Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Minor histocompatibility antigens as determinants for graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

Minor histocompatibility antigens (minor H antigens) are genetically polymorphic peptides that have been shown to elicit immune response when mismatched between donor and recipient of haematopoietic stem cell transplantation (HSCT). Depending on the expression profiles, mismatches in these genes may either lead to harmful graft-versus-host (GvH) reaction or desired graft-versus-leukaemia (GvL) effect. We analysed retrospectively the effect of HLA-restricted matching 11 established autosomal minor H antigens on the risk of graft-versus-host disease and relapse in 311 HLA-matched sibling HSCT of a single centre. Increased incidence of chronic GvH disease was shown to be associated with mismatches in the HA-8 and ACC-1. The mRNA expression profiles in a large set of healthy and malignant tissue samples of minor H antigen genes demonstrated in silico that the expression profiles of HA-8 and ACC-1 were surprisingly different: HA-8 gene was expressed in practically all tissues, whereas ACC-1 gene had a restricted profile. The results demonstrated that mismatches in minor H antigens HA-8 and ACC-1 predisposed to chronic graft-versus-host disease (GvHD).

Recruitment of the tumour suppressor protein p73 by Kaposi's Sarcoma Herpesvirus latent nuclear antigen contributes to the survival of primary effusion lymphoma cells.

Kaposi's Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi's Sarcoma (KS) and two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease (MCD). The KSHV latency-associated nuclear antigen-1 (LANA), required for the replication and maintenance of latent viral episomal DNA, is involved in the transcriptional regulation of viral and cellular genes and interacts with different cellular proteins, including the tumour suppressor p53. Here, we report that LANA also recruits the p53-related nuclear transcription factor p73, which influences cellular processes like DNA damage response, cell cycle progression and apoptosis. Both the full-length isoform TAp73α, as well as its dominant negative regulator ΔNp73α, interact with LANA. LANA affects TAp73α stability and sub-nuclear localisation, as well as TAp73α-mediated transcriptional activation of target genes. We observed that the small-molecule inhibitor Nutlin-3, which disrupts the interaction of p53 and p73 with MDM2, induces apoptotic cell death in p53 wild-type, as well as p53-mutant PEL cell lines, suggesting a possible involvement of p73. The small-molecule RETRA, which activates p73 in the context of mutant p53, leads to the induction of apoptosis in p53-mutant PEL cell lines. RNAi-mediated knockdown of p73 confirmed that these effects depend on the presence of the p73 protein. Furthermore, both Nutlin-3 and RETRA disrupt the LANA-p73 interaction in different PEL cell lines. These results suggest that LANA modulates p73 function and that the LANA-p73 interaction may represent a therapeutic target to interfere with the survival of latently KSHV-infected cells.

Kaposi's sarcoma herpesvirus lytic replication compromises apoptotic response to p53 reactivation in virus-induced lymphomas.

Primary effusion lymphomas (PELs) are aggressive Kaposi's sarcoma herpesvirus (KSHV)-induced malignancies with median survival time <6 months post-diagnosis. Mutations in the TP53 gene seldom occur in PELs, suggesting that genetic alterations in the TP53 are not selected during PEL progression. We have reported that p53 reactivation by an inhibitor of the p53-MDM2 interaction, Nutlin-3, induces selective and massive apoptosis in PEL cells leading to efficient anti-tumor activity in a subcutaneous xenograft model for PEL. Here, we show compelling anti-tumor activity of Nutlin-3 in the majority of intraperitoneal PEL xenografts in vivo. Interestingly, our results demonstrate that spontaneous induction of viral lytic replication in tumors could drastically attenuate the p53-dependent apoptotic response to Nutlin-3. Moreover, viral reactivation compromised p53-dependent apoptosis in PEL cells treated with genotoxic anti-cancer agents doxorubicin and etoposide. We have recently demonstrated that the Ser/Thr kinases Pim 1 and 3 are required to trigger induction of the lytic replication cascade of KSHV. We have now assessed the ability of a novel Pim kinase inhibitor to restore the Nutlin-3-induced cytotoxicity in lytic PEL cells. PEL cells induced to lytic replication by phorbol esters showed 50% inhibition of active viral replication following treatment with the Pim kinase inhibitor. Importantly, co-treatment of these cells with the kinase inhibitor and Nutlin-3 resulted in a robust restoration of the Nutlin-3-induced cell death. These results highlight the potential impact of activation of viral lytic replication on disease progression and response to treatment in KSHV-induced lymphomas.

