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 RL13 gene grow slowly in cell culture, release little extracellular virus, and form small foci. By contrast, viruses carrying disruptive mutations in the RL13 gene form larger foci and release higher amounts of cell-free infectious virions. Such mutations invariably arise during cell culture passage of clinical isolates and are consistently found in highly adapted strains. The potential existence in such strains of other mutations with roles in mitigating RL13's restrictive effects, however, has not been explored. To this end, a mutation that frame shifts the RL13 gene in the highly cell culture-adapted laboratory strain Towne was repaired, and a C-terminal FLAG epitope was added. Compared to the frame-shifted parental virus, viruses encoding wild-type or FLAG-tagged wild-type RL13 produced small foci and replicated poorly. Within six to ten cell culture passages, mutations emerged in RL13 that restored replication and focus size to those of the RL13-frame-shifted parental virus, implying that none of the numerous adaptive mutations acquired by strain Towne during more than 125 cell culture passages mitigate the temperance activity of RL13. Whilst RL13-FLAG expressed by passage zero stocks was localized exclusively within the virion assembly compartment, RL13-FLAG with a E208K substitution that emerged in one lineage was mostly dispersed into the cytoplasm, suggesting that localization to the virion assembly compartment is likely required for RL13 to exert its growth-restricting activities. Changes in localization also provided a convenient way to assess the emergence of RL13 mutations during serial passage, highlighting the usefulness of RL13-FLAG Towne variants for elucidating the mechanisms underlying RL13's temperance functions.

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. Which cell types and neutralizing activities are important for protective efficacy in vivo remain unclear. Here, we present evidence that certain CMV strains have evolved polymorphisms in gO to evade trimer-specific neutralizing antibodies. Using luciferase-tagged variants of strain TB40/E in which the native gO is replaced by gOs from other strains, we tested the effects of gO polymorphisms on neutralization by monoclonal antibodies (mAbs) targeting four independent epitopes in gH/gL that are common to both trimer and pentamer. Neutralization of fibroblast entry by three mAbs displayed a range of potencies that depended on the gO type, a fourth mAb failed to neutralize fibroblast entry regardless of the gO type, while neutralization of epithelial cell entry by all four mAbs was potent and independent of the gO type. Thus, specific polymorphisms in gO protect the virus from mAb neutralization in the context of fibroblast but not epithelial cell entry. No influence of gO type was observed for protection against CMV hyperimmune globulin or CMV-seropositive human sera, suggesting that antibodies targeting protected gH/gL epitopes represent a minority of the polyclonal neutralizing repertoire induced by natural 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. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell-cell fusion, and ensuing viral spread and pathogenesis in different organs.

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. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

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 de novo assembly of long reads generated from viral genomes on the PacBio platform. LoReTTA exploits a reference genome to guide the assembly process, an approach that has been successful with short reads. The tool was designed to deal with reads originating from viral genomes, which feature high genetic variability, possible multiple isoforms, and the dominant presence of additional organisms in clinical or environmental samples. LoReTTA was tested on a range of simulated and experimental datasets and outperformed established long-read assemblers in terms of assembly contiguity and accuracy. The software runs under the Linux operating system, is designed for easy adaptation to alternative systems, and features an automatic installation pipeline that takes care of the required dependencies. A command-line version and a user-friendly graphical interface version are available under a GPLv3 license at https://bioinformatics.cvr.ac.uk/software/ with the manual and a test dataset.

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.

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. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

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. That certain sera exhibit similar discordances between neutralizing potencies measured using epithelial vs. endothelial cells (Gerna G. et al.J Gen Virol, 89:853-865, 2008) suggested that additional mechanistic differences may also exist between epithelial and endothelial cell entry. To further explore this issue, neutralizing potencies using epithelial and endothelial cells were simultaneously determined for eight CMV-positive human sera, CMV-hyperimmune globulin, and a panel of monoclonal or anti-peptide antibodies targeting specific epitopes in gB, gH, gH/gL, or the PC. No significant differences were observed between epithelial and endothelial neutralizing potencies of epitope-specific antibodies, CMV-hyperimmune globulin, or seven of the eight human sera. However, one human serum exhibited a six-fold higher potency for neutralizing entry into epithelial cells vs. endothelial cells. These results suggest that epitopes exist that are important for epithelial entry but are less critical, or perhaps dispensable, for endothelial cell entry. Their existence should be considered when developing monoclonal antibody therapies or subunit vaccines representing limited epitopes.

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. Here, we profile the transcriptomes of ∼7000 individual cells at day 1 post-infection using the 10× genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit - Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells permissive to CMV replication, and provide a possible rationale for their preferential infection.

