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PubMed2002

Importance of compartment formation for a self-encoding system.

A self-encoding system designed to have strict "compartition" of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.

Amino Acid Substitution↗
PubMed1989

The role of viral and cellular nuclear proteins in herpes simplex virus replication.

Following infection of cells by herpes simplex virus, the cell nucleus is subverted for transcription and replication of the viral genome and assembly of progeny nucleocapsids. The transition from host to viral transcription involves viral proteins that influence the ability of the cellular RNA polymerase II to transcribe a series of viral genes. The regulation of RNA polymerase II activity by viral gene products seems to occur by several different mechanisms: (1) viral proteins complex with cellular proteins and alter their transcription-promoting activity (e.g., alpha TIF), (2) viral proteins bind to specific DNA sequences and alter transcription (e.g., ICP4), and (3) viral proteins affect the posttranslational modification of viral or cellular transcriptional regulatory proteins (e.g., possibly ICP27). Thus, HSV may utilize several different approaches to influence the ability of host-cell RNA polymerase II to transcribe viral genes. Although it is known that viral transcription uses the host-cell polymerase II, it is not known whether viral infection causes a change in the structural elements of the nucleus that promote transcription. In contrast, HSV encodes a new DNA polymerase and accessory proteins that complex with and reorganize cellular proteins to form new structures where viral DNA replication takes place. HSV may encode a large number of DNA replication proteins, including a new polymerase, because it replicates in resting cells where these cellular gene products would never be expressed. However, it imitates the host cell in that it localizes viral DNA replication proteins to discrete compartments of the nucleus where viral DNA synthesis takes place. Furthermore, there is evidence that at least one specific viral gene protein can play a role in organizing the assembly of the DNA replication structures. Further work in this system may determine whether assembly of these structures is essential for efficient viral DNA replication and if so, why assembly of these structures is necessary. Thus, the study of the localization and assembly of HSV DNA replication proteins provides a system to examine the mechanisms involved in morphogenesis of the cell nucleus. Therefore, several critical principles are apparent from these discussions of the metabolism of HSV transcription and DNA replication. First, there are many ways in which the activity of RNA polymerase II can be regulated, and HSV proteins exploit several of these in controlling the transcription of a single DNA molecule. Second, the interplay of these multiple regulatory pathways is likely to control the progress of the lytic cycle and may play a role in determining the lytic versus latent infection decision.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA Replication↗
PubMed2006

Patterns of human cytomegalovirus infection in term placentas: a preliminary analysis.

BACKGROUND: Primary maternal CMV infection is the major risk factor for symptomatic congenital infection as maternal immunity reduces the risk of transmission to the fetus. Analysis of first trimester placentas showed that virus replicates in the uterus and is transmitted to the placenta causing focal infection. OBJECTIVES AND STUDY DESIGN: We examined 78 term placentas from uncomplicated deliveries for the presence of CMV DNA and evaluated evidence of infection by means of immunohistological and serological analysis. RESULTS: PCR analysis of villus biopsy samples and decidua showed that CMV DNA was present in 62% of tissues. Seven placentas with neutralizing titers were further examined by immunohistology for expression of viral proteins. In placentas with high levels of CMV DNA, fetal blood vessels in the villus core contained neutrophils with viral replication proteins, and macrophages/dendritic cells with glycoprotein B (gB). Cord blood samples from 1 of 11 placentas contained CMV DNA, an indication of replication in the fetal compartment. In placentas with low levels of viral DNA, macrophage/dendritic cells in the villus core contained CMV gB. This pattern was comparable to that seen in early gestation placentas from women with strong neutralizing antibodies. CONCLUSIONS: The results show CMV replication proteins in focal areas of the placenta, implying virus transmission to the fetal circulation. These preliminary results suggest that the incidence of asymptomatic congenital CMV infection might be higher than currently estimated.

Cytomegalovirus↗
PubMed2005

Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA.

BACKGROUND: West Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed. Recent studies have suggested that RNA interference (RNAi) has therapeutic potential as a sequence specific inhibitor of viral infection. Here, we examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of West Nile virus in human cells. RESULTS: WNV replication and infection was greatly reduced when siRNA were introduced by cytoplasmic-targeted transfection prior to but not after the establishment of viral replication. WNV appeared to evade rather than actively block the RNAi machinery, as sequence-specific reduction in protein expression of a heterologous transgene was still observed in WNV-infected cells. However, sequence-specific decreases in WNV RNA were observed in cells undergoing active viral replication when siRNA was transfected by an alternate method, electroporation. CONCLUSION: Our results suggest that actively replicating WNV RNA may not be exposed to the cytoplasmic RNAi machinery. Thus, conventional lipid-based siRNA delivery systems may not be adequate for therapy against enveloped RNA viruses that replicate in specialized membrane compartments.

