Complex Membrane Remodeling during Virion Assembly of the 30,000-Year-Old Mollivirus Sibericum.

Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique.IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae," which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

p27T187A knockin identifies Skp2/Cks1 pocket inhibitors for advanced prostate cancer.

SCFSkp2/Cks1 ubiquitinates Thr187-phosphorylated p27 for degradation. Overexpression of Skp2 coupled with underexpression of p27 are frequent characteristics of cancer cells. When the role of SCFSkp2/Cks1-mediated p27 ubiquitination in cancer was specifically tested by p27 Thr187-to-Ala knockin (p27T187A KI), it was found dispensable for KrasG12D-induced lung tumorigenesis but essential for Rb1-deficient pituitary tumorigenesis. Here we identify pRb and p53 doubly deficient (DKO) prostate tumorigenesis as a context in which p27 ubiquitination by SCFSkp2/Cks1 is required for p27 downregulation. p27 protein accumulated in prostate when p27T187A KI mice underwent DKO prostate tumorigenesis. p27T187A KI or Skp2 knockdown (KD) induced similar degrees of p27 protein accumulation in DKO prostate cells, and Skp2 KD did not further increase p27 protein in DKO prostate cells that contained p27T187A KI (AADKO prostate cells). p27T187A KI activated an E2F1-p73-apoptosis axis in DKO prostate tumorigenesis, slowed disease progression and significantly extended survival. Querying co-occurrence relationships among RB1, TP53, PTEN, NKX3-1 and MYC in TCGA of prostate cancer identified co-inactivation of RB1 and TP53 as the only statistically significant co-occurrences in metastatic castration-resistant prostate cancer (mCRPC). Together, our study identifies Skp2/Cks1 pocket inhibitors as potential therapeutics for mCRPC. Procedures for establishing mCRPC organoid cultures from contemporary patients were recently established. An Skp2/Cks1 pocket inhibitor preferentially collapsed DKO prostate tumor organoids over AADKO organoids, which spontaneously disintegrated over time when DKO prostate tumor organoids grew larger, setting the stage to translate mouse model findings to precision medicine in the clinic on the organoid platform.

Femoral nerve infusion after primary total knee arthroplasty: a prospective, double-blind, randomised and placebo-controlled trial.

OBJECTIVES: Effective analgesia after total knee arthroplasty (TKA) improves patient satisfaction, mobility and expedites discharge. This study assessed whether continuous femoral nerve infusion (CFNI) was superior to a single-shot femoral nerve block in primary TKA surgery completed under subarachnoid blockade including morphine. METHODS: We performed an adequately powered, prospective, randomised, placebo-controlled trial comparing CFNI of 0.125% bupivacaine versus normal saline following a single-shot femoral nerve block and subarachnoid anaesthesia with intrathecal morphine for primary TKA. Patients were randomised to either treatment (CFNI 0 ml to 10 ml/h 0.125% bupivacaine) or placebo (CFNI 0 ml to 10 ml/h normal saline). Both groups received a single-shot femoral nerve block (0.25% 20 ml bupivacaine) prior to placement of femoral nerve catheter and subarachnoid anaesthesia with intrathecal morphine. All patients had a standardised analgesic protocol. The primary end point was post-operative visual analogue scale (VAS) pain score over 72 hours post-surgery. Secondary outcomes were morphine equivalent dose, range of movement, side effects, and length of stay. RESULTS: A total of 86 patients were recruited. Treatment and placebo groups were comparable. No significant difference was found in VAS pain scores, total morphine equivalent requirements, side effects, range of movement, motor block, or length of hospital stay. CONCLUSION: No significant advantage was found for CFNI over a single-shot femoral block and subarachnoid anaesthesia after TKA. Cite this article: Bone Joint Res 2015;4:11-16.

Antibacterial validation of electrogenerated hypochlorite using carbon-based electrodes.

