Biomedical subjects

Thomas Shenk

Publications and source records attributed to Thomas Shenk.

At least 19 recordsLinked to original sources

PubMed2007

Human cytomegalovirus UL38 protein blocks apoptosis.

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.

Apoptosis↗

PubMed2007

An HIV feedback resistor: auto-regulatory circuit deactivator and noise buffer.

Animal viruses (e.g., lentiviruses and herpesviruses) use transcriptional positive feedback (i.e., transactivation) to regulate their gene expression. But positive-feedback circuits are inherently unstable when turned off, which presents a particular dilemma for latent viruses that lack transcriptional repressor motifs. Here we show that a dissipative feedback resistor, composed of enzymatic interconversion of the transactivator, converts transactivation circuits into excitable systems that generate transient pulses of expression, which decay to zero. We use HIV-1 as a model system and analyze single-cell expression kinetics to explore whether the HIV-1 transactivator of transcription (Tat) uses a resistor to shut off transactivation. The Tat feedback circuit was found to lack bi-stability and Tat self-cooperativity but exhibited a pulse of activity upon transactivation, all in agreement with the feedback resistor model. Guided by a mathematical model, biochemical and genetic perturbation of the suspected Tat feedback resistor altered the circuit's stability and reduced susceptibility to molecular noise, in agreement with model predictions. We propose that the feedback resistor is a necessary, but possibly not sufficient, condition for turning off noisy transactivation circuits lacking a repressor motif (e.g., HIV-1 Tat). Feedback resistors may be a paradigm for examining other auto-regulatory circuits and may inform upon how viral latency is established, maintained, and broken.

Feedback, Physiological↗

PubMed2006

Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores.

The human CMV UL37x1-encoded protein, also known as the viral mitochondria-localized inhibitor of apoptosis, traffics to the endoplasmic reticulum and mitochondria of infected cells. It induces the fragmentation of mitochondria and blocks apoptosis. We demonstrate that UL37x1 protein mobilizes Ca(2+) from the endoplasmic reticulum into the cytosol. This release is accompanied by cell rounding, cell swelling, and reorganization of the actin cytoskeleton, and these morphological changes can be substantially blocked by a Ca(2+) chelating agent. The UL37x1-mediated release of Ca(2+) from the endoplasmic reticulum likely has multiple consequences, including induction of the unfolded protein response, modulation of mitochondrial function, induction of mitochondrial fission, and protection against apoptotic stimuli.

Actins↗

PubMed2006

Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse.

The human cytomegalovirus immediate-early 5-kb RNA previously has been shown to be a stable intron that is not required for efficient replication of the virus in cultured fibroblasts. Here we describe a murine cytomegalovirus 7.2-kb ortholog of the human cytomegalovirus 5-kb RNA. The 5' end of the 7.2-kb transcript maps to a consensus splice-donor site that is conserved among all cytomegaloviruses. We constructed mutant viruses lacking the entire 7.2-kb coding domain, the splice-donor site predicted to function in the generation of the intron or a hairpin predicted to stabilize the intron. All three mutant viruses failed to produce the 7.2-kb RNA, supporting our conclusion that it is a stable intron. Each of the mutants replicated with normal kinetics in cultured fibroblasts, but the mutants exhibited a clear defect within infected mice. Although the initial acute phase at 4 days after infection appeared to be normal, none of the mutant viruses progressed to the persistent phase, i.e., little virus was detected in the salivary gland at 14 days after infection. The intron functions as an in vivo virulence factor, facilitating progression from the acute to persistent phase of infection.

Animals↗

PubMed2006

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins.

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway.

Capsid Proteins↗

PubMed2006

Human cytomegalovirus pUS24 is a virion protein that functions very early in the replication cycle.

We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

Cells, Cultured↗

PubMed2006

UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells.

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

Blotting, Western↗

PubMed2006

Dynamics of the cellular metabolome during human cytomegalovirus infection.

Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host cell. Despite this reliance, the effect of viral infection on host cell metabolic composition remains poorly understood. Here we applied liquid chromatography-tandem mass spectrometry to measure the levels of 63 different intracellular metabolites at multiple times after human cytomegalovirus (HCMV) infection of human fibroblasts. Parallel microarray analysis provided complementary data on transcriptional regulation of metabolic pathways. As the infection progressed, the levels of metabolites involved in glycolysis, the citric acid cycle, and pyrimidine nucleotide biosynthesis markedly increased. HCMV-induced transcriptional upregulation of specific glycolytic and citric acid cycle enzymes mirrored the increases in metabolite levels. The peak levels of numerous metabolites during infection far exceeded those observed during normal fibroblast growth or quiescence, demonstrating that HCMV markedly disrupts cellular metabolic homeostasis and institutes its own specific metabolic program.

