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

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.

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Polymorphisms in Human Cytomegalovirus Glycoprotein O (gO) Exert Epistatic Influences on Cell-Free and Cell-to-Cell Spread and Antibody Neutralization on gH Epitopes

Journal of Virology ◽

10.1128/jvi.02051-19 ◽

2020 ◽

Vol 94 (8) ◽

Cited By ~ 4

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

ABSTRACT Human cytomegalovirus (HCMV) glycoproteins H and L (gH/gL) can be bound by either gO or the UL128 to UL131 proteins (referred to here as UL128-131) 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 study, 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 strains 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) [referred to here as ADgO(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 that (i) there are mechanistically distinct roles for gH/gL/gO in cell-free and cell-to-cell spread, (ii) gO isoforms can differentially shield the virus from neutralizing antibodies, and (iii) effects of gO polymorphisms are epistatically dependent on other variable loci. IMPORTANCE Advances 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 showed 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.

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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

Journal of Virology ◽

10.1128/jvi.00034-20 ◽

2020 ◽

Vol 94 (13) ◽

Cited By ~ 3

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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Pathogen at the Gates: Human Cytomegalovirus Entry and Cell Tropism

10.20944/preprints201811.0378.v2 ◽

2018 ◽

Cited By ~ 2

Author(s):

Christopher C. Nguyen ◽

Jeremy P. Kamil

Keyword(s):

Human Cytomegalovirus ◽

Wide Spectrum ◽

Growth Factor Receptor ◽

Cell Types ◽

Stable Complex ◽

Cell Tropism ◽

The Past ◽

Factor Receptor Alpha ◽

Glycoprotein H ◽

Substantial Progress

The past few years have brought substantial progress toward understanding how human cytomegalovirus (HCMV) enters the remarkably wide spectrum of cell types and tissues that the virus infects. Neuropilin-2 and platelet-derived growth factor receptor alpha (PDGFRa) were identified as receptors, respectively, for the trimeric and pentameric glycoprotein H/glycoprotein L (gH/gL) complexes that in large part govern HCMV cell tropism, while CD90 and CD147 were also found to play roles during entry. X-ray crystal structures for the proximal viral fusogen, glycoprotein B (gB), and for the pentameric gH/gL complex (pentamer) have been solved. A novel virion gH complex consisting of gH bound to UL116 instead of gL was described, and findings supporting the existence of a stable complex between gH/gL and gB were reported. Additional work indicates that the pentamer promotes a mode of cell-associated spread that resists antibody neutralization, as opposed to the trimeric gH/gL complex (trimer), which appears to be broadly required for the infectivity of cell-free virions. Finally, viral factors such as UL148 and US16 were identified that can influence the incorporation of the alternative gH/gL complexes into virions. We will review these advances and their implications for understanding HCMV entry and cell tropism.

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Immunomodulatory Properties of a Viral Homolog of Human Interleukin-10 Expressed by Human Cytomegalovirus during the Latent Phase of Infection

Journal of Virology ◽

10.1128/jvi.02173-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3736-3750 ◽

Cited By ~ 79

Author(s):

