scholarly journals Mutagenesis of human cytomegalovirus glycoprotein L disproportionately disrupts gH/gL/gO over gH/gL/pUL128-131.

2021 ◽  
Author(s):  
Eric P. Schultz ◽  
Qin Yu ◽  
Cora Stegmann ◽  
Le Zhang Day ◽  
Jean-Marc Lanchy ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Transient Expression ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Cell Types ◽  
Regulatory Factor ◽  
Bac Clones ◽  
Dependent Cell ◽  
Different Cell Types ◽  
Cell Spread

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but the how the gH/gL portion of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB dependent cell-cell fusion, but were still able to form gH/gL/pUL128-131 and induce receptor-interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. Effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRa, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a “hyperactive” gH/gL/gO. Recently published crystallography and cryo-EM studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB.

scholarly journals Mutagenesis of human cytomegalovirus glycoprotein L disproportionately disrupts gH/gL/gO over gH/gL/pUL128-131.

2021 ◽  
Author(s):  
Eric P. Schultz ◽  
Qin Yu ◽  
Cora Stegmann ◽  
Le Zhang Day ◽  
Jean-Marc Lanchy ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Neutralizing Antibodies ◽  
Complex Mixture ◽  
Cell Types ◽  
Human Populations ◽  
Persistent Infections ◽  
Bac Clones ◽  
Dependent Cell ◽  
Different Cell Types ◽  
Significant Disease

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but the how the gH/gL portion of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB dependent cell-cell fusion, but were still able to form gH/gL/pUL128-131 and induce receptor-interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. Effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a “hyperactive” gH/gL/gO. Recently published crystallography and cryo-EM studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCE The endemic beta -herpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes, and may provide a basis for more refined evaluations of neutralizing antibodies.

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

2019 ◽  
Vol 116 (14) ◽  
pp. 7043-7052 ◽  
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.

scholarly journals Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

2020 ◽  
Vol 21 (20) ◽  
pp. 7747
Author(s):  
Ananth Kumar Kammala ◽  
Samantha Sheller-Miller ◽  
Enkhtuya Radnaa ◽  
Talar Kechichian ◽  
Hariharan Subramanian ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Cell Types ◽  
Fetal Membrane ◽  
Regulatory Factor ◽  
Rna Targeting ◽  
Sodium Hydrogen Exchanger ◽  
Functional Relevance ◽  
Interfering Rna ◽  
Different Cell Types ◽  
Phosphate Buffered Saline

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

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

2008 ◽  
Vol 82 (20) ◽  
pp. 9994-10007 ◽  
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.

scholarly journals The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3072
Author(s):  
Natalia Landázuri ◽  
Jennifer Gorwood ◽  
Ylva Terelius ◽  
Fredrik Öberg ◽  
Koon Chu Yaiw ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Cell Types ◽  
Productive Infection ◽  
Endothelin Receptor ◽  
Viral Control ◽  
Congenital Birth Defects ◽  
Specific Antagonist ◽  
Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

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

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.

scholarly journals Polymorphisms in Human Cytomegalovirus gO Exert Epistatic Influences on Cell-Free and Cell-To-Cell Spread, and Antibody Neutralization on gH Epitopes

2019 ◽  
Author(s):  
Le Zhang Day ◽  
Cora Stegmann ◽  
Eric P. Schultz ◽  
Jean-Marc Lanchy ◽  
Qin Yu ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Genetic Background ◽  
Neutralizing Antibodies ◽  
Important Determinant ◽  
Cell Types ◽  
Cell Tropism ◽  
Laboratory Studies ◽  
Antibody Neutralization ◽  
Cell Spread

