja Stögerer ◽

Julia Kalser ◽

Barbara Adler ◽

Ines Kunz ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Envelope Glycoprotein ◽

Hcmv Infection ◽

Therapeutic Option ◽

Cell Types ◽

Complete Inhibition ◽

Dependent Manner ◽

Response Curves ◽

Entry Receptor

ABSTRACT Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 μg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 μg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner. IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

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OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814850116 ◽

2019 ◽

Vol 116 (14) ◽

pp. 7043-7052 ◽

Cited By ~ 37

Author(s):

Xiaofei E ◽

Paul Meraner ◽

Ping Lu ◽

Jill M. Perreira ◽

Aaron M. Aker ◽

...

Keyword(s):

Epithelial Cells ◽

Adenylate Cyclase ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Cell Types ◽

Akt Activation ◽

Antiviral Therapies ◽

Glycoprotein Complex ◽

Genome Wide ◽

Different Cell Types

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH–gL–UL128–UL130–UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT–mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies.

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Neutralizing Monoclonal Antibodies Reduce Human Cytomegalovirus Infection and Spread in Developing Placentas

Vaccines ◽

10.3390/vaccines7040135 ◽

2019 ◽

Vol 7 (4) ◽

pp. 135 ◽

Cited By ~ 6

Author(s):

Takako Tabata ◽

Matthew Petitt ◽

June Fang-Hoover ◽

Daniel C. Freed ◽

Fengsheng Li ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Human Cytomegalovirus ◽

Virus Transmission ◽

Passive Immunization ◽

First Trimester ◽

Clinical Strain ◽

Stromal Fibroblasts ◽

Virion Assembly ◽

Effective Strategies ◽

Hyperimmune Globulin

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects worldwide, yet the most effective strategies for preventing virus transmission during pregnancy are unknown. We measured the efficacy of human monoclonal antibodies (mAbs) to HCMV attachment/entry factors glycoprotein B (gB) and the pentameric complex, gH/gL-pUL128–131, in preventing infection and spread of a clinical strain in primary placental cells and explants of developing anchoring villi. A total of 109 explants from five first-trimester placentas were cultured, and infection was analyzed in over 400 cell columns containing ~120,000 cytotrophoblasts (CTBs). mAbs to gB and gH/gL, 3-25 and 3-16, respectively, neutralized infection in stromal fibroblasts and trophoblast progenitor cells. mAbs to pUL128-131 of the pentameric complex, 1-103 and 2-18, neutralized infection of amniotic epithelial cells better than mAbs 3-25 and 3-16 and hyperimmune globulin. Select mAbs neutralized infection of cell column CTBs, with mAb 2-18 most effective, followed by mAb 3-25. Treatment of anchoring villi with mAbs postinfection reduced spread in CTBs and impaired formation of virion assembly compartments, with mAb 2-18 achieving better suppression at lower concentrations. These results predict that antibodies generated by HCMV vaccines or used for passive immunization have the potential to reduce transplacental transmission and congenital disease.

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Towards Physiologically and Tightly Regulated Vectored Antibody Therapies

Cancers ◽

10.3390/cancers12040962 ◽

2020 ◽

Vol 12 (4) ◽

pp. 962 ◽

Cited By ~ 2

Author(s):

Audrey Page ◽

Floriane Fusil ◽

François-Loïc Cosset

Keyword(s):