Cells, Cultured↗

PubMed2005

Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism.

Human cytomegalovirus replicates in many different cell types, including epithelial cells, endothelial cells, and fibroblasts. However, laboratory strains of the virus, many of which were developed as attenuated vaccine candidates by serial passage in fibroblasts, have lost the ability to infect epithelial and endothelial cells. Their growth is restricted primarily to fibroblasts, due to mutations in the UL131-UL128 locus. We now demonstrate that two products of this locus, pUL130 and pUL128, form a complex with gH and gL, but not gO. The AD169 laboratory strain, which lacks a functional UL131 protein, produces virions containing only the gH-gL-gO complex. An epithelial and endothelial cell tropic AD169 variant in which the UL131 ORF has been repaired, termed BADrUL131, produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130 complexes. Antibodies against pUL130 and pUL128 block infection of epithelial and endothelial cells by BADrUL131 and the fusion-inducing factor X clinical human cytomegalovirus isolate but do not affect the efficiency with which fibroblasts are infected.

Cytomegalovirus↗

PubMed2005

Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein.

The human cytomegalovirus UL99-coded pp28 is a myristoylated phosphoprotein located in the virion tegument domain, which resides between the capsid and envelope. A previous study has demonstrated that BADsubUL99, a pp28-deficient mutant virus, fails to assemble enveloped virus particles. Capsids, coated with tegument proteins, accumulate in the cytoplasm of mutant virus-infected cells. This phenotype indicates that pp28 is required for the acquisition of an envelope; it presumably acts by directing tegument-associated capsids to bud through an intracellular membrane derived from the cell's secretory apparatus that has been modified to contain viral transmembrane glycoproteins. Here we demonstrate that BADsubUL99 can spread from cell to cell, even though highly sensitive assays fail to detect infectious virus progeny in cultures of infected fibroblasts. We propose that, in the absence of pp28, tegument-coated capsids might nevertheless bud through cellular membranes, including the plasma membrane. If this suggestion is correct, the enveloped particle could potentially infect an adjacent cell to mediate the cell-to-cell spread that is observed. This mode of spread might also occur after infection with wild-type virus, and it could facilitate immune evasion, assuming that the resulting particles do not have a normal complement of virus-coded envelope glycoproteins.

Antiviral Agents↗

PubMed2005

Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism.

Epithelial cells are one of the prominent cell types infected by human cytomegalovirus (HCMV) within its host. However, many cultured epithelial cells, such as ARPE-19 retinal pigmented epithelial cells, are poorly infected by laboratory-adapted strains in cell culture, and little is known about the viral factors that determine HCMV epithelial cell tropism. In this report, we demonstrate that the UL131 open reading frame (ORF), and likely the entire UL131-128 locus, is required for efficient infection of epithelial cells. Repair of the mutated UL131 gene in the AD169 laboratory strain of HCMV restored its ability to infect both epithelial and endothelial cells while compromising its ability to replicate in fibroblasts. ARPE-19 epithelial cells support replication of the repaired AD169 virus as well as clinical isolates of HCMV. Productive infection of cultured epithelial cells, endothelial cells, and fibroblasts with the repaired AD169 virus leads to extensive membrane fusion and syncytium formation, suggesting that the virus may spread through cell-cell fusion.

Amino Acid Sequence↗

PubMed2004

Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation.

The human cytomegalovirus 72-kDa immediate-early (IE)1 and 86-kDa IE2 proteins are expressed at the start of infection, and they are believed to exert much of their function through promiscuous transcriptional activation of viral and cellular gene expression. Here, we show that the impaired growth of an IE1-deficient mutant virus in human fibroblasts is efficiently rescued by histone deacetylase (HDAC) inhibitors of three distinct chemical classes. In the absence of IE1 expression, the viral major IE and UL44 early promoters exhibited decreased de novo acetylation of histone H4 during the early phase of infection, and the hypoacetylation correlated with reduced transcription and accumulation of the respective gene products. Consistent with these findings, IE1 interacts specifically with HDAC3 within infected cells. We also demonstrate an interaction between IE2 and HDAC3. We propose that the ability to modify chromatin is fundamental to transcriptional activation by IE1 and, likely, IE2 as well.

Acetylation↗

PubMed2004

Human cytomegalovirus encodes a highly specific RANTES decoy receptor.