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Integration of electrokinetics and chemical oxidation for the remediation of creosote-contaminated clay.

Remediation of clayey soils that are contaminated with polycyclic aromatic hydrocarbons (PAHs) is a challenging task that may require integration of several technologies. The benefits of integrating in situ electrokinetic remediation with chemical oxidation were evaluated in laboratory-scale experiments lasting for 8 weeks. A voltage gradient of 48 V/m of direct current and 4.7 V/m of alternating current and periodic additions of chemical oxidants were applied to creosote-contaminated soil. Electrokinetically enhanced oxidation with sodium persulphate resulted in better PAH removal (35%) than either electrokinetics (24%) or persulphate oxidation (12%) alone. However, the improvement was shown only within 1/3 (5 cm) of the soil compartment. Electrokinetics did not improve the performance of Fenton oxidation. Both chemical oxidants created more positive oxidation-reduction potential than electrokinetic treatment alone. On the other hand, persulphate treatment impaired the electroosmotic flow rate. Elemental analyses showed reduction in the natural Al and Ca concentrations, increase in Zn, Cu, P and S concentrations and transfer of several metal cations towards the cathode. In conclusion, the results encourage to further optimisation of an integrated remediation technology that combines the beneficial effects of electrokinetics, persulphate oxidation and Fenton oxidation.

Electrophoretic signal comparison applied to mRNA differential display analysis.

Gene expression analysis by electrophoretic methods is currently limited by the labor-intensive visual evaluation of the electrophoretic signal profiles. For this purpose, we present a flexible approach to computer-assisted comparison of quantitative electrophoretic patterns between multiple expression signals. Gaussian curves are first fitted to the complex peak mixtures, and the resulting approximate signals are then aligned and compared on a peak-by-peak basis with respect to specific patterns defined by the investigator. The rationale of the method is to produce a compressed list of exceptional expression patterns quantified by a set of associated numeric features. A score value is attached to each pattern in such a way that large values identify the most potential findings to be focused on in visual analysis instead of the vast amount of original electrophoretic results. The validity of the method is demonstrated by analyzing a large set of electrophoretic data from mRNA differential display experiments monitoring changes in gene expression patterns in human colonic carcinoma. The automated identification of variously defined gene expression patterns agrees well with the visual evaluation of the same electropherograms. The general comparison approach may also be found useful with other gene expression profiling instruments.

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.

CAK-independent activation of CDK6 by a viral cyclin.

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Automated detection of differently expressed fragments in mRNA differential display.

We present a novel method for the automated detection of fragments showing dissimilar expression in mRNA differential display. The analysis is based on aligning the numerical electrophoretic lane data in respect of a given distance function defined on a set of fragments, or signal peaks in general. We presume that significant dissimilarities between peaks result in extreme score values computed for aligned peak pairs. Whereas in sequence comparison, an overall sequence similarity score is conventionally used, the current method defines a special dissimilarity score for searching the peak pairs showing the largest relative differences between the lanes. The output of the analysis is a highly reduced list of peak pairs, along with a set of associated features extracted from the lanes. Only the peaks of this list need to be visually confirmed instead of the vast amount of peaks in the original electrophoretic results. The results obtained by the algorithm correlate well with results of visual evaluation of the same electropherograms. The current algorithm may be applied to the study of complex expression patterns in multiple lanes and, in general, to automated recognition of variously defined patterns of quantitative electrophoretic data.