C. Jenkins ◽

W. Garcia ◽

M. J. Godwin ◽

J. V. Spencer ◽

J. Lewis Stern ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Neutralizing Antibodies ◽

Latent Infection ◽

Cell Types ◽

Interleukin 10 ◽

Immune Recognition ◽

Necrosis Factor Alpha ◽

Mhc Ii ◽

Immunosuppressive Cytokine ◽

The Impact

ABSTRACT Human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells, from which it can reactivate to cause significant disease in immunocompromised individuals. HCMV expresses a functional homolog of the immunosuppressive cytokine interleukin-10 (termed cmvIL-10), and alternate splicing of the cmvIL-10 transcript results in expression of a latency-associated cmvIL-10 transcript (LAcmvIL-10). To determine whether LAcmvIL-10 encodes immunosuppressive functions, recombinant LAcmvIL-10 protein was generated, and its impact on major histocompatibility complex class II (MHC-II) expression was examined on granulocyte macrophage progenitor cells (GM-Ps) and monocytes. LAcmvIL-10 (and cmvIL-10) downregulated MHC-II on the surfaces of both cell types. This downregulation was associated with a decrease in total MHC-II protein and transcription of components of the MHC-II biosynthesis pathway. Unlike cmvIL-10, LAcmvIL-10 did not trigger phosphorylation of Stat3, and its ability to downregulate MHC-II was not blocked by neutralizing antibodies to the human IL-10 receptor, suggesting that LAcmvIL-10 either does not engage the cellular IL-10 receptor or utilizes it in a different manner from cmvIL-10. The impact of LAcmvIL-10 on dendritic cell (DC) maturation was also assessed. In contrast to cmvIL-10, LAcmvIL-10 did not inhibit the expression of costimulatory molecules CD40, CD80, and CD86 and the maturation marker CD83 on DCs, nor did it inhibit proinflammatory cytokines (IL-1α, IL-1β, IL-6 and tumor necrosis factor alpha). Thus, LAcmvIL-10 retains some, but not all, of the immunosuppressive functions of cmvIL-10. As GM-Ps and monocytes support latent infection, expression of LAcmvIL-10 may enable HCMV to avoid immune recognition and clearance during latency.

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Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus

Virology ◽

10.1016/j.virol.2013.06.002 ◽

2013 ◽

Vol 444 (1-2) ◽

pp. 140-147 ◽

Cited By ~ 24

Author(s):

Christian L. Jacob ◽

Louie Lamorte ◽

Eliud Sepulveda ◽

Ivo C. Lorenz ◽

Annick Gauthier ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Neutralizing Antibodies ◽

Cell Spread

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

Journal of Virology ◽

10.1128/jvi.00612-21 ◽

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.

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The Molecular Tweezer CLR01 Inhibits Antibody-Resistant Cell-to-Cell Spread of Human Cytomegalovirus

Viruses ◽

10.3390/v13091685 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1685

Author(s):

Sina Brenner ◽

Berenike Braun ◽

Clarissa Read ◽

Tatjana Weil ◽

Paul Walther ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Neutralizing Antibodies ◽

Resistant Cell ◽

Viral Envelope ◽

Free Virus ◽

Virus Particles ◽

Virus Dissemination ◽

Direct Cell ◽

Molecular Tweezer ◽

Cell Spread

Human cytomegalovirus (HCMV) uses two major ways for virus dissemination: infection by cell-free virus and direct cell-to-cell spread. Neutralizing antibodies can efficiently inhibit infection by cell-free virus but mostly fail to prevent cell-to-cell transmission. Here, we show that the ‘molecular tweezer’ CLR01, a broad-spectrum antiviral agent, is not only highly active against infection with cell-free virus but most remarkably inhibits antibody-resistant direct cell-to-cell spread of HCMV. The inhibition of cell-to-cell spread by CLR01 was not limited to HCMV but was also shown for the alphaherpesviruses herpes simplex viruses 1 and 2 (HSV-1, -2). CLR01 is a rapid acting small molecule that inhibits HCMV entry at the attachment and penetration steps. Electron microscopy of extracellular virus particles indicated damage of the viral envelope by CLR01, which likely impairs the infectivity of virus particles. The rapid inactivation of viral particles by CLR01, the viral envelope as the main target, and the inhibition of virus entry at different stages are presumably the key to inhibition of cell-free virus infection and cell-to-cell spread by CLR01. Importance: While cell-free spread enables the human cytomegalovirus (HCMV) and other herpesviruses to transmit between hosts, direct cell-to-cell spread is thought to be more relevant for in vivo dissemination within infected tissues. Cell-to-cell spread is resistant to neutralizing antibodies, thus contributing to the maintenance of virus infection and virus dissemination in the presence of an intact immune system. Therefore, it would be therapeutically interesting to target this mode of spread in order to treat severe HCMV infections and to prevent dissemination of virus within the infected host. The molecular tweezer CLR01 exhibits broad-spectrum antiviral activity against a number of enveloped viruses and efficiently blocks antibody-resistant cell-to-cell spread of HCMV, thus representing a novel class of small molecules with promising antiviral activity.