The human cytomegalovirus pUL21.5 protein is a small, secreted glycoprotein whose mRNA is packaged into virions. We demonstrate that pUL21.5 protein is a soluble CC chemokine receptor that functions as a decoy to modulate the host immune response to infection. In contrast to other viral chemokine-binding proteins, which interact promiscuously with multiple chemokines, pUL21.5 selectively binds RANTES (regulated upon activation, normal T cell expressed and secreted) with high affinity. By binding RANTES, pUL21.5 blocks RANTES interaction with its cellular receptors. We propose that human cytomegalovirus directs the synthesis of a secreted, virus-coded protein that modulates the host antiviral response even before the newly infecting viral genome becomes transcriptionally active.

Cells, Cultured↗

PubMed2004

Evaluation of the host transcriptional response to human cytomegalovirus infection.

Gene expression data from human cytomegalovirus (HCMV)-infected cells were analyzed using DNA-Chip Analyzer (dChip) followed by singular value decomposition (SVD) and compared with a previous analysis of the same data that employed GeneChip software and a fold change filtering approach. dChip and SVD analysis revealed two clusters of coexpressed human genes responding differently to HCMV infection: one containing some genes identified previously, and another that was largely unique to this analysis. Annotating these genes, we identified several functional categories important to host cell responses to HCMV infection. These categories included genes involved in transcriptional regulation, oncogenesis, and cell cycle regulation, which were more prevalent in cluster 1, and genes involved in immune system regulation, signal transduction, and cell adhesion, which were more prevalent in cluster 2. Within these categories, we found genes involved in the host response to HCMV infection (mainly in cluster 1), as well as genes targeted by HCMV's immune evasion strategies (mainly in cluster 2). As the second group of genes identified by the dChip and SVD approach was statistically and biologically significant, our results point out the advantages of using different methods to analyze gene expression data.

Cytomegalovirus Infections↗

PubMed2004

Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations.

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment, and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells, viral gene expression was undetectable by 10 days after infection. Importantly, viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly, a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency.

ADP-ribosyl Cyclase↗

PubMed2004

SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication.

The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus (BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72 kDa due to a single amino acid exchange in the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity of IE1-72 kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72 kDa, including its ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient IE1-72 kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to modify IE1-72 kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein (IE2-86 kDa). These observations suggest that SUMO modification of IE1-72 kDa contributes to efficient HCMV replication by promoting the accumulation of IE2-86 kDa.

Cell Line↗

PubMed2004

Human cytomegalovirus TRS1 protein is required for efficient assembly of DNA-containing capsids.

The human cytomegalovirus tegument protein, pTRS1, appears to function at several discrete stages of the virus replication cycle. We previously demonstrated that pTRS1 acts during the late phase of infection to facilitate the production of infectious virions. We now have more precisely identified the late pTRS1 function by further study of a mutant virus lacking the TRS1 region, ADsubTRS1. We observed a significant reduction in the production of capsids, especially DNA-containing C-capsids, in mutant virus-infected cells. ADsubTRS1 exhibited normal cleavage of DNA concatemers, so the defect in C-capsid production must occur after DNA cleavage and before DNA is stably inserted into a capsid. Further, the normal virus-induced morphological reorganization of the nucleus did not occur after infection with the pTRS1-deficient mutant.

Capsid↗

PubMed2004

RNAs are packaged into human cytomegalovirus virions in proportion to their intracellular concentration.

The assembly of human cytomegalovirus (HCMV) virions is a complex process and involves the incorporation of viral transcripts. These RNAs are delivered to the newly infected cells and have the potential to be translated in the absence of HCMV gene expression. We have quantified the relative amount of RNAs in HCMV virions and infected cells with real-time reverse transcription-PCR and observed that viral and cellular RNAs are packaged in proportion to the amount of RNA within the cell at the time of assembly. To determine whether cis elements influenced RNA packaging, we constructed a recombinant HCMV mutant virus that expressed the yellow fluorescence protein (YFP) gene fused to the virion RNA UL21.5. We also constructed a mutant virus in which the UL21.5 transcription unit was replaced with the YFP gene. YFP RNA was incorporated into both viruses, indicating that RNA is incorporated in the absence of a virus-specific signal motif. Furthermore, with in situ hybridization, packaged transcripts were observed throughout the cytoplasm of the infected cells, including the site of virus assembly. Several proteins that nonspecifically interact with RNA, including the tegument protein pp28, were found within HCMV virions. These studies demonstrate that both viral and cellular RNAs are nonspecifically incorporated into HCMV, potentially through interactions with several virion proteins.

Bacterial Proteins↗