Nuclei Of Uninfected Cells Research Articles

Co-expression of four baculovirus proteins, IE1, LEF3, P143, and PP31, elicits a cellular chromatin-containing reticulate structure in the nuclei of uninfected cells

Baculovirus DNA replication, transcription, and nucleocapsid assembly occur within a subnuclear structure called the virogenic stroma (VS) that consists of two subcompartments. Specific components of the VS sub-compartments have not been identified except for PP31, a DNA-binding protein that localizes specifically to the electron-dense region of VS. Here, we investigate the dynamic structure of VS using a GFP-tagged PP31 molecule (GFP-PP31). GFP-PP31 localizes to the VS throughout the course of infection. At later times post-infection, a PP31 reticulum distributed within VS was also apparent, indicating that VS sub-compartments compose a reticulate structure. Transient expression of PP31 with the viral proteins, IE1, LEF3, and P143, in uninfected cells resulted in the formation of a reticulate structure containing cellular chromatin and the spatial arrangements of the four proteins within the induced reticulum were the same as those within VS reticulum, suggesting that the two reticula are formed by a similar mechanism.

Alpha-Herpesvirus Infection Induces the Formation of Nuclear Actin Filaments

Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process.

Interferon‐inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication†

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis

Recombinant human cytomegaloviruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly refractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.

Nuclear perturbations following adenovirus infection.

Adenoviruses are processed and assembled in the nuclei of infected cells and thereby produce significant perturbations to their structure and function. As the complex interactions that occur in the nuclei of uninfected cells are not yet fully understood many of the changes seen on infection have been described mainly in morphological terms. This chapter attempts to place more recent findings into this context and demonstrates that adenoviruses are able to hijack many cellular processes and enzymes to their advantage. In particular, modifications to nuclear PODs and nucleoli have more recently been explored in greater detail.

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.

Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.

HIV1 infection of human monocytes and macrophages promotes induction or translocation of NF-KB-related factors

In 1991, we demonstrated, using electrophoretic mobility shift assays, that 3 different factors (termed B1, B2 and B3) with affinity for the KB-enhancer target sequence were specifically detected in nuclear extracts from HIV1-infected monocytes and macrophages. The B2 factor was induced in the nuclei of these cells only upon HIV1 infection. The B3 factor was only slightly evident in nuclei of uninfected cells but was readily detectable in nuclei of infected monocytes. Its expression remained very low in nuclei of HIV1-infected macrophages. In this paper, we demonstrate that the B2 factor is expressed in the cytosol of monocytes and macrophages as a DNA-binding protein, indicating that it is not associated with an inhibitor (IKB). This factor remained clustered in the cytosol and was translocated to the nuclei only after HIV1 infection. The B3 factor is detected in the cytosol only when cells are HIV1-infected. The role of HIV1 infection in the expression and the translocation of these factors is discussed.

Epitope Mapping of Japanese Encephalitis Virus Envelope Protein Using Monoclonal Antibodies against an Indian Strain

A panel of monoclonal antibodies (MAbs) raised against an Indian strain of Japanese encephalitis (JE) virus was used to map topographically the epitopes on the envelope protein. Two separate clusters of epitopes were revealed. On the basis of reactivity in haemagglutination inhibition (HI), neutralization (NT), passive protection and antibody-dependent plaque enhancement (ADPE) assays with the MAbs, five functional domains (A, B, C, D and E) were delineated. The flavivirus cross-reactive domain for HI (A) was distinct. The JE virus-specific domain for HI (B) was in continuum with those domains representing non-HI JE virus-specific MAbs (C) and flavivirus cross-reactive MAbs (D). Domain E, which mapped close to domain D was represented by two MAbs that reacted with both JE virus and uninfected cell nuclei. Four conclusions can be drawn. (i) Two distinct antigenic domains were associated with HI, (ii) HI and NT in vivo and in vitro were dissociated functions, (iii) ADPE activity was solely linked with the A domain and (iv) all MAbs reacting with epitopes in the B domain had HI/NT/protective activity but failed to show ADPE. The B domain might therefore be considered the most suitable for development of synthetic or genetically engineered vaccines.

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.

Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B.

Nuclei which were isolated from SV40 infected cells with a hypotonic detergent-free buffer were used to establish in vitro conditions which lead to transcription-termination at the attenuation site of SV40. This system allowed us to identify regulatory elements involved in transcription-termination by RNA polymerase B transcribing SV40. Transcription-termination at the attenuation site was found to be ionic strength dependent. Efficient termination occurred at low (100 mM NaCl) but not at high (100 mM (NH4)2 SO4 or 300 mM NaCl) ionic strength. When nuclei were prewashed with 300 mM NaCl, the efficiency of transcription-termination was low even when transcription was carried out at low ionic strength (100 mM NaCl). Efficient transcription-termination in the high salt prewashed nuclei was reconstituted by complementation with a high salt (300 mM NaCl) soluble factor extracted from nuclei of uninfected cells. In addition, the efficiency of transcription-termination was significantly reduced when ITP replaced GTP in the transcription reaction mixture. Our data indicate that a nuclear factor and RNA secondary structure are essential regulatory elements involved in transcription-termination by RNA polymerase B.

DNA methyltransferase induced by frog virus 3.

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin.

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.