Cell Line↗
PubMed2001

Thymic lesions in cats infected with a pathogenic molecular clone or an ORF-A/2-deficient molecular clone of feline immunodeficiency virus.

Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (ORF-A/2) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518-8526, 1996; E. E. Sparger, et al., Virology 205:546-553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an ORF-A/2-deficient mutant, JSY3DeltaORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3DeltaORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3DeltaORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3DeltaORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4(-) CD8(+) thymocytes in JSY3DeltaORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4(-) CD8(+) thymocyte subpopulation.

Animals↗
PubMed2004

The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the alga Scenedesmus quadricauda.

The courses of rRNA accumulation, DNA replication, and nuclear division were followed both in the chloroplast and the nucleocytosolic compartments during the cell cycle in synchronized populations of the chlorococcal alga Scenedesmus quadricauda. Control and nalidixic acid-treated cultures were compared. Nalidixic acid (150 mg/L) was added either at the beginning of the cell cycle or consecutively during the cell cycle to subcultures transferred into the dark. If the inhibitor was applied at the beginning of the cell cycle, chloroplast DNA did not replicate and nucleoids did not divide. Chloroplast division, however, was coordinated in a timely fashion with cytokinesis even under conditions of blocked chloroplast DNA replication. While the growth rate was slowed down, the courses of reproductive processes in the nucleocytosolic compartment were not affected and their timing and the number of rounds were coordinated with growth rate as in the control culture. The rate of cytosolic rRNA synthesis was lower but no apparent effect was seen on the amount of rRNA that accumulated during the cell cycle. In contrast, lower levels of chloroplast rRNA were found at the end of the cell cycle compared with the control culture. Experiments in which cells were transferred to the dark during the cell cycle showed that the inhibitor affected none of the reproductive events in the nucleocytosolic compartment. In the chloroplast compartment, DNA replication was inhibited in inhibitor-treated cultures, but was unaffected in controls. The chloroplast nucleoids themselves divided even in the presence of the inhibitor, reducing their DNA content to a level which corresponded to that in freshly formed control daughter cells.

Chloroplasts↗
PubMed2005

Host cell nuclear proteins are recruited to cytoplasmic vaccinia virus replication complexes.

The initiation and termination of vaccinia virus postreplicative transcription have been reported to require cellular proteins, some of which are believed to be nuclear proteins. Vaccinia virus replicates in the cytoplasmic compartment of the cell, raising questions as to whether vaccinia virus has access to nuclear proteins. This was addressed here by following the fate of several nuclear proteins after infection of cells with vaccinia virus. The nuclear transcription factors YY1, SP1, and TATA binding protein were found to colocalize with virus replication complexes in the cytoplasm of infected cells. In addition, the nuclear proteins RNA polymerase II, TAFIIp32, and histone deacetylase 8, but not the structural protein lamin B, also were found in the cytoplasm of the cell. The association of YY1 with replication complexes was dependent on DNA replication and required only the DNA binding domain of the protein, indicating that DNA binding alone may be responsible for the association of nuclear transcription factors with viral replication complexes in the cytoplasm. The cytoplasmic localization of YY1 was resistant to the nuclear export inhibitor leptomycin B. Evidence is presented indicating that nuclear import and export pathways were not adversely affected by vaccinia virus infection. These observations indicate that vaccinia virus replication complexes have ready access to nuclear proteins by allowing leakage from the nucleus.

Animals↗
PubMed2003

Differences in HIV-1 pol sequences from female genital tract and blood during antiretroviral therapy.

