Infectious hepatitis A virus particles produced in cell culture consist of three distinct types with different buoyant densities in CsCl.

Although hepatitis A virus (HAV) released by infected BS-C-1 cells banded predominantly at 1.325 g/cm3 (major component) in CsCl, smaller proportions of infectious virions banded at 1.42 g/cm3 (dense HAV particles) and at 1.27 g/cm3 (previously unrecognized light HAV particles). cDNA-RNA hybridization confirmed the banding of viral RNA at each density, and immune electron microscopy demonstrated apparently complete viral particles in each peak fraction. The ratio of the infectivity (radioimmunofocus assay) titer to the antigen (radioimmunoassay) titer of the major component was approximately 15-fold greater than that of dense HAV particles and 4-fold that of light HAV particles. After extraction with chloroform, the buoyant density of light and major component HAV particles remained unchanged, indicating that the lower density of the light particles was not due to association with lipids. Light particles also banded at a lower density (1.21 g/cm3) in metrizamide than did the major component (1.31 g/cm3). Dense HAV particles, detected by subsequent centrifugation in CsCl, were indistinguishable from the major component when first banded in metrizamide (1.31 g/cm3). However, dense HAV particles recovered from CsCl subsequently banded at 1.37 g/cm3 in metrizamide. Electrophoresis of virion RNA under denaturing conditions demonstrated that dense, major-component, and light HAV particles all contained RNA of similar length. Thus, infectious HAV particles released by BS-C-1 cells in vitro consist of three distinct types which band at substantially different densities in CsC1, suggesting different capsid structures with varied permeability to cesium or different degrees of hydration.     

RNA associated with murine intracisternal type A particles codes for the main particle protein.

Intracisternal type A particles were isolated from MOPC-104E myeloma grown subcutaneously and from N 4 neuroblastoma cells in culture. Polyadenylated RNA was prepared from the particles and tested in a cell-free translation system derived from rabbit reticulocytes. RNA from the two sