Inhibition of human cytomegalovirus entry into mucosal epithelial cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A.

The RL13 Temperance Factor Represses Replication of the Highly Cell Culture-Adapted Towne Strain of Human Cytomegalovirus.

Human cytomegalovirus (CMV) has evolved to replicate while causing minimal damage, maintain life-long latency, reactivate sub-clinically, and, in spite of robust host immunity, produce and shed infectious virus in order to transmit to new hosts. The CMV temperance factor RL13 may contribute to this strategy of coexistence with the host by actively restricting viral replication and spread. Viruses with an intact gene grow slowly in cell culture, release little extracellular virus, and form small foci.

Polymorphic Forms of Human Cytomegalovirus Glycoprotein O Protect against Neutralization of Fibroblast Entry by Antibodies Targeting Epitopes Defined by Glycoproteins H and L.

Human cytomegalovirus (CMV) utilizes different glycoproteins to enter into fibroblast and epithelial cells. A trimer of glycoproteins H, L, and O (gH/gL/gO) is required for entry into all cells, whereas a pentamer of gH/gL/UL128/UL130/UL131A is selectively required for infection of epithelial, endothelial, and some myeloid-lineage cells, but not of fibroblasts. Both complexes are of considerable interest for vaccine and immunotherapeutic development but present a conundrum: gH/gL-specific antibodies have moderate potency yet neutralize CMV entry into all cell types, whereas pentamer-specific antibodies are more potent but do not block fibroblast infection.

Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts.

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts.

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus.

LoReTTA, a user-friendly tool for assembling viral genomes from PacBio sequence data.

Long-read, single-molecule DNA sequencing technologies have triggered a revolution in genomics by enabling the determination of large, reference-quality genomes in ways that overcome some of the limitations of short-read sequencing. However, the greater length and higher error rate of the reads generated on long-read platforms make the tools used for assembling short reads unsuitable for use in data assembly and motivate the development of new approaches. We present LoReTTA (Long Read Template-Targeted Assembler), a tool designed for performing assembly of long reads generated from viral genomes on the PacBio platform.

Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells.

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells.

Genome Sequence of Human Cytomegalovirus Ig-KG-H2, a Variant of Strain KG Propagated in the Presence of Neutralizing Antibodies.

Human cytomegalovirus shed in infant urine was isolated and serially passaged in fibroblasts in the presence or absence of neutralizing antibodies. Comparison of the genome sequences of representative viruses Ig-KG-H2 (passed with antibody) and ϕ-KG-B5 (passed without antibody) revealed the presence of several mutations in each virus.

Comparative neutralizing potencies of antibodies suggest conservation as well as mechanistic differences in human cytomegalovirus entry into epithelial and endothelial cells.

Antibody neutralization of cytomegalovirus (CMV) entry into diverse cell types is a key consideration for development of vaccines and immunotherapeutics. CMV entry into fibroblasts differs significantly from entry into epithelial or endothelial cells: fibroblast entry is mediated by gB and gH/gL/gO, whereas both epithelial and endothelial cell entry require an additional pentameric complex (PC) comprised of gH/gL/UL128/UL130/UL131A. Because PC-specific antibodies in CMV-seropositive human sera do not affect fibroblast entry but potently block entry into epithelial or endothelial cells, substantially higher neutralizing potencies for CMV-positive sera are observed when assayed using epithelial cells as targets than when using fibroblasts.

Single-Cell Transcriptome Analysis of CD34 Stem Cell-Derived Myeloid Cells Infected With Human Cytomegalovirus.

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34 hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of resistant and permissive cell types, and the cellular features characterizing the latter, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic.

Inclusion of Antibodies to Cell Culture Media Preserves the Integrity of Genes Encoding RL13 and the Pentameric Complex Components During Fibroblast Passage of Human Cytomegalovirus.

Propagation of human cytomegalovirus (CMV) in cultured cells results in genetic adaptations that confer improved growth in vitro and significant attenuation in vivo. Mutations in arise quickly, while mutations in the locus emerge later during fibroblast passage and disrupt formation of a glycoprotein complex that is important for entry into epithelial and endothelial cells. As CMV replicates in the context of host antibodies in vivo, we reasoned that antibodies might mitigate the accumulation of adaptive mutations during cell culture passage.

Activation of Langerhans-Type Dendritic Cells Alters Human Cytomegalovirus Infection and Reactivation in a Stimulus-Dependent Manner.

Oral mucosal Langerhans cells (LC) are likely to play important roles in host defense against infection by human cytomegalovirus (CMV). We previously showed that in vitro-differentiated immature LC (iLC) populations contain smaller amounts of infected cells but produce higher yields than mature LC (mLC) cultures, obtained by iLC stimulation with fetal bovine serum (FBS), CD40 ligand (CD40L) and lipopolysaccharide (LPS). Here, we sought to determine if exposure to select stimuli can improve LC permissiveness to infection, if specific components of the mLC cocktail are responsible for lowering viral yields, if this is due to defects in progeny production or release, and if these restrictions are also effective against reactivated virus.

Dynamics of Human Cytomegalovirus Infection in CD34+ Hematopoietic Cells and Derived Langerhans-Type Dendritic Cells.

Unlabelled: Acquisition of human cytomegalovirus (CMV) usually occurs by contact between contaminated bodily fluids, such as urine and saliva, and host mucosal cells. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of the oral mucosae, where they not only provide a first line of defense against CMV but can easily be targeted by orally administered vaccines, while their bone marrow resident progenitors are important sites of virus latency. In this work, we tracked the progress of infection in CD34(+) progenitor cells, immature LC (iLC), and mature LC (mLC) exposed to the clinical-like strain TB40-BAC4 or to the vaccine strain AD169varATCC, prior to their long-term maintenance under either immature or mature conditions.

Human cytomegalovirus tropism for mucosal myeloid dendritic cells.

Human CMV infections are a serious source of morbidity and mortality for immunocompromised patients and for the developing fetus. Because of this, the development of new strategies to prevent CMV acquisition and transmission is a top priority. Myeloid dendritic cells (DC) residing in the oral and nasal mucosae are among the first immune cells to encounter CMV during entry and greatly contribute to virus dissemination, reactivation from latency, and horizontal spread.

Human cytomegalovirus infection of langerhans-type dendritic cells does not require the presence of the gH/gL/UL128-131A complex and is blocked after nuclear deposition of viral genomes in immature cells.

Human cytomegalovirus (CMV) enters its host via the oral and genital mucosae. Langerhans-type dendritic cells (LC) are the most abundant innate immune cells at these sites, where they constitute a first line of defense against a variety of pathogens. We previously showed that immature LC (iLC) are remarkably resistant to CMV infection, while mature LC (mLC) are more permissive, particularly when exposed to clinical-strain-like strains of CMV, which display a pentameric complex consisting of the viral glycoproteins gH, gL, UL128, UL130, and UL131A on their envelope.

Herpesviruses and intermediate filaments: close encounters with the third type.

Intermediate filaments (IF) are essential to maintain cellular and nuclear integrity and shape, to manage organelle distribution and motility, to control the trafficking and pH of intracellular vesicles, to prevent stress-induced cell death, and to support the correct distribution of specific proteins. Because of this, IF are likely to be targeted by a variety of pathogens, and may act in favor or against infection progress. As many IF functions remain to be identified, however, little is currently known about these interactions.

Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels.

Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively.

RASCAL is a new human cytomegalovirus-encoded protein that localizes to the nuclear lamina and in cytoplasmic vesicles at late times postinfection.

The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear rim-associated cytomegaloviral protein, or RASCAL.

Onset of human cytomegalovirus replication in fibroblasts requires the presence of an intact vimentin cytoskeleton.

Like all viruses, herpesviruses extensively interact with the host cytoskeleton during entry. While microtubules and microfilaments appear to facilitate viral capsid transport toward the nucleus, evidence for a role of intermediate filaments in herpesvirus entry is lacking. Here, we examined the function of vimentin intermediate filaments in fibroblasts during the initial phase of infection of two genotypically distinct strains of human cytomegalovirus (CMV), one with narrow (AD169) and one with broad (TB40/E) cell tropism.

Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis.

The human cytomegalovirus (CMV) US2-US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS).

Comparisons of CD8+ T cells specific for human immunodeficiency virus, hepatitis C virus, and cytomegalovirus reveal differences in frequency, immunodominance, phenotype, and interleukin-2 responsiveness.

To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8(+) T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8(+) T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP.

Viral and cell cycle-regulated kinases in cytomegalovirus-induced pseudomitosis and replication.

A process of pseudomitosis occurs during human cytomegalovirus infection that appears similar to cellular mitosis but involves the formation of multiple spindle poles, abnormal condensation, and mislocalization of chromosomal DNA. The relationship of this process to viral replication and cell cycle regulation during infection has been poorly understood. Pseudomitosis consistently peaks at late times of infection in all viral strains examined but at overall highest frequencies (30% to 35% of cells) using one common laboratory strain variant (AD169varATCC).

Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC.

Global analysis of host cell gene expression late during cytomegalovirus infection reveals extensive dysregulation of cell cycle gene expression and induction of Pseudomitosis independent of US28 function.

Replication of human cytomegalovirus (CMV) depends on host cell gene products working in conjunction with viral functions and leads to a dramatic dysregulation of cell cycle gene expression. Comprehensive transcriptional profiling was used to identify pathways most dramatically modulated by CMV at late times during infection and to determine the extent to which expression of the viral chemokine receptor US28 contributed to modulating cellular gene expression. Cells infected with the AD169 strain of virus or a fully replication competent US28-deficient derivative (RV101) were profiled throughout the late phase of infection (50, 72, and 98 h postinfection).

Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus.

Human cytomegalovirus (CMV) infection initiates in mucosal epithelia and disseminates via leukocytes throughout the body. Langerhans cells (LCs), the immature dendritic cells (DCs) that reside in epithelial tissues, are among the first cells to encounter virus and may play important roles in the immune response, as well as in pathogenesis as hosts for viral replication and as vehicles for dissemination. Here, we demonstrate that CD34(+) progenitor cell-derived LC-type DCs exhibit a differentiation state-dependent susceptibility to CMV infection.