Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts

Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole-mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity.     

控制人巨细胞病毒感染的策略

人巨细胞病毒（HCMV）感染对免疫受损人群危害极大,本综述了目前控制HCMV感染的策略,其中包括药物控制和免疫控制。阐述了使用药物控制HCMV感染的效果,疫苗在控制HCMV感染及引发疾病方面的作用及通过被动免疫控制HCMV感染的现状与进展。对这些策略的了解对控制HCMV感染具有重要意义。     

Stimulation of cellular DNA synthesis by human cytomegalovirus

Human cytomegalovirus (CMV) is able to induce cellular DNA synthesis in both permissive (human embryonic lung) and nonpermissive (Vero) cells. The induction of cell DNA synthesis was assayed by the incorporation of [methyl-³H]thymidine into macromolecules having the buoyant density characteristics of cell DNA. The DNA synthesis induced by CMV infection appears to represent normal semiconservative replication as opposed to repair synthesis. Both strains of CMV tested were capable of inducing cell DNA synthesis. Virus exposed to heat or UV light prior to infection lost the ability to induce DNA synthesis, indicating that a virus-coded function expressed after infection is responsible for stimulation of cell DNA synthesis.     

The human serum metabolome

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.     

AMPK-mediated inhibition of mTOR kinase is circumvented during immediate-early times of human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection increases synthetic rates in infected cells. The resulting increase in energy utilization could potentially increase the AMP:ATP ratio, causing activation of 5'-AMP-activated protein kinase (AMPK). Activated AMPK promotes inhibition of mammalian target of rapamycin (mTOR) kinase, which could be deleterious to the viral infection. Using the AMPK-activating drug 5-amino-4-imidazolecarboxamide ribose (AICAR), we showed that, by 12 h post-HCMV infection, inhibition of mTOR by AMPK is circumvented. However, growth curves showed that progeny virion production is inhibited when AICAR is added, suggesting other inhibitory effects of AICAR or activated AMPK.     

The changing role of mTOR kinase in the maintenance of protein synthesis during human cytomegalovirus infection

The mammalian target of rapamycin (mTOR) kinase occurs in mTOR complex 1 (mTORC1) and complex 2 (mTORC2), primarily differing by the substrate specificity factors raptor (in mTORC1) and rictor (in mTORC2). Both complexes are activated during human cytomegalovirus (HCMV) infection. mTORC1 phosphorylates eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) and p70S6 kinase (S6K) in uninfected cells, and this activity is lost upon raptor depletion. In infected cells, 4E-BP1 and S6K phosphorylation is maintained when raptor or rictor is depleted, suggesting that either mTOR complex can phosphorylate 4E-BP1 and S6K. Studies using the mTOR inhibitor Torin1 show that phosphorylation of 4E-BP1 and S6K in infected cells depends on mTOR kinase. The total levels of 4E-BP1 and viral proteins representative of all temporal classes were lowered by Torin1 treatment and by raptor, but not rictor, depletion, suggesting that mTORC1 is involved in the production of all classes of HCMV proteins. We also show that Torin1 inhibition of mTOR kinase is rapid and most deleterious at early times of infection. While Torin1 treatment from the beginning of infection significantly inhibited translation of viral proteins, its addition at later time points had far less effect. Thus, with respect to mTOR's role in translational control, HCMV depends on it early in infection but can bypass it at later times of infection. Depletion of 4E-BP1 by use of short hairpin RNAs (shRNAs) did not rescue HCMV growth in Torin1-treated human fibroblasts as it has been shown to in murine cytomegalovirus (MCMV)-infected 4E-BP1(-/-) mouse embryo fibroblasts (MEFs), suggesting that during HCMV infection mTOR kinase has additional roles other than phosphorylating and inactivating 4E-BP1. Overall, our data suggest a dynamic relationship between HCMV and mTOR kinase which changes during the course of infection.     

Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.     