ABSTRACTThe human cytomegalovirus (HCMV) glycoproteins H and L (gH/gL) can be bound by either gO, or the UL128-131 proteins to form complexes that facilitate entry and spread and the complexes formed are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131 and this can impact infectivity and cell tropism. In this report, we investigated how natural interstrain variation in the amino acid sequence of gO influences the biology of HCMV. Heterologous gO recombinants were constructed in which 6 of the 8 alleles or genotypes (GT) of gO were analyzed in the backgrounds of strain TR and Merlin (ME). The levels of gH/gL complexes were not affected, but there were impacts on entry, spread and neutralization by anti-gH antibodies. AD169 (AD) gO (GT1a) drastically reduced cell-free infectivity of both strains on fibroblasts and epithelial cells. PHgO(GT2a) increased cell-free infectivity of TR in both cell types, but spread in fibroblasts was impaired. In contrast, spread of ME in both cell types was enhanced by Towne (TN) gO (GT4), despite similar cell-free infectivity. TR expressing TNgO(GT4) was resistant to neutralization by anti-gH antibodies AP86 and 14-4b, whereas ADgO(GT1a) conferred resistance to 14-4b, but enhanced neutralization by AP86. Conversely, ME expressing ADgO(GT1a) was more resistant to 14-4b. These results suggest; 1) mechanistically distinct roles for gH/gL/gO in cell-free and cell-to-cell spread, 2) gO isoforms can differentially shield the virus from neutralizing antibodies, and 3) effects of gO polymorphisms are epistatically dependent on other variable loci.IMPORTANCEAdvances in HCMV population genetics have greatly outpaced understanding of the links between genetic diversity and phenotypic variation. Moreover, recombination between genotypes may shuffle variable loci into various combinations with unknown outcomes. UL74(gO) is an important determinant of HCMV infectivity, and one of the most diverse loci in the viral genome. By analyzing interstrain heterologous UL74(gO) recombinants, we show that gO diversity can have dramatic impacts on cell-free and cell-to-cell spread as well as on antibody neutralization and that the manifestation of these impacts can be subject to epistatic influences of the global genetic background. These results highlight the potential limitations of laboratory studies of HCMV biology that use single, isolated genotypes or strains.

scholarly journals Murine Cytomegalovirus m41 Open Reading Frame Encodes a Golgi-Localized Antiapoptotic Protein

2003 ◽  
Vol 77 (21) ◽  
pp. 11633-11643 ◽  
Author(s):  
Wolfram Brune ◽  
Michael Nevels ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Cell Types ◽  
Open Reading Frame ◽  
Host Cells ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Transposon Insertion ◽  
Cellular Genes ◽  
Exon 1 ◽  
Different Cell Types

ABSTRACT Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.

Human cytomegalovirus infection stimulates Cl−/ HCO 3 − exchanger activity in human fibroblasts

1998 ◽  
Vol 275 (2) ◽  
pp. C515-C526 ◽  
Author(s):  
Lilia M. Maglova ◽  
William E. Crowe ◽  
Aníbal A. Altamirano ◽  
John M. Russell
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Human Lung ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Recovery Phase ◽  
Lung Fibroblasts ◽  
Continuous Exposure ◽  
Infected Cells ◽  
Human Cytomegalovirus Infection

The effects of human cytomegalovirus (HCMV) infection on Cl−/[Formula: see text]exchanger activity in human lung fibroblasts (MRC-5 cells) were studied using fluorescent, ion-sensitive dyes. The intracellular pH (pHi) of mock- and HCMV-infected cells bathed in a solution containing 5% CO2-25 mM[Formula: see text] were nearly the same. However, replacement of external Cl−with gluconate caused an H2DIDS-inhibitable (100 μM) increase in the pHi of HCMV-infected cells but not in mock-infected cells. Continuous exposure to hyperosmotic external media containing CO2/[Formula: see text]caused the pHi of both cell types to increase. The pHi remained elevated in mock-infected cells. However, in HCMV-infected cells, the pHi peaked and then recovered toward control values. This pHirecovery phase was completely blocked by 100 μM H2DIDS. In the presence of CO2/[Formula: see text], there was an H2DIDS-sensitive component of net Cl− efflux (external Cl− was substituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl− (in the nominal absence of CO2/[Formula: see text]), the H2DIDS-sensitive net Cl− efflux was much greater from HCMV- than from mock-infected cells. In mock-infected cells, H2DIDS-sensitive, net Cl− efflux decreased as pHi increased, whereas for HCMV-infected cells, efflux increased as pHi increased. All these results are consistent with an HCMV-induced enhancement of Cl−/[Formula: see text]exchanger activity.

scholarly journals Specialization for Cell-Free or Cell-to-Cell Spread of BAC-Cloned Human Cytomegalovirus Strains Is Determined by Factors beyond the UL128-131 and RL13 Loci

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Eric P. Schultz ◽  
Jean-Marc Lanchy ◽  
Le Zhang Day ◽  
Qin Yu ◽  
Christopher Peterson ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Neutralizing Antibodies ◽  
Early Time ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Conceptual Approach ◽  
Bac Clone ◽  
Intact Cells ◽  
A Cell ◽  
Cell Spread

ABSTRACT It is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The bacterial artificial chromosome (BAC) clone Merlin (ME) expresses abundant UL128-131, is RL13 impaired, and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, are RL13 intact, and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than did TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptionally repressed, cell-to-cell spread of ME was still more efficient than that of TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread, and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci. IMPORTANCE Both cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether “cell-associated” phenotypes are simply the result of poor cell-free spread or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

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