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

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

10.1101/2021.04.09.439256 ◽

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

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Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2

Journal of Virology ◽

10.1128/jvi.72.1.309-319.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 309-319 ◽

Cited By ~ 318

Author(s):

Elizabeth A. Rutledge ◽

Christine L. Halbert ◽

David W. Russell

Keyword(s):

Neutralizing Antibodies ◽

Cell Types ◽

Human Populations ◽

Cloning And Sequencing ◽

Adeno Associated Virus ◽

Nucleotide Sequence Level ◽

Infectious Clones ◽

Almost All ◽

Different Cell Types

ABSTRACT Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.

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Uncovering the cell type specificity of blood sample derived gene signatures using RNA expression data

10.1101/684159 ◽

2019 ◽

Author(s):

Mikhail Pomaznoy ◽

Brendan Ha ◽

Bjoern Peters

Keyword(s):

Blood Cell ◽

Web Application ◽

Cell Lineage ◽

Complex Mixture ◽

Cell Types ◽

Cell Type Specificity ◽

Signature Genes ◽

Expression Of Genes ◽

Biological Interpretation ◽

Different Cell Types

AbstractAnalysis of transcriptomic data derived from blood samples is complicated by the complex mixture of cell types such samples contain. Transcriptomic signatures derived from such samples are often driven by a particular cell lineage within the mixture. Identifying this most contributing lineage can help to provide a biological interpretation of the signature. We created a web application CellTypeScore which quantifies and visually represents the expression level of signature genes in common blood cell types. This is done by constructing an interactive stacked bar plot with the bars representing expression of genes across blood cell types. Summed scores serve as a measure of how highly the combined signature is expressed in different cell types. An online version of the application can be found at https://tools.dice-database.org/celltypescore/.

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

10.1101/867234 ◽

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.

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

Journal of Virology ◽

10.1128/jvi.77.21.11633-11643.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11633-11643 ◽

Cited By ~ 46

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.

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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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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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Major Tegument Protein pp65 of Human Cytomegalovirus Is Required for the Incorporation of pUL69 and pUL97 into the Virus Particle and for Viral Growth in Macrophages

Journal of Virology ◽

10.1128/jvi.01818-08 ◽

2008 ◽

Vol 83 (6) ◽

pp. 2480-2490 ◽

Cited By ~ 50

Author(s):

Meike Chevillotte ◽

Sandra Landwehr ◽

Leonhard Linta ◽

Giada Frascaroli ◽

Anke Lüske ◽

...

Keyword(s):

Virus Particle ◽

Human Cytomegalovirus ◽

Cell Types ◽

Viral Proteins ◽

Tegument Protein ◽

Viral Growth ◽

Virus Particles ◽

Infected Cells ◽

Different Cell Types

ABSTRACT The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.

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