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PubMed1989

Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1, encoded by gene UL42, is required for herpes simplex virus origin-dependent DNA replication (C.A. Wu, N.J. Nelson, D.J. McGeoch, and M.D. Challberg, J. Virol. 62:435-443, 1988). We found by indirect immunofluorescence with monoclonal antibody to 65KDBP that the protein was first detectable at 3 h postinfection. It localized first to the inner periphery of the nucleus, but accumulated in large globular compartments within the nucleus by 6 h postinfection in a pattern similar to that displayed by the major DNA-binding protein ICP8. Immune electron microscopy revealed that 65KDBP was associated with the marginated heterochromatin at the early times, but migrated further into the nucleus at late times when the only discernible areas devoid of 65KDBP were the nucleoli and heterochromatin. The 65KDBP gene is a member of the beta kinetic class as determined by the ability of the mRNA to be expressed at significant levels even in the absence of viral DNA synthesis. Furthermore, in the presence or absence of the DNA polymerase inhibitor phosphonoacetic acid, the patterns of accumulation of protein as well as mRNA were virtually indistinguishable from those displayed by the model beta genes encoding ICP8 and thymidine kinase. Nuclear run-on experiments demonstrated that maximum rates of 65KDBP gene transcription occurred prior to the maximum rate of progeny viral DNA synthesis and confirmed that the expression of the 65KDBP gene is regulated at the level of transcriptional initiation.

Animals↗

PubMed1979

Assembly of vesicular stomatitis virus nucleocapsids in vivo: a kinetic analysis.

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.

Actins↗

PubMedDate not supplied

Estimating inestimable standard errors in population pharmacokinetic studies: the bootstrap with Winsorization.

A simulation study was performed to determine how inestimable standard errors could be obtained when population pharmacokinetic analysis is performed with the NONMEM software on data from small sample size phase I studies. Plausible sets of concentration-time data for nineteen subjects were simulated using an incomplete longitudinal population pharmacokinetic study design, and parameters of a drug in development that exhibits two compartment linear pharmacokinetics with single dose first order input. They were analyzed with the NONMEM program. Standard errors for model parameters were computed from the simulated parameter values to serve as true standard errors of estimates. The nonparametric bootstrap approach was used to generate replicate data sets from the simulated data and analyzed with NONMEM. Because of the sensitivity of the bootstrap to extreme values, winsorization was applied to parameter estimates. Winsorized mean parameters and their standard errors were computed and compared with their true values as well as the non-winsorized estimates. Percent bias was used to judge the performance of the bootstrap approach (with or without winsorization) in estimating inestimable standard errors of population pharmacokinetic parameters. Winsorized standard error estimates were generally more accurate than non-winsorized estimates because the distribution of most parameter estimates were skewed, sometimes with heavy tails. Using the bootstrap approach combined with winsorization, inestimable robust standard errors can be obtained for NONMEM estimated population pharmacokinetic parameters with > or = 150 bootstrap replicates. This approach was also applied to a real data set and a similar outcome was obtained. This investigation provides a structural framework for estimating inestimable standard errors when NONMEM is used for population pharmacokinetic modeling involving small sample sizes.

Clinical Trials, Phase I as Topic↗

PubMed1984

Semliki Forest virus penetration from endosomes: a morphological study.

The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest virus genome into the host cell for replication was visualized by electron microscopy. In order to increase the frequency at which fusion images could be detected a reversible inhibitor, ammonium chloride, was used to synchronize endosomal acidification, and 20 degrees C incubation was employed to concentrate virus particles into the endosomal compartment.

Animals↗

PubMed1988

Angiogenesis in the developing corpus luteum of pregnant rats: a stereologic and autoradiographic study.