The apoptotic v-cyclin-CDK6 complex phosphorylates and inactivates Bcl-2.

v-cyclin encoded by Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV or HHV8) associates with cellular cyclin-dependent kinase 6 (CDK6) to form a kinase complex that promotes cell-cycle progression, but can also induce apoptosis in cells with high levels of CDK6. Here we show that whereas HHV8-encoded v-Bcl-2 protects against this apoptosis, cellular Bcl-2 has lost its anti-apoptotic potential as a result of an inactivating phosphorylation in its unstructured loop region. Moreover, we identify Bcl-2 as a new substrate for v-cyclin-CDK6 in vitro, and show that it is present in a complex with CDK6 in cell lysates. A Bcl-2 mutant with a S70A S87A double substitution in the loop region is not phosphorylated and provides resistance to apoptosis, indicating that inactivation of Bcl-2 by v-cyclin-CDK6 may be required for the observed apoptosis. Furthermore, the identification of phosphorylated Bcl-2 in HHV8-positive Kaposi's sarcoma indicates that HHV8-mediated interference with host apoptotic signalling pathways may encourage the development of Kaposi's sarcoma.

Distribution of group II phospholipase A2 protein and mRNA in rat tissues.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.

Analysis of similarity of electrophoretic patterns in mRNA differential display.

We present a novel method of statistical analysis for the comparison of electrophoretic data. The method is based on the squared Euclidian distance of normalized signal data vectors of electrophoretic lanes. The differences in the electrophoretic patterns are evaluated by a statistical test based on Hubert's statistics which measures the significance of the signal grouping. We demonstrate the validity and applicability of the method in a large data set derived from automated fluorescent mRNA differential display analysis of the expression of acute-phase proteins during experimental Escherichia coli infection in mice. The current testing method is capable of finding theoretically similar natural groupings to be similar in a statistically significant way whereas theoretically dissimilar or random groupings can be recognized to be artifactual. We also show how the calculated pairwise signal distances can be utilized in methodological problem solving. These analytical methods can be applied to the study of other related problems of similarity analysis of electrophoretic patterns, and also provide useful tools for the development of automated recognition of differentially expressed mRNAs.

mRNA differential display of acute-phase proteins in experimental Escherichia coli infection.

We present a modification of mRNA differential display in which increased throughput results from the use of an automated fluorescent sequencer. The sequence analysis is performed directly on purified fragments without further cloning. The amplified fragments carry a T7 RNA polymerase promoter sequence tag for in vitro transcription of riboprobes for nonradioactive in situ hybridization. We compared changes in gene expression in the liver and colon of group II phospholipase A2 transgenic and group II phospholipase A2 deficient mice during the course of experimental Escherichia coli infection. Fluorescent mRNA differential display comprising a 7 x 24 set of primers was used to study a total of 31,257 amplified cDNA fragments. Sequence analysis of the displayed fragments associated with infection identified classical acute-phase proteins in the liver and host defense proteins in the colon. The displayed mRNAs associated to transgenicity were the transgene itself, i.e., human group II phospholipase A2, and glutathione-S-transferase in the liver. In the colon, the displayed mRNAs associated with transgenicity were the pancreatitis-associated protein and mucin. The results show that fluorescent mRNA differential display is a reliable method to identify differences in the expression of the genes of acute-phase proteins.

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro.

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.

Kaposi's sarcoma-associated herpesvirus-encoded v-cyclin triggers apoptosis in cells with high levels of cyclin-dependent kinase 6.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a key etiological role in development of Kaposi's sarcoma (KS). v-Cyclin is a KSHV-encoded homologue to D-type cyclins that associates with cellular cyclin-dependent kinase 6 (CDK6). v-Cyclin promotes S-phase entry of quiescent cells and has been suggested to execute functions of both D- and E-type cyclins. In this study, expression of v-cyclin in cells with elevated levels of CDK6 led to apoptotic cell death after the cells entered S phase. The cell death required the kinase activity of CDK6 because cells expressing a kinase-deficient form of CDK6 did not undergo apoptosis upon v-cyclin expression. Studies on the mechanisms involved in this caspase-3-mediated apoptosis indicated that it was independent of cellular p53 or pRb status, and it was not suppressed by Bcl-2. In contrast, the KSHV-encoded v-Bcl-2 efficiently suppressed v-cyclin-/CDK6-induced apoptosis, demonstrating a marked difference in the antiapoptotic properties of c-Bcl-2 and v-Bcl-2. In KS lesions, high CDK6 expression was confined to a subset of cells, some of which displayed signs of apoptosis. These results suggest that v-cyclin may exert both growth-promoting and apoptotic functions in KS, depending on factors regulating CDK6 and v-Bcl-2 levels.

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.