B Cells ◽

Viral Vectors ◽

Checkpoint Inhibitors ◽

Ex Vivo ◽

Passive Immunization ◽

Cell Types ◽

Cell Receptor ◽

Short Period ◽

Antibody Secretion

Cancers represent highly significant health issues and the options for their treatment are often not efficient to cure the disease. Immunotherapy strategies have been developed to modulate the patient’s immune system in order to eradicate cancerous cells. For instance, passive immunization consists in the administration at high doses of exogenously produced monoclonal antibodies directed either against tumor antigen or against immune checkpoint inhibitors. Its main advantage is that it provides immediate immunity, though during a relatively short period, which consequently requires frequent injections. To circumvent this limitation, several approaches, reviewed here, have emerged to induce in vivo antibody secretion at physiological doses. Gene delivery vectors, such as adenoviral vectors or adeno-associated vectors, have been designed to induce antibody secretion in vivo after in situ cell modification, and have driven significant improvements in several cancer models. However, anti-idiotypic antibodies and escape mutants have been detected, probably because of both the continuous expression of antibodies and their expression by unspecialized cell types. To overcome these hurdles, adoptive transfer of genetically modified B cells that secrete antibodies either constitutively or in a regulated manner have been developed by ex vivo transgene insertion with viral vectors. Recently, with the emergence of gene editing technologies, the endogenous B cell receptor loci of B cells have been modified with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas-9) system to change their specificity in order to target a given antigen. The expression of the modified BCR gene hence follows the endogenous regulation mechanisms, which may prevent or at least reduce side effects. Although these approaches seem promising for cancer treatments, major questions, such as the persistence and the re-activation potential of these engineered cells, remain to be addressed in clinically relevant animal models before translation to humans.

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Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL

Journal of Virology ◽

10.1128/jvi.01875-15 ◽

2015 ◽

Vol 90 (5) ◽

pp. 2294-2305 ◽

Cited By ~ 16

Author(s):

Eric P. Schultz ◽

Jean-Marc Lanchy ◽

Erin E. Ellerbeck ◽

Brent J. Ryckman

Keyword(s):

Epithelial Cells ◽

Membrane Fusion ◽

Human Cytomegalovirus ◽

Vaccine Development ◽

Cell Types ◽

Receptor Interaction ◽

Surface Phenomenon ◽

Barr Virus ◽

The Core ◽

Receptor Interference

ABSTRACTThe core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO.IMPORTANCEInterest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

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Biotin uptake by human intestinal and liver epithelial cells: role of the SMVT system

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00059.2003 ◽

2003 ◽

Vol 285 (1) ◽

pp. G73-G77 ◽

Cited By ~ 60

Author(s):

Krishnaswamy Balamurugan ◽

Alvaro Ortiz ◽

Hamid M. Said

Keyword(s):

Epithelial Cells ◽

Hepg2 Cells ◽

Pantothenic Acid ◽

Cell Types ◽

Uptake System ◽

Small Interfering Rnas ◽

Uptake Mechanism ◽

High Affinity ◽

Liver Epithelial Cells ◽

Relative Contribution

It has been well established that human intestinal and liver epithelial cells transport biotin via an Na+-dependent carrier-mediated mechanism. The sodium-dependent multivitamin transport (SMVT), a biotin transporter, is expressed in both cell types. However, the relative contribution of SMVT toward total carrier-mediated uptake of physiological (nanomolar) concentrations of biotin by these cells is not clear. Addressing this issue is important, especially in light of the recent identification of a second human high-affinity biotin uptake mechanism that operates at the nanomolar range. Hence, we employed a physiological approach of characterizing biotin uptake by human-derived intestinal Caco-2 and HepG2 cells at the nanomolar concentration range. We also employed a molecular biology approach of selectively silencing the endogenous SMVT of these cells with specific small interfering RNAs (siRNAs), then examining carrier-mediated biotin uptake. The results showed that in both Caco-2 and HepG2 cells, the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. Furthermore, we found that the addition of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid to the incubation medium had no effect on the uptake of 2.6 nM [3H]biotin. Pretreatment of Caco-2 and HepG2 cells with SMVT specific siRNAs substantially reduced SMVT mRNA and protein levels. In addition, carrier-mediated [3H]biotin (2.6 nM) uptake by Caco-2 and HepG2 cells was severely ( P 0.01) inhibited by the siRNAs pretreatment. These results demonstrate that the recently described human high-affinity biotin uptake system is not functional in intestinal and liver epithelial cells. In addition, the results provide strong evidence that SMVT is the major (if not the only) biotin uptake system that operates in these cells.