Within the adult mammalian ovary, angiogenesis is associated with development of the corpus luteum (CL). In this study, developing luteal tissue was examined to determine whether its vascularization involves endothelial cell replication and to what extent this proliferation contributes to forming new capillaries. Five rats each at 12, 24, 36, 48, 60, 72, 84, and 96 hr of gestation were given a subcutaneous injection of tritiated thymidine (specific activity 5 Ci/mM; 1 uCi/g body weight). One hour later they were anesthetised with sodium pentobarbitone, and the left ovary was processed for light microscopy. Sections were cut through each ovary until three newly formed CL were recognized; a 1-micron section was taken from the maximum diameter of each CL and processed for autoradiographic demonstration of thymidine labeling in endothelial cell nuclei. The same sections were also examined with stereological techniques to quantitate growth of the vascular compartment. The results show that 36.1 +/- 5.7% of endothelial cells of invading capillary sprouts divide within 12 hr of ovulation; at 24 hr, 29.0 +/- 2.8% are dividing. Within 12 hr after ovulation, blood vessels occupied 5.9 +/- 1.4% of the peripheral space of the ruptured follicle but only 1.6 +/- 0.5% in the center. However, by 36 hr these respective values were 9.3 +/- 1.6% and 8.4 +/- 1.9%. A further peak in endothelial-cell replication (31.2 +/- 5.4%), early on the 3rd day of gestation, corresponded to the very extensive anastomoses within the capillary bed established between this time (13.6% vascularity) and late on the 4th day (about 23%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Liposome-mediated transfection of intact viral particles reveals that plasma membrane penetration determines permissivity of tissue culture cells to rotavirus.

Rotaviruses are an important cause of gastroenteritis in human infants. In vivo, rotavirus displays striking cell tropism with viral replication generally restricted to the villus tip enterocytes of the small intestine. We studied a panel of cell lines that vary significantly in their permissivity to rotavirus infection. L cells and HEp2 cells were relatively resistant to rotavirus infection compared with permissive Ma104 cells and HT29 cells. RNA transcription among the cell lines was proportional to antigen synthesis making a translational or posttranslational block an unlikely source of observed differences in susceptibility. All of the cell lines bound and internalized radiolabeled virus equally well, as measured by escape from surface protease treatment. Analysis of the escape of cell bound virus from neutralizing monoclonal antibody revealed that rotavirus did not immediately enter an eclipse phase in nonpermissive cells, but was internalized in an infectious form for several hours, possibly sequestered within endocytic vacuoles. L cells and HEp2 cells were as permissive as Ma104 and HT29 cells when rotavirus infection was mediated by transfection of single- or double-shelled rotavirus particles with cationic liposomes (Lipofectin). Rotavirus cell tropism in tissue culture cells is determined by the ability of infecting virions to traverse the plasma membrane of the cells into the cytoplasmic compartment.

Animals↗

PubMed1992

Aging in the T lymphocyte compartment. A developmental view.

A decline in the capacity of bone marrow cells to differentiate to T lymphocytes was found when cells from young and old donors were seeded onto an alymphoid fetal thymus. A step-by-step analysis of cell-cell interactions of the lymphohemopoietic cells and the thymic stroma indicated an effect of age on a variety of cell differentiation parameters. These included a decrease in the affinity of bone marrow cells to the stroma, and in their capacity to compete with the thymic lymphoid resident cells on colonization of the thymus. There was a significant decrease in the ability of cells of old donors to replicate sequentially within the thymic microenvironment. There was a reduced capacity of bone marrow cells from aging mice to express a developmental preference after seeding onto a syngeneic fetal thymus in a mixture with cells from allogeneic donors. We addressed the question whether the aging thymus contains increased levels of immature cells that fail to differentiate in the involuted thymic microenvironment by seeding thymocytes from young and old donors onto the fetal thymic stroma. The values of T cells that developed from the old donor inoculum were lower under these conditions. Our studies suggest that at least some of the manifestations of aging in the T cell compartment are related to developmentally programmed events in the lymphohemopoietic cell compartment.

Aging↗

PubMed1997

Reproducibility and validity of electric source localisation with high-resolution electroencephalography.