UNLABELLED: This proof-of-concept study explores the novel use of carbon-based electrodes for the electrochemical generation of hypochlorite and compares the antimicrobial efficacy against commercial hypochlorite solution. Antimicrobial concentrations of hypochlorite were generated using pad-printed carbon and carbon fibre electrodes, yielding up to 0·027% hypochlorite in 60 min and 0·1% hypochlorite in 15 min, respectively, in a nondivided assembly. The minimum inhibitory concentration (MIC) of the electrogenerated hypochlorite produced using carbon fibre electrodes was established for four medically important bacteria (Pseudomonas aeruginosa and Staphylococcus aureus approx. 0·025%, Escherichia coli and Enterococcus faecalis approx. 0·012%) and found to be in agreement with those determined using commercial hypochlorite solution. Therefore, carbon-based electrodes, particularly carbon fibre, have proven effective for the generation of antimicrobial concentrations of hypochlorite. The similarity of the MIC values to commercial hypochlorite solutions suggests that the antimicrobial efficacy is derived from the quantified hypochlorite generated and not due to marked cogeneration of reactive oxygen species, as identified for other assemblies. As such, the application of carbon electrodes may be suitable for the local production of hypochlorite for healthcare antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: Carbon fibre electrodes can rapidly generate antimicrobial concentrations of hypochlorite; as such, these cheap and commercially available electrodes are proposed for the local production of hypochlorite for healthcare antisepsis. Importantly, the antimicrobial properties of the electrochemically generated hypochlorite mirror those of commercial hypochlorite, suggesting this is not enhanced by the cogeneration of reactive oxygen species. This illustrates the potential use of disposable carbon electrodes for localized small-volume production of hypochlorite for surface and skin cleansing, and opens a broader scope of research into the exploitation of carbon electrodes for this application.

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.

p21CIP1 mediates reciprocal switching between proliferation and invasion during metastasis.

Cell proliferation and invasion are critical for malignant progression, yet how these processes relate to each other and whether they regulate one another during metastasis is unknown. We show that invasiveness of breast cancer cells is associated with growth arrest due to p21CIP1 upregulation. Knockdown of p21CIP1 increases cell proliferation and suppresses invasion. Since p21CIP1 acts to inhibit cyclin E during cell-cycle progression, we demonstrated that a constitutively active form of cyclin E had similar effects to p21CIP1 inhibition resulting in enhanced cell growth and suppressed invasiveness. We tested these findings in vivo in the Polyoma middle T mammary tumor model in which p21CIP1 was deleted. p21CIP1 knockout mice exhibited dramatic suppression of metastasis, independent of tumor growth, which was rescued by p21CIP1. Metastasis suppression by p21CIP1 ablation was associated with striking cytoskeletal reorganization leading to a non-invasive and highly proliferative state. Thus, p21CIP1 regulates metastasis by mediating reciprocal switching between invasion and proliferation.

Triiodothyronine stimulates hepatocyte proliferation in two models of impaired liver regeneration.

OBJECTIVES: Liver regeneration is attenuated in old age and is substantially slower after 90% than after 70% partial hepatectomy (PH). We have previously demonstrated that the proliferative response to a primary mitogen is intact in aged mice, indicating that impaired liver regeneration is not due to loss of proliferative capacity. Here, we have investigated whether mitogenic effects of triiodothyronine (T3) could reverse the impaired regeneration of ageing or 90% hepatectomy, in the rat. MATERIALS AND METHODS: T3 (20 microg/100 g body weight) was administered to 14-month-old rats subjected to 70% PH or to young rats subjected to 90% PH. Cell-proliferative capacity was determined by bromodeoxyuridine incorporation and microscopy and changes of cell cycle-related proteins were analysed by Western blot analysis. RESULTS: Treatment of old intact rats with T3 increased cyclin D(1) expression that was followed by an enhanced proliferative response, the labelling index (LI), being 7.8% versus 1.3% of controls. T3 given before 70% PH stimulated regenerative response (LI was 10.8% versus 2.28%), and expression of cyclin D(1) and proliferating cell nuclear antigen (PCNA) 24 h after PH. Pre-treatment with T3 also improved the regenerative response of the liver after 90% hepatectomy (LI was 27.9% versus 14.2%). CONCLUSIONS: These findings show in principle that mitogen-induced hyperplasia could be applied to human therapy in patients with reduced regenerative capacity or massive loss of hepatocytes.