OBJECTIVE: To determine whether HIV-1 replicates locally in the female genital tract during therapy, and to study whether endocervix is the dominant source of virus in cervicovaginal lavage fluid. DESIGN: Sequence analyses of HIV-1 pol were performed from cervicovaginal secretions and blood plasma of HIV-infected women failing antiretroviral therapy with detectable viral load in both compartments, as well as from drug-naive subjects. METHODS: Viral RNA was extracted from cervicovaginal lavage fluid, endocervical secretions collected by Sno-strips, and blood plasma. Population sequencing of HIV-1 pol was performed using cycle sequencing. Drug resistance mutations were analyzed. Phylogenies were constructed based on synonymous positions in the sequences. RESULTS: Resistant virus was detected concordantly in blood and genital tract specimens, consistent with drug selection pressure in both compartments. However, drug-selected mutations often differed in each compartment, and phylogenetic analysis showed differences in virus lineage in these compartments, consistent with local replication in female genital tract. Viruses in cervicovaginal lavage and endocervical secretions were genetically distinguishable, suggesting that endocervix is not the only source of virus found in cervicovaginal lavage. CONCLUSION: These data support the hypothesis that HIV replication is compartmentalized within the female genital tract during antiretroviral therapy, which has implications for pathogenesis and for epidemiologic surveillance of drug-resistant virus.

Adult↗
PubMed2001

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis.

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.

Bacillus subtilis↗
PubMed2002

The antiviral protein human lactoferrin is distributed in the body to cytomegalovirus (CMV) infection-prone cells and tissues.

PURPOSE: Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. METHODS AND RESULTS: After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels. was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs. mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. CONCLUSIONS: The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.

Animals↗
PubMed2004

Nef binds p6* in GagPol during replication of human immunodeficiency virus type 1.

The atypical Nef protein (NefF12) from human immunodeficiency virus type 1 strain F12 (HIV-1(F12)) interferes with virion production and infectivity via a mysterious mechanism. The correlation of these effects with the unusual perinuclear subcellular localization of NefF12 suggested that the wild-type Nef protein could bind to assembly intermediates in late stages of viral replication. To test this hypothesis, Nef from HIV-1(NL4-3) was fused to an endoplasmic reticulum (ER) retention signal (NefKKXX). This mutant NefKKXX protein recapitulated fully the effects of NefF12 on on Gag processing and virion production, either alone or as a CD8 fusion protein. Importantly, the mutant NefKKXX protein also localized to the intermediate compartment, between the ER and the trans-Golgi network. Furthermore, Nef bound the GagPol polyprotein in vitro and in vivo. This binding mapped to the C-terminal flexible loop in Nef and the transframe p6* protein in GagPol. The significance of this interaction was demonstrated by a genetic assay in which the release of a mutant HIV-1 provirus lacking the PTAP motif in the late domain that no longer binds Tsg101 was rescued by a Nef.Tsg101 chimera. Importantly, this rescue as well as incorporation of Nef into HIV-1 virions correlated with the ability of Nef to interact with GagPol. Our data demonstrate that the retention of Nef in the intermediate compartment interferes with viral replication and suggest a new role for Nef in the production of HIV-1.

Amino Acid Sequence↗
PubMed2005

Organization of supercoil domains and their reorganization by transcription.

During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription-dependent chromosome remodelling are also emerging from eukaryotic cell systems.

Bacterial Proteins↗
PubMed2004

Stimulation of Kaposi's sarcoma-associated herpesvirus viremia during hematopoietic stem cell mobilization with filgrastim.

The effects of hematopoietic stem cell (HSC) mobilization on Kaposi's sarcoma-associated herpesvirus (KSHV) were evaluated in three KSHV and human immunodeficiency virus type 1 co-infected subjects. KSHV DNA was not detected in purified CD34+ cell preparations from the period of filgrastim treatment. However, two of 3 subjects had transiently increased cell-free plasma KSHV DNA during filgrastim treatment. Peak plasma KSHV DNA (2,600 and 4,300 copies/mL) occurred on day 4 and declined to below the limit of detection by day 7. These findings suggest that, although CD34+ cell preparations do not have evidence of KSHV infection, HSC mobilization may stimulate KSHV replication in other cellular compartments that contribute to KSHV viremia.

DNA, Viral↗
PubMed2002

The host cytosol: front-line or home front?

Intracellular pathogens replicate in modified vacuolar compartments or in the cytosol of host cells. Many pathogenic bacterial species have evolved to modify the host vacuolar environment, but little is known about the mammalian cytosol as a medium for bacterial growth. Recent studies indicate that the cytosol is restrictive for the growth of bacteria other than cytosolic pathogens in contrast to earlier research that provided evidence that any bacteria with access to the cytosol can replicate there. Comparison of these studies suggests that the cytosolic contents of various host cell types can be differentially permissive for bacterial growth, and that both host and bacterial factors are important in determining the ability of particular bacteria to replicate in the cytosol.

Bacteria↗
PubMed1987

A model for the spatio-temporal organization of DNA replication in mammalian cells.

The spatio-temporal organization of chromosomal DNA replication was analyzed using a model based on a "DNA unit" (or decondensation unit) hypothesis. The model is an extension of the fork movement theory of Huberman & Riggs (1968) and can account for a partially deterministic and partially stochastic order of DNA replication in chromosomes. It presumes that each chromosome is composed of DNA units that are arranged in sequence and that are replicated in parallel. A deterministic wave of chromatin decondensation propagates along the DNA unit continuously and progressively providing a field for the random activation of replication origin. Assignment of replication times to DNA compartments by a Monte Carlo method was programmed based on the model and the program was used to stimulate DNA synthesis rate curves that can be measured by the method of Dolbeare et al. (1983, 1985). The shape of the curve is shown to constrain possible parameter values of the model, which include the rate of fork movement, the fraction of chromatin that is decondensed at the start of S-phase, the initial number of origins activated, the rate at which new origins are activated, etc. The chromosomal organization that controls the molecular level of DNA replication is briefly reviewed and its relevance to the model is also discussed.

Algorithms↗
PubMed2003

Monitoring HIV disease with new and clinically useful surrogate markers.

PURPOSE OF REVIEW: This review aims to identify which patient based observations could enable the development of new surrogate markers for widespread clinical use. RECENT FINDINGS: Anti-retroviral drug therapy reduces but does not abolish HIV transmission and replication in all body compartments. It is now clear that monitoring plasma HIV RNA does not help to predict drug failure or to define the existence of persistent viral reservoirs. SUMMARY: New surrogate markers are required for long-term patient monitoring and to enable the evaluation of additional therapeutic strategies.

Biomarkers↗
PubMed1990

Presentation of Listeria monocytogenes to CD8+ T cells requires secretion of hemolysin and intracellular bacterial growth.

Cell-mediated immunity to Listeria monocytogenes (LM) involves both CD4+ and CD8+ T cell responses. An important virulence factor in the pathogenesis of infection and development of protective immunity to LM is secretion of the sulfhydryl-activated hemolysin (Hly), listeriolysin. Listeria secretion of Hly allows LM to escape the endosomal compartment and enter the cytosol of the cell where intracellular growth can occur. We developed a system using bone marrow macrophages cultured in CSF-1 or IFN-gamma for comparing the response of CD4+ or CD8+ T cells to heat-killed, live Hly-, and live Hly+ LM. Macrophages grown in CSF were permissive for intracellular growth of Hly+ but not Hly- LM. CD8+ T cells recognized Hly+ LM but not HK or Hly- LM pulsed macrophages. However, CD4+ T cells recognized all three Listeria preparations fed to IFN-gamma-treated macrophages. These results suggest that both heat-killed LM and live LM efficiently enter the exogenous pathway for class II Ag processing and presentation. In contrast, only Hly+ LM activates the class I pathway, probably as a result of Hly+ bacterial replication within the cytosolic compartment.

Animals↗
PubMed1976

Pluripotential stem cell differentiation in hemopoietic colonies.

To determine if mononuclear cells proliferating in murine hemopoietic spleen colonies were pluripotential in addition to possessing kinetic features of stem cells, we performed sequential studies of mice during their recovery from a split-dose irradiation regimen of 850 roentgens leg shielded-3-hr interval-850 roentgens leg irradiated (850R L.S. 3- L.I.). Injecting tritiated thymidine during stem cell compartment repletion 3 and 4 days after 850R L.S. 3- L.I. resulted in heavily labeled mononuclear cells resembling medium to large leptochromatic lymphocytes in the portion of spleen removed an hour after injection. The splenic remnant obtained from the same mouse 24-48 hr later contained lightly labeled erythroblasts, myeloid cells, and lymphoid cells. Grain counts suggested that erythroblasts and their precursors had undergone about four divisions, myeloid cells and their precursors two to three divisions, and lymphoid cells and their precursors two to three divisions during the 48-hr period. Similar studies in plethoric mice demonstrated the labeling of mononuclear cells on day 4 and their differentiation to myeloid and lymphoid cells by day 6. This finding confirmed that the labeled mononuclear cells were not exclusively erythroblast progenitors. On the basis of these and previous studies of post-irradiation survival and erythropoietic recovery, we conclude that these endogenous monomuclear cells, which resemble medium to large leptochromatic lymphocytes and replicate during stem cell compartment repletion, are pluripotential hemopoietic stem cells.

Animals↗