The present study investigates the reproducibility and validity of the EEG source localisation of somatosensory evoked potentials (SEPs) using high-resolution EEG (61 scalp electrodes) and a source reconstruction on the basis of the individual brain morphology as obtained from magnetic resonance images (MRIs). The somatosensory evoked potentials (SEPs) to electrical stimulation of the right median nerve were repeatedly collected from the scalp of one healthy subject in 9 replications run on 9 different days. The source reconstruction for the 19 ms SEP component was performed by using a single moving dipole model as a source model. Two different head models were used: a spherical 3 shell model and a more realistically shaped 3 compartment model computed using the boundary element method (BEM). The source locations of the 19 ms SEP component were found to be highly reproducible using both head models: the mean standard deviation of the dipole locations was found to be 2.6 mm for the 3 shell model and 4 mm for the more realistically shaped head model. By projection into the individual MRI, the dipoles resulting from either head models were found to be located within the postcentral gyrus. The electric source locations were consistent with the maximum of the task-specific changes seen in a functional magnetic resonance imaging (fMRI) experiment when using the same somatosensory stimulation protocol.

Brain Mapping↗

PubMed2002

Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.

Cell Cycle↗

PubMed1998

Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotide in vitro.

We have used a ribonuclease protection assay to investigate RNase H cleavage of HIV-1 mRNA mediated by phosphorothioate antisense oligonucleotides complementary to the gag region of the HIV-1 genome in vitro. Cell lysate experiments in H9 and U937 cells chronically infected with HIV-1 IIIB showed RNase H cleavage of unspliced gag message but no cleavage of spliced message which did not contain the target gag region. RNase H cleavage products were detected at oligonucleotide concentrations as low as 0.01 microM and the RNase H activity was seen to be concentration dependent. Similar experiments with 1-, 3- and 5-mismatch oligonucleotides demonstrated sequence specificity at low concentrations, with cleavage of gag mRNA correlating with the predicted activities of the parent and mismatch oligonucleotides based on their hybridization melting temperatures. Experiments in living cells suggested that RNase H-specific antisense activity was largely determined by the amount of oligonucleotide taken up by the different cell lines studied. RNase H cleavage products were detected in antisense oligonucleotide treated MT-4 cells acutely infected with HIV-1 IIIB, but not in infected H9 cells treated with oligonucleotide under the same conditions. The data presented demonstrate potent and specific RNase H cleavage of HIV-1 mRNA mediated by an antisense oligonucleotide targeted to HIV-1 gag mRNA, and are in agreement with previous reports that the major obstacle to demonstrating antisense activity in living cells remains the lack of penetration of these agents into the desired cellular compartment.

Gene Products, gag↗

PubMed2026

Architectural logic of the 3D genome: mechanisms of dysregulation and emerging cancer therapeutics.

The three-dimensional (3D) genome provides an essential layer of organization that shapes genome function in space and time. Chromatin compartments and topologically associating domains (TADs) arise from the interplay between intrinsic properties of chromatin and architectural factors, including cohesin and CTCF. Despite substantial progress in defining these structural features, whether 3D genome architecture plays a causal role in regulating processes such as transcription, DNA replication, and DNA repair, or instead reflects underlying regulatory activity, remains unresolved. Here, we use the distinction between chromatin-intrinsic features and architectural factors as a framework to evaluate evidence for causality in genome structure-function relationships. We extend this framework to cancer, where both intrinsic alterations (including noncoding mutations, structural variants, and changes in chromatin state) and architectural factor perturbations (such as mutations in architectural proteins and dysregulation of transcriptional machinery) disrupt genome organization and contribute to disease progression. These findings suggest that alterations in genome structure can, in some contexts, actively reshape oncogenic programs. A major limitation in applying 3D genome insights to cancer biology is the cost and complexity of omics assays. Recent advances in artificial intelligence (AI) and machine learning (ML) enable inference and prediction of 3D genome organization from sequence and epigenomic features, providing insight into the extent to which genome folding is encoded intrinsically versus dynamically regulated in architectural factors. This perspective provides a unified view of how genome structure is established, how it relates to function, and how its disruption contributes to tumorigenesis.