Inhibition of drug metabolism by blocking the activation of nuclear receptors by ketoconazole.

Individual variation in drug metabolism is a major cause of unpredictable side effects during therapy. Drug metabolism is controlled by a class of orphan nuclear receptors (NRs), which regulate expression of genes such as CYP (cytochrome)3A4 and MDR-1 (multi-drug resistance-1), that are involved in this process. We have found that xenobiotic-mediated induction of CYP3A4 and MDR-1 gene transcription was inhibited by ketoconazole, a commonly used antifungal drug. Ketoconazole mediated its effect by inhibiting the activation of NRs, human pregnenolone X receptor and constitutive androstene receptor, involved in regulation of CYP3A4 and MDR-1. The effect of ketoconazole was specific to the group of NRs that control xenobiotic metabolism. Ketoconazole disrupted the interaction of the xenobiotic receptor PXR with the co-activator steroid receptor co-activator-1. Ketoconazole treatment resulted in delayed metabolism of tribromoethanol anesthetic in mice, which was correlated to the inhibition of PXR activation and downmodulation of cyp3a11 and mdr-1 genes and proteins. These studies demonstrate for the first time that ketoconazole represses the coordinated activation of genes involved in drug metabolism, by blocking activation of a specific subset of NRs. Our results suggest that ketoconazole can be used as a pan-antagonist of NRs involved in xenobiotic metabolism in vivo, which may lead to novel strategies that improve drug effect and tolerance.

Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures.

Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 microm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.

Use of recombinant human erythropoietin in two-stage total knee arthroplasty for infection.

A multicenter prospective study was conducted to determine whether epoetin alfa could be used to lower transfusion requirements after two-stage exchange arthroplasty for infection. Forty-one consecutive patients undergoing successful two-stage exchange arthroplasty for an infected total knee arthroplasty were enrolled in a prospective study. Epoetin alfa (40,000 units) was administered subcutaneously after prosthesis resection and antibiotic spacer placement. Although there was no difference in the hemoglobin levels before resection arthroplasty or on postoperative Day 3 between the study group and the control group, hemoglobin levels before reimplantation were higher in the patients who received epoetin alfa (12.4 mg/dL; range, 9.3-15.1 mg/dL) compared with the control group (11.3 mg/dL; range, 8.1-14.4 mg/dL). Average increase in hemoglobin level in the interval between stages was higher in the treatment group (3.2 mg/dL; range, -0.7-6.8 mg/dL) than the control group (1.7 mg/dL; range, -1.9-6 mg/dL). The transfusion rate decreased from 83% of patients in the control group to 34% in the study group during reimplantation. In addition, overall incidence of transfusion for either stage improved from 89% in the control group to 44% in the patients treated with epoetin alfa. Perioperative epoetin alfa statistically increased the hemoglobin levels and decreased transfusion rates for patients undergoing two-stage revision for infected total knee arthroplasty.

Structure and assembly of intracellular mature vaccinia virus: isolated-particle analysis.

In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503-1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

Structure and assembly of intracellular mature vaccinia virus: thin-section analyses.

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper.

Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei.

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

The A17L gene product of vaccinia virus is exposed on the surface of IMV.

The p21 membrane protein of vaccinia virus (VV), encoded by the A17L gene, has been reported to localize on the inner of the two membranes of the intracellular mature virus (IMV). It has also been shown that p21 acts as a membrane anchor for the externally located fusion protein p14 (A27L gene). Since p14 is located on the surface of IMVs, it is hard to envision that p21 should be located only on the inner membrane. Our results from (i) immunoelectron microscopy, (ii) biotinylation, and (iii) protease treatment of purified IMVs showed that the N-terminus of p21 is exposed on the surface of virus particles, while the C-terminus is embedded in the membrane. Mono-specific antibodies to the N-terminus of p21 neutralize infection of VV while antibodies to the C-terminal domain do not. We suggest that p21 molecules are located both in the inner and in the outer membrane of IMV.