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Checks and balances between human cytomegalovirus replication and indoleamine-2,3-dioxygenase

Journal of General Virology ◽

10.1099/vir.0.061994-0 ◽

2014 ◽

Vol 95 (3) ◽

pp. 659-670 ◽

Cited By ~ 9

Author(s):

Albert Zimmermann ◽

Sebastian Hauka ◽

Marco Maywald ◽

Vu Thuy Khanh Le ◽

Silvia K. Schmidt ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Cell Types ◽

Interferon Γ ◽

Counter Measures ◽

Indoleamine 2,3 Dioxygenase ◽

Infected Cells ◽

Hcmv Replication ◽

Ifn Γ ◽

The Impact

Despite a rigorous blockade of interferon-γ (IFN-γ) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-γ induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-γ-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-γ treatment in both cell types. These findings highlight the effectiveness of this IFN-γ induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

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Maternal Fc-mediated non-neutralizing antibody responses correlate with protection against congenital human cytomegalovirus infection

10.1101/2021.12.05.21267312 ◽

2021 ◽

Author(s):

Eleanor C. Semmes ◽

Itzayana G. Miller ◽

Jennifer A. Jenks ◽

Courtney E. Wimberly ◽

Stella J. Berendam ◽

...

Keyword(s):

Cord Blood ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Passive Immunization ◽

Congenital Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

High Avidity ◽

Igg Binding ◽

Congenital Hcmv Infection

AbstractHuman cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital infection has been hindered by limited knowledge of the immune responses that protect against placental HCMV transmission in maternal primary and nonprimary infection. To identify protective antibody responses, we measured anti-HCMV IgG binding and anti-viral functions in maternal and cord blood sera from HCMV transmitting (n=41) and non- transmitting (n=40) mother-infant dyads identified via a large U.S.-based public cord blood bank. In a predefined immune correlate analysis, maternal monocyte-mediated antibody-dependent cellular phagocytosis (ADCP) and high avidity IgG binding to HCMV envelope glycoproteins were associated with decreased risk of congenital HCMV infection. Moreover, HCMV-specific IgG engagement of FcγRI and FcγRIIA, which mediate non-neutralizing antibody responses, was enhanced in non-transmitting mother-infant dyads and strongly correlated with ADCP. These findings suggest that Fc effector functions including ADCP protect against placental HCMV transmission. Taken together, our data indicate that future active and passive immunization strategies to prevent congenital HCMV infection should target Fc-mediated non-neutralizing antibody responses.

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Neonatal Neural Progenitor Cells and Their Neuronal and Glial Cell Derivatives Are Fully Permissive for Human Cytomegalovirus Infection

Journal of Virology ◽

10.1128/jvi.00943-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 9994-10007 ◽

Cited By ~ 61

Author(s):

Min Hua Luo ◽

Philip H. Schwartz ◽

Elizabeth A. Fortunato

Keyword(s):

Progenitor Cells ◽

Human Cytomegalovirus ◽

Neural Progenitor Cells ◽

Hcmv Infection ◽

Full Range ◽

Cell Types ◽

Neural Progenitor ◽

Clinical Strain ◽

Cytopathic Effects ◽

Β Tubulin

ABSTRACT Congenital human cytomegalovirus (HCMV) infection causes central nervous system structural abnormalities and functional disorders, affecting both astroglia and neurons with a pathogenesis that is only marginally understood. To better understand HCMV's interactions with such clinically important cell types, we utilized neural progenitor cells (NPCs) derived from neonatal autopsy tissue, which can be differentiated down either glial or neuronal pathways. Studies were performed using two viral isolates, Towne (laboratory adapted) and TR (a clinical strain), at a multiplicity of infection of 3. NPCs were fully permissive for both strains, expressing the full range of viral antigens (Ags) and producing relatively large numbers of infectious virions. NPCs infected with TR showed delayed development of cytopathic effects (CPE) and replication centers and shed less virus. This pattern of delay for TR infections held true for all cell types tested. Differentiation of NPCs was carried out for 21 days to obtain either astroglia (>95% GFAP+) or a 1:5 mixed neuron/astroglia population (β-tubulin III+/GFAP+). We found that both of these differentiated populations were fully permissive for HCMV infection and produced substantial numbers of infectious virions. Utilizing a difference in plating efficiencies, we were able to enrich the neuron population to ∼80% β-tubulin III+ cells. These β-tubulin III+-enriched populations remained fully permissive for infection but were very slow to develop CPE. These infected enriched neurons survived longer than either NPCs or astroglia, and a small proportion were alive until at least 14 days postinfection. These surviving cells were all β-tubulin III+ and showed viral Ag expression. Surprisingly, some cells still exhibited extended processes, similar to mock-infected neurons. Our findings strongly suggest neurons as reservoirs for HCMV within the developing brain.

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