3D genome↗

PubMed1994

Expression of alpha/beta interferons (IFN-alpha/beta) and their relationship to IFN-alpha/beta-induced genes in lymphocytic choriomeningitis.

Expression of alpha interferon (IFN-alpha)-, IFN-beta-, and IFN-alpha/beta-induced genes was monitored during the development of lymphocytic choriomeningitis (LCM) to assess whether a restricted influence of these antiviral cytokines could be found in the central nervous system (CNS). High levels of IFN-alpha (83 +/- 42 U/ml) were present in the blood of LCM virus-infected mice 3 days postinfection, whereas IFN-beta was not detected (< 1.0 U/ml) at any time point. Spleens contained high levels of IFN-alpha and IFN-beta mRNAs at days 1 and 3 postinfection, whereas no IFN-alpha mRNA and only low levels of IFN-beta mRNA were detected in brains. In situ hybridization showed IFN-alpha mRNA-expressing cells in the marginal zones of the spleen and in the subcapsular sinus and outer cortex of cervical lymph nodes. The expression of 2',5'-oligoadenylate synthetase (2',5'-OAS) mRNA followed the expression of IFN-beta mRNA in the brain, whereas 2',5'-OAS mRNA in the periphery was associated with systemic IFN-alpha. The localization of IFN-alpha-expressing cells in the spleen and lymph nodes in proximity to T- and B-cell compartments is consistent with a role for these cytokines in immune regulation. Furthermore, the absence of IFN-alpha and the relatively low level and delayed expression of IFN-beta in the brain suggest that the CNS is an especially vulnerable organ for virus replication. With certain strains of LCM virus, the absence of early antiviral IFN-alpha/beta activity and preferential virus growth in the brain might lead to targeted T-cell inflammation of the CNS, resulting in death of the animal.

2',5'-Oligoadenylate Synthetase↗

PubMed1997

Vectorial production of interleukin 1 and interleukin 6 by rat Sertoli cells cultured in a dual culture compartment system.

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 production appeared equally distributed between both poles, while IL-6 and transferrin constitutive production was predominantly directed apically. Two activators of macrophages, lipopolysaccharides and zymosan, were found to induce marked increases of IL-1 in the compartment where they had been added: basal if added to the lower compartment and vice versa. In contrast, after a basal stimulation, IL-6 production was mainly increased in the upper compartment that corresponds to a Sertoli cell apical flux. In this system, IL-1 and IL-6 levels were not modified by FSH; they were not also affected by residual bodies and latex beads, probably due to the fact that, in the bicameral system, phagocytosis is restricted to the Sertoli cells situated at the surface of the inner compartment. IL-1beta, but not IL-1alpha, induced IL-6 secretion in the compartment of stimulation. In conclusion, the present study demonstrates that vectorial secretory patterns of IL-1 and IL-6 production greatly differ and that these cytokines are also differently regulated. These results suggest that Sertoli IL-1 and IL-6 have different targets within the testis and that, in normal and pathophysiological conditions, both the tubular and the interstitial compartments may be influenced by the action of these paracrine factors.

Animals↗

PubMed2004

Avoidance of copper contamination by field populations of Daphnia longispina.