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Ebola Virus Glycoprotein: Proteolytic Processing, Acylation, Cell Tropism, and Detection of Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.75.3.1576-1580.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1576-1580 ◽

Cited By ~ 115

Author(s):

Hiroshi Ito ◽

Shinji Watanabe ◽

Ayato Takada ◽

Yoshihiro Kawaoka

Keyword(s):

Endothelial Cells ◽

Neutralizing Antibodies ◽

Ebola Virus ◽

Cell Types ◽

Proteolytic Processing ◽

Cell Tropism ◽

Transmembrane Region ◽

Virus Glycoprotein ◽

Vesicular Stomatitis ◽

Neutralizing Epitopes

ABSTRACT Using the vesicular stomatitis virus (VSV) pseudotype system, we studied the functional properties of the Ebola virus glycoprotein (GP). Amino acid substitutions at the GP cleavage site, which reduce glycoprotein cleavability and viral infectivity in some viruses, did not appreciably change the infectivity of VSV pseudotyped with GP. Likewise, removal of two acylated cysteine residues in the transmembrane region of GP showed no discernible effects on infectivity. Although most filoviruses are believed to target endothelial cells and hepatocytes preferentially, the GP-carrying VSV showed greater affinity for epithelial cells than for either of these cell types, indicating that Ebola virus GP does not necessarily have strong tropism toward endothelial cells and hepatocytes. Finally, when it was used to screen for neutralizing antibodies against Ebola virus GP, the VSV pseudotype system allowed us to detect strain-specific neutralizing activity that was inhibited by secretory GP (SGP). This finding provides evidence of shared neutralizing epitopes on GP and SGP molecules and indicates the potential of SGP to serve as a decoy for neutralizing antibodies.

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HCMV trimer- and pentamer-specific antibodies synergize for virus neutralization but do not correlate with congenital transmission

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814835116 ◽

2019 ◽

Vol 116 (9) ◽

pp. 3728-3733 ◽

Cited By ~ 16

Author(s):

Adam L. Vanarsdall ◽

Andrea L. Chin ◽

Jing Liu ◽

Theodore S. Jardetzky ◽

James O. Mudd ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Cell Types ◽

The Other ◽

Major Focus ◽

Specific Antibodies ◽

Transplant Patients ◽

Human Sera ◽

Cell Spread ◽

Major Target ◽

Pregnant Mothers

Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131. In some studies, most of the NAbs in animal or human sera were found to recognize the pentamer, which mediates HCMV entry into endothelial and epithelial cells. It was also reported that pentamer-specific antibodies correlate with protection against transmission from mothers to babies. One problem with the studies on pentamer-specific NAbs to date has been that the studies did not compare the pentamer to the other major form of gH/gL, the gH/gL/gO trimer, which is essential for entry into all cell types. Here, we demonstrate that both trimer and pentamer NAbs are frequently found in human transplant patients’ and pregnant mothers’ sera. Depletion of human sera with trimer caused reductions in NAbs similar to that observed following depletion with the pentamer. The trimer- and pentamer-specific antibodies acted in a synergistic fashion to neutralize HCMV and also to prevent virus cell-to-cell spread. Importantly, there was no correlation between the titers of trimer- and pentamer-specific NAbs and transmission of HCMV from mothers to babies. Therefore, both the trimer and pentamer are important targets of NAbs. Nevertheless, these antibodies do not protect against transmission of HCMV from mothers to babies.

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Specialization for cell-free or cell-to-cell spread of BAC-cloned HCMV strains is determined by factors beyond the UL128-131 and RL13 loci

10.1101/760611 ◽

2019 ◽

Cited By ~ 1

Author(s):

Eric P. Schultz ◽

Jean-Marc Lanchy ◽

Le Zhang Day ◽

Qin Yu ◽

Christopher Peterson ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Early Time ◽

Cell Types ◽

Conceptual Approach ◽

Bac Clone ◽

Intact Cells ◽

A Cell ◽

Infected Cells ◽

Kinetics Of ◽

Cell Spread

ABSTRACTIt 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 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, 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 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 transcriptional repressed, cell-to-cell spread of ME was still more efficient than 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.IMPORTANCEBoth 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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