The human cerebrospinal fluid metabolome

With continuing improvements in analytical technology and an increased interest in comprehensive metabolic profiling of biofluids and tissues, there is a growing need to develop comprehensive reference resources for certain clinically important biofluids, such as blood, urine and cerebrospinal fluid (CSF). As part of our effort to systematically characterize the human metabolome we have chosen to characterize CSF as the first biofluid to be intensively scrutinized. In doing so, we combined comprehensive NMR, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) Fourier transform-mass spectrometry (FTMS) methods with computer-aided literature mining to identify and quantify essentially all of the metabolites that can be commonly detected (with today's technology) in the human CSF metabolome. Tables containing the compounds, concentrations, spectra, protocols and links to disease associations that we have found for the human CSF metabolome are freely available at http://www.csfmetabolome.ca.     

The effects of human cytomegalovirus infection on cytoskeleton-associated polysomes

Human cytomegalovirus (HCMV) infection induces disruption of the host cell's cytoskeleton (CSK). This disruption is accompanied by three transient phases of actin depolymerization that occur at 20 min, 5 to 10 h and 48 to 72 h post infection (pi). During the 20 min peak of actin depolymerization, the level of cellular polysomes associated with the CSK was reduced, due to release of ribosomes from CSK-associated polysomes. Cellular mRNAs previously existing in these polysomes, however, remained associated with the CSK. Also during this period, nuclear to cytoplasmic transport of host cellular mRNA as well as the association of newly synthesized mRNA with the CSK was temporarily delayed. By 60 min pi, ribosomes, preexisting host cellular mRNA, and newly synthesized mRNAs (host and viral) had reestablished a distribution in the infected cell comparable to that of uninfected cells. Sedimentation profiles of soluble and CSK fractions at various times throughout the viral infection indicated that, although the amount of polysomes associated with the CSK at 20 min pi was reduced, essentially all HCMV and all host cell polysomes present were associated with the CSK. The majority of HCMV DNA hybridizable poly(A)+ RNAs were associated with the CSK throughout the viral infection. These early events appear to correlate with a transient interruption of host cellular mRNA translation early in infection and may represent a process whereby HCMV gene expression becomes competitive with that of the host cell.     

Dynamics of HIV infection studied with cellular automata and conformon-P systems

Recently a cellular automaton (CA) has been used to model the dynamics of HIV infection, with interesting results. We replicate and further test this model, and we introduce an alternative model based on conformon-P (cP) systems. We find (in common with other recently published comments) that the CA model is very sensitive to initial conditions and produces appropriate qualitative dynamics only for a narrow range of rule probabilities. In contrast, the cP system model is robust to a wide range of conditions and parameters, with more reproducible qualitative agreement to the overall dynamics and to the densities of healthy and infected cells observed in vivo.     

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.     

Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.     

The relationship between human cytomegalovirus infection and atherosclerosis development

Transport characteristics of essential trace elements as zinc, copper, selenium and iron have been studied in maternal–fetal direction in normal pregnancies, using in vitro perfusion of human placental lobules. Solutions of trace elements corresponding to twice the physiological concentrations were injected (100 l bolus) into the maternal arterial perfusate. Serial perfusate samples were collected every 30 sec from venous outflows for a study period of 5 min. Concentrations of these trace elements and their transport kinetics were determined. Transport fractions (TF) of zinc, copper, selenium and iron averaged 0.21, 0.49, 0.55 and 0.10% of maternal load respectively. Other parameters such as area under the curve, clearance, elimination constant, absorption and elimination rates showed some significant differences between the various elements. Copper and selenium appear to be transported passively in maternal–fetal direction, while for iron and zinc, role of active transport for transfer across the human placental membrane cannot be discounted. We speculate that alterations in copper: zinc TR50 (transport rate for 50% efflux) and TF ratios could serve as useful indicators for assessing placental transport status of these essential elements in complicated pregnancy states.