Transient enhanced synthesis of a cellular protein following infection of Aedes albopictus cells with Sindbis virus

Sindbis or Chikungunya virus infection of Aedes albopictus cells led to the synthesis of maximum amounts of intracellular virus structural protein at approximately 24 hr postinfection. Subsequently, viral protein synthesis declined and by 3 days postinfection little capsid protein was synthesized in infected cells. Starting at 2 days postinfection and for a period of 2–3 days, infected cells synthesized large amounts of a predominantly nuclear protein with molecular weight 43K (P43). In addition, smaller quantities of four other nuclear proteins with molecular weights 80K, 71K, 41K, and 26K were also transiently synthesized. Several lines of evidence showed that P43 is very closely related to, if not identical to a 43K protein found in the nuclei of uninfected cells. First, the 43K protein from uninfected cells and P43 were indistinguishable on the basis of their electrophoertic mobilities in both one- and two-dimensional separations in denaturing poly-acrylamide gels. Second, both P43 and the 43K protein generated the same digestion products after treatment with Staphylococcus aureus V8 protease. Heterogeneous nuclear ribonucleoprotein (hnRNP) particles were isolated from the nuclei of both uninfected and virus infected A. albopictus cells 3 days postinfection. HnRNP particles sedimented at 39 S and contained, as their major constituent, a protein which comigrated in both one- and two-dimensional gels with P43. These data indicate that alphavirus infection of A. albopictus cells leads to a transient enhancement in the synthesis of a 43K hnRNA binding protein.

Accumulation of poliovirus proteins in the host cell nucleus.

Poliovirus (type 1, Mahoney) proteins were transported into the nuclei of HEp-2 cells, as demonstrated by means of electron microscopic autoradiography. Quantitative determinations of the proteins by electrophoresis showed that the relative amounts of precursor proteins (1a, 3b, 1b), of some of the intermediate polypeptides (3a, 3c, 5b, 2), and of one end product (VP1) were higher in the nucleus than in the cytoplasm. Other proteins (VPO, VP2, X, 4, 6a) were found in the same relative amounts in the nucleus as in the cytoplasm, whereas two proteins (VP3 and 6b) were excluded from the nucleus. The findings are discussed in view of the recently demonstrated inhibitory activity exerted by cytoplasmic extracts of poliovirus-infected cells on transcription in isolated nuclei of uninfected cells.

The problem of host DNA synthesis in murine cytomegalovirus-infected cells

Murine cytomegalovirus (MCMV) infection in mouse fibroblasts resulted in an early inhibition in thymidine incorporation into cell DNA. In contrast [32P]phosphate incorporation was only slightly inhibited, while the incorporation of deoxycytidine, deoxyadenosine, and deoxyguanosine continued unabated. The limiting factor in thymidine incorporation was a decrease in uptake by the infected cells. Uptake of the other nucleosides, however, was either normal or enhanced. Nuclear monolayers, derived from infected cells, incorporated thymidine triphosphate into cell DNA at the same rate as nuclei of uninfected cells. Measurement of total DNA content of the cells revealed no difference between infected and control cultures. Thus MCMV neither inhibits nor stimulates cellular DNA synthesis.

Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: Some characteristics of the reaction products

Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2(+)D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [gamma-(32)P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50 degrees C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that (32)P is incorporated into O-phosphoserine or O-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase, and that cell-specified nuclear proteins other than histones may serve as substrates for the enzyme.

Effect of Rimantadine on Influenza Virus Replication

SummaryRimantadine hydrochloride in concentrations up to 50 μg/ml inhibits the replication of A/WSN influenza virus in MDCK cells without causing visible cytopathic effects. Virus replication was inhibited when the drug was added immediately following infection, but not when added late in the replicative cycle. Rimantadine does not alter the structural integrity of influenza virus as measured by sedimentation profiles of purified virus in linear sucrose gradients. Addition of drug following the viral adsorption period blocked the stimulation of cellular DNA-dependent RNA polymerase II activity observed in untreated infected cells, but had no effect on RNA polymerase II activity isolated from uninfected cell nuclei. Rimantadine present 0-8 hr postinfection inhibited the formation of viral RNA-dependent RNA polymerase activity in infected cells, but when this enzyme was assayed in vitro, rimantadine at concentrations as high as 250 μg/ml did not significantly inhibit polymerase activity. Finally, rimantadin...

Inhibition of RNA synthesis in mouse myeloma cells infected with vesicular stomatitis virus

Infection of mouse myeloma cells (MPC-11) with vesicular stomatitis (VS) virus resulted in rapid and marked reduction in cellular RNA synthesis considerably before cell viability was compromised. Mouse myeloma cells responded maximally to viral infection at a multiplicity of 1 and were considerably more se;sitive to shut-off of RNA synthesis than were mouse L cells or BHK-21 cells. This inhibition of cellular RNA synthesis was shown not to be caused by differential membrane permeability of infected and uninfected MPC-11 cells to [3H]uridine, nor was it due to greater degradation of previously synthesized RNA. VS viral infection appeared not to impede transport of newly synthesized nuclear RNA to the cytoplasm; moreover, infected cells accumulated polyadenylated mRNA at the same rate as did uninfected cells. Polyacrylamide gel electrophoresis of newly synthesized nuclear RNA demonstrated that the polydisperse nature and size distribution were not affected by VS viral infection. Isolated nuclei of infected MPC-11 cells also inhibited greatly impaired capacity to synthesize RNA despite the absence of cytoplasmic factors. Infected-cell cytosol did not inhibit transcription by uninfected-cell nuclei, nor did uninfected-cell cytosol reverse viral inhibition of nuclear transcription. Studies with alpha-amanitin revealed that VS viral infection inhibited the activity of polymerases I, II, and III, but only polymerase II was affected progressively throughout infection and to a much greater extent. These data suggest that, even at low multiplicities of infection, VS virus rapidly shuts off cellular RNA synthesis at the level of nuclear transcription.

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.