Almost all terrestrial and aquatic assays that are accepted as standardized, or that have been proposed, involve the exposure of confined organisms to toxicants. If avoidance (sensu evasion, displacement) of contaminants occurs in real situations, then bioassays involving forced exposure severely underestimate pernicious effects of contamination. Two main objectives were achieved in this study: To verify the occurrence of avoidance of copper by cladocerans; to determine the association between avoidance and other toxicity endpoints (lethality and feeding depression), and therefore, to ascertain if fully acclimated individuals from a reference field population exhibited a genetically determined avoidance markedly different from those belonging to a historically metal-stressed population. Twelve cloned lineages of the cladoceran Daphnia longispina, collected from two field populations, were selected according to their lethal sensitivity to copper and acclimated to controlled conditions for more than 30 generations. A 1.1-m test chamber with five compartments was built, allowing the establishment of a dissolved toxicant gradient and the free movement of individuals. In the absence of any toxicant, juveniles from each cloned lineage distributed themselves randomly along the test chamber and furthermore, no significant differences were observed between the two replicates, attesting the repeatability of this novel assay. All lineages showed significant avoidance to copper when exposed to a gradient from 3 to 87 microg/L. The most sensitive lineages to lethal levels of copper began avoiding this metal earlier than resistant ones. An intense association was observed between other endpoints and avoidance; furthermore, avoidance was determined to be much more sensitive than lethality. Therefore, avoidance assays should be recommended as a complementary tool in ecological risk assessments and effluent biomonitoring because such assays can provide cost-effective and ecologically relevant information.

Animals↗

PubMed2026

Epigenetic alterations in rheumatoid arthritis: multilayer mechanisms and translational opportunities.

Rheumatoid arthritis (RA) is a chronic inflammatory disease driven by immune dysregulation, in which genetic susceptibility and environmental exposures promote persistent synovitis, progressive joint damage, and systemic comorbidities. Recent epigenomic studies show several recurring abnormalities. Many RA susceptibility variants lie outside protein-coding sequence and map to immune-cell and synovial fibroblast regulatory elements, linking inherited risk to enhancer activity, methylation quantitative trait effects, and distal gene control. Blood-based epigenome-wide association studies identify disease-associated DNA methylation signatures, but these signals require careful control for leukocyte composition, smoking, treatment exposure, and disease stage. RA fibroblast-like synoviocytes also display stable methylome remodeling, including relative hypomethylation at loci involved in inflammation, migration, matrix degradation, and apoptosis resistance, while TET3-associated 5-hydroxymethylcytosine has emerged as a functional contributor to chemokine production and invasive stromal behavior. Histone modifications, chromatin accessibility, and 3D genome organization define pathogenic regulatory states and connect non-coding risk loci to effector genes in immune and stromal compartments. Finally, miRNAs, lncRNAs, circRNAs, snoRNAs, extracellular RNAs, and m6A-related pathways add post-transcriptional and chromatin-linked layers with potential biomarker value. We synthesize these findings and discuss translational opportunities for diagnosis, stratification, flare monitoring, and therapeutic targeting, while emphasizing incomplete replication, uneven evidence across epigenetic layers, biospecimen variability, and the need for causal, longitudinal, cell-type-resolved validation.

Humans↗

PubMed1996

Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction.

Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by "tagged" RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.

Adult↗

PubMed1997

Preclinical, pharmacologic, and phase I studies of gemcitabine.

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel nucleoside analogue that exerts its antitumor activity via multiple mechanisms of action. These include (1) incorporation of gemcitabine into replicating DNA, which inhibits DNA replication and cell growth, (2) masked DNA chain termination, and (3) several self-potentiation mechanisms that serve to increase intracellular levels of the active compound. Preclinical experiments in various cell lines and animal models demonstrate a broad range of cytotoxic activity. Pharmacokinetic studies of gemcitabine delivered by its usual schedule (30-minute weekly infusion) reveal a short plasma half-life and a high clearance into central and peripheral compartments (two-compartment model). The drug is excreted almost completely in the urine as the parent compound and primary metabolite (difluorodeoxyuridine). Phase I trials demonstrate that pharmacokinetics are schedule dependent and that, in general, gemcitabine is well tolerated. Dose-limiting toxicities are primarily myelosuppression, with other toxicities being rash, flu-like symptoms, and transient elevations in liver function tests.

Animals↗

PubMed2006

Localization of ERK/MAP kinase is regulated by the alphaherpesvirus tegument protein Us2.

Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.

Animals↗