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 RL13 arise quickly, while mutations in the UL128-131A 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. To test this, CMV in infant urine was used to infect replicate fibroblast cultures. One lineage was passaged in the absence of CMV-hyperimmuneglobulin (HIG) while the other was passaged with HIG in the culture medium. The former lost epithelial tropism and acquired mutations disrupting RL13 and UL131A expression, whereas the latter retained epithelial tropism and both gene loci remained intact after 22 passages. Additional mutations resulting in single amino acid changes also occurred in UL100 encoding glycoprotein M, UL102 encoding a subunit of the helicase/primase complex, and UL122 encoding the Immediate Early 2 protein. An epitheliotropic RL13+/UL131A+ virus was isolated by limiting dilution in the presence of HIG and expanded to produce a working stock sufficient to conduct cell tropism experiments. Thus, production of virus stocks by culture in the presence of antibodies may facilitate in vitro experiments using viruses that are genetically more authentic than previously available.

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. None of the stimuli tested extended the proportion of infected cells to 100%, suggesting that the block to infection onset cannot be fully removed. While CD40L and FBS exerted positive effects on viral progeny production per cell, stimulation with LPS alone or in combination with CD40L was detrimental. Reductions in viral titers were not due to defects in progeny release, and the permissive or restrictive intracellular environment established upon exposure to each stimulus appeared to act in a somewhat similar way toward lytic and latent infections.

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. We show that the genomes of both strains are efficiently maintained in CD34(+) cells during their differentiation into iLC, although this requires the presence of larger amounts of input AD169varATCC DNA. Lipopolysaccharide- and CD40 ligand-induced maturation of iLC derived from latently infected progenitors was not associated with robust viral genome replication and progeny production, while maturation of directly infected iLC increased and prolonged expression of the viral immediate early proteins. While effective replication of viral genomes from both strains occurred only in mLC, both iLC and mLC produced viral progeny, suggesting that both types of LC may contribute to CMV horizontal transmission in vivo. IMPORTANCE: Human CMV is usually acquired via the oral and nasal mucosae. Langerhans-type dendritic cells (LC) are the only type of immune cells found in the outermost layers of these tissues. Understanding how CMV interacts with LC and their hematopoietic progenitors is thus essential to develop innovative means of defense against this virus. Here we show that the genomes of a virulent and an attenuated strain of CMV are maintained in hematopoietic progenitor cells during their differentiation into immature LC and that maturation of these cells by exposure to lipopolysaccharide and CD40 ligand is not sufficient to trigger virus reactivation. While the extents of viral protein expression and genome replication were broadest in directly infected mature LC populations, similar amounts of viral progeny were detected in the supernatants of immature and mature LC, suggesting that these immune cells of the oral mucosa are likely to be important for CMV transmission within the human population.

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. Albeit affected by the immunoevasive tactics of CMV, mucosal DC remain potent inducers of cellular and humoral immune responses against this virus. Their natural functions could thus be exploited to generate long-lasting protective immunity against CMV by vaccination via the oronasal mucosae. Although related, epithelial Langerhans-type DC and dermal monocyte-derived DC interact with CMV in dramatically different ways. Whereas immature monocyte-derived DC are fully permissive to infection, for instance, immature Langerhans-type DC are completely resistant. Understanding these differences is essential to design innovative vaccines and new antiviral compounds to protect these cells from CMV infection in vivo.

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. This complex was recently shown to be required for the infection of immature monocyte-derived dendritic cells. We thus sought to establish if the presence of this complex is also necessary for virion penetration of LC and if defects in entry might be the source of iLC resistance to CMV. Here we report that the efficiency of LC infection is reduced, but not completely abolished, in the absence of the pentameric complex. While virion penetration and nuclear deposition of viral genomes are not impaired in iLC, the transcription of the viral immediate early genes UL122 and UL123 and of the delayed early gene UL50 is substantially lower than that in mLC. Together, these data show that the UL128, UL130, and UL131A proteins are dispensable for CMV entry into LC and that progression of the viral cycle in iLC is restricted at the step of viral gene expression.

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. Herpesviruses can infect a wide variety of cell types, and are thus bound to encounter the different types of IF expressed in each tissue. The analysis of these interrelationships can yield precious insights into how IF proteins work, and into how viruses have evolved to exploit these functions. These interactions, either known or potential, will be the focus of this review.

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. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV.

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. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

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. Chemical disruption of the vimentin network with acrylamide, intermediate filament bundling in cells from a patient with giant axonal neuropathy, and absence of vimentin in fibroblasts from vimentin(-/-) mice severely reduced entry of either strain. In vimentin null cells, viral particles remained in the cytoplasm longer than in vimentin(+/+) cells. TB40/E infection was consistently slower than that of AD169 and was more negatively affected by the disruption or absence of vimentin. These findings demonstrate that an intact vimentin network is required for CMV infection onset, that intermediate filaments may function during viral entry to facilitate capsid trafficking and/or docking to the nuclear envelope, and that maintenance of a broader cell tropism is associated with a higher degree of dependence on the vimentin cytoskeleton.

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). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2-US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-alpha, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection.

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. In contrast to HIV-specific CD8(+) T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8(+) T cells were predominantly CD27(+)45RO(+) for HIV and CD27(-)45RA(+) for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8(+) T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8(+) T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8(+) T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.