The RNA replication complexes of mammalian positive-stranded RNA viruses are generally associated with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the associated membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products containing different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphological studies and labeling experiments argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is associated and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP 2b (previously named G S ), GP 3 , GP 4 , and GP 5 (previously named G L ). Proteins N, M, and GP 5 are major virion components, E occurs in virus particles in intermediate amounts, and GP 4 , GP 3 , and GP 2b are minor structural proteins. The M and GP 5 proteins occur in virus particles as disulfide-linked heterodimers while the GP 4 , GP 3 , and GP 2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP 2b , GP 3 , or GP 4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP 2b , GP 3 , and GP 4 proteins into viral particles. EAV particles lacking GP 2b , GP 3 , GP 4 , and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP 2b /GP 3 /GP 4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.Equine arteritis virus (EAV) is the prototypic member of the Arteriviridae family (order Nidovirales), which also includes lactate dehydrogenase-elevating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), and simian hemorrhagic fever virus (4). Virus particles have a diameter of approximately 60 nm and consist of a 12.7-kb RNA genome of positive polarity that is packaged by the 14-kDa nucleocapsid protein (N) into a putatively icosahedral core, which is surrounded by a lipid-containing envelope with small surface projections (17,20).In the EAV envelope six viral proteins have been identified (6, 27, 43). The 16-kDa unglycosylated membrane protein M and the heterogeneously glycosylated GP 5 (previously named G L ) protein of 30 to 42 kDa are the most abundant envelope proteins and occur in virions as covalently linked heterodimers (7, 26). The membrane topology of the EAV M protein is unknown but its hydropathy profile resembles that of the LDV M protein. The latter protein was previously shown to be a triple-spanning membrane protein having its amino terminus at the outside of the virion and its carboxy terminus at the inside (12). The EAV M protein is, therefore, assumed to contain three internal transmembrane segments...

Pit cells--a new type of cell first described here and so named because they contain highly characteristic granules--are situated in the wall of rat liver sinusoids, and have hyaloplasmic pseudopodia intermingling with the microvilli of the parenchymal cells. The characteristic granules are mainly situated at one side of the nucleus, the other side showing organelle-free hyaloplasm. Pit cells are also found in portal tracts and in granuloma-like cellular aggregates. They also occur in rat peripheral blood, although there are morphological differences between cells in these two sites. Pit cells can be regarded as regular inhabitants of the sinusoidal wall, and therefore belong to the series of sinusoidal cells, i.e., the endothelial (Wisse, 1972), Kupffer (Widmann et al., 1972; Wisse and Daems, 1970; Wisse, 1974a, b), and fat-storing cells (Ito, 1973). Pit cells do not phagocytose and do not react to a great number of experimental conditions, to which endothelial and Kupffer cells do react (Wisse, 1972, 1974b). Mitosis has been observed in a pit cell. The function of pit cells remains obscure, but an endocrine function is suggested by the morphology of their highly characteristic granules.

Degradation of three types of sintered calcium phosphate ceramic spheres was investigated in vitro at low pH conditions (LPC) and in an in vivo model, that is, injection into a mouse peritoneal cavity. Degradation was observed under both conditions. The rate of degradation depended on the type of ceramic, with beta-TCP degrading faster than HA and HA degrading faster than FA. Degradation was characterized by dissolution of the necks and the formation of cracks and irregularities in the grains. Intraperitoneal injection of the spheres into a mouse peritoneal cavity led to the formation of foreign body granulomas in which degradation could be observed. The in vivo degradation pattern was similar to that observed in vitro, but longer implantation times resulted in a further degradation. Small fragments rich in Ca and P were present in inclusion bodies. Calcium phosphate crystals sometimes also were observed in mitochondria, many of which were subject to lysis. We observed that ceramic type and implantation period also were related to the number of dead cells in the granulomas. Furthermore, extracellular deposits were seen between cells and ceramic spheres. Ca and P and also Fe were detected in these deposits. The presence of Fe is indicative of a lysosomal origin and thus of exocytotic activity.

The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.

The 5 UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C⅐C and C⅐A mismatches. In this article, evidence is presented indicating that the 5 proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C⅐C and C⅐A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5 proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C⅐C and͞or C⅐A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.T urnip yellow mosaic virus (TYMV) is a nonenveloped plant virus and the type member of the genus Tymovirus. The virion is an icosahedral particle with T ϭ 3 symmetry, and the protein shell consists of 180 identical subunits (1). The genomic RNA is 6,318 nt long and has an unusually high cytosine content (38%) and a rather low guanine content (18%). The genome carries three ORFs coding for the overlapping protein, which is thought to be involved in cell-to-cell transport, the RNAdependent RNA polymerase polyprotein, and the coat protein, which is expressed from a subgenomic messenger RNA. The 3Ј UTR has been shown to contain a tRNA-like structure whose function is still unclear (2).The 5Ј UTR of TYMV RNA is 90 nt long and has been shown, by means of structure probing and sequence comparison, to harbor two simple hairpins containing symmetric internal loops, which consist of C⅐C and C⅐A mismatches (ref. 3; see Fig. 1A). Under slightly acidic conditions these mismatches can form rather stable base pairs. NMR and UV melting experiments have confirmed that protonation of the C⅐C and C⅐A mismatches in hairpin 2 (HP2) stabilizes the structure when the pH is lowered from 7 to 5 (refs. 3 and 4; see Fig. 1B). A preliminary study of the possible function of the protonatable cytosines in the two 5Ј UTR hairpins suggested a dependence of TYMV on these nucleotides to establish a successful infection (5). A role for these hairpins in encapsidation was proposed. However, conclusions about the apparent requirement of C⅐C and͞or C⅐A mismatches were preliminary, as this study described one single mutant for which only a few revertants were recovered after passaging in Chinese cabbage. Moreover, this construct could not solve the question of whether an A⅐C pair instead of C⅐A was also allowed, because the chosen mutation rather forced the recovery of C⅐C and C⅐A mismatches through single ...

A case of well-documented and -illustrated megakaryoblastic transformation is described in a patient with thrombocythemia passing through a stage of myelofibrosis without features of chronic granulocytic leukemia. Immunocytologic studies with the use of conventional and monoclonal antibodies against platelet membrane glycoproteins and electron microscopic investigations, demonstrating bull's-eye granules and platelet peroxidase positivity, proved the megakaryocytic differentiation of the blast cells. From the onset of the disease as well as during the megakaryoblastic transformation, the Philadelphia (Ph1) karyotype, 46XX t(9:22) (q34:q11), was found in peripheral blood and bone marrow cells as the only clonal abnormality. Southern blot analysis of DNA extracted from the blast cells revealed a rearrangement within the bcr on chromosome 22 similar to findings in chronic granulocytic leukemia. The presentation with excessive small and abnormal megakaryocytes in the initial and subsequent bone marrow and the rapid progressive myelofibrosis and splenomegaly differentiate the Ph1 chromosome-positive thrombocythemia from the chronic myeloproliferation of thrombocythemia in its primary form or associated with polycythemia vera.