Immunophenotypic and genotypic markers of follicular center cell neoplasia in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCL) are a biologically and clinically heterogeneous entity. Although some DLBCL represent transformation of follicular lymphomas (FL), the proportion that is of follicular center cell (FCC) origin remains uncertain. Immunophenotypic and genotypic markers used to suggest a FCC origin for a lymphoma (bcl-6 and CD10 expression, lack of CD138 expression, bcl-2 rearrangements [R]) or to subdivide DLBCL (bcl-2 expression, bcl-6 R) were therefore investigated in 22 FL and 44 DLBCL using paraffin section immunostains and Southern blot/polymerase chain reaction analysis. All FL tested were bcl-6+ (19) and CD138- (22) with 16/19 also bcl-2 and CD10+ (classic phenotype), one bcl2+, CD10- (grade III) and two bcl2-, CD10+ (grade II or III). Bcl-2R was identified in 4/5 FL-GrI, 3/6 FL-GrII, and 1/3 FL-GrIII. Bcl-6R was found in 0/5, 2/4, and 0/3 FL, respectively. All but 3/41 DLBCL were bcl-6+ with 17/37 also bcl-2+ and CD10+. Three of these cases were also CD138+. Twelve bcl-6+ cases were bcl-2+, CD10-, six bcl-2-, CD10+, and two bcl-2-, CD10-. The three bcl-6- cases were bcl-2+, CD138- and two were CD10+. Bcl-2R was identified in 5/27 DLBCL with 4/5 bcl-2+, 3/4 tested CD10+ and 4/4 bcl-6+. Bcl-6R was identified in 7/26 including three with a classic FL phenotype. The vast majority of DLBCL in this study have an immunophenotype that supports a FCC origin. Although the proportion of DLBCL that co-expressed bcl-6, CD10 and bcl-2 was lower than for the FL, absence of bcl-2 or CD10 may be associated with higher grade FL It is also possible that bcl-6 expression is not completely specific for a FCC origin. Only a minority of cases suggested postfollicular differentiation. Only a minority of DLBCL show bcl-2R, suggesting that many have a different molecular pathogenesis than most low-grade FL. Bcl-6R did not exclude a FCC origin.

Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line.

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV.

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice.

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.

Distant enhancers stimulate the albumin promoter through complex proximal binding sites.

The albumin-alpha-fetoprotein locus epitomizes the main features of transcriptional regulation of fetal and adult hepatocyte-specific genes: developmentally regulated promoters and strong distant enhancers. Full enhancer activity required only a proximal albumin-promoter region containing the TATA box, hepatic nuclear factor 1 (HNF1), and nuclear factor Y (NF-Y) sites. Deletion of the HNF1 site abrogated enhancer and promoter activity, whereas methylation of the site reduced all activity by about 3-fold. Deletion of the NF-Y site attenuated activity by about half, but much of the activity could be replaced by juxtaposition of an upstream region (designated distal element IV). Gel shift and competition analysis demonstrated that binding of architectural factors overlapped NF-Y binding. Moreover, a mutation that eliminated NF-Y binding but only minimally perturbed the surrounding region did not affect enhancer function. In plasmids with a second promoter, the enhancers simultaneously stimulated both albumin and alpha-fetoprotein promoters with minimal competition, but surprisingly some mutations in the albumin promoter attenuated expression from both promoters, whereas another uncoupled their expression. With single promoters, the function of the proximal promoter region was controlled by three parameters in the following hierarchy: HNF1 binding > local architecture > NF-Y binding, but integrated two-promoter function had a much greater dependence on NF-Y.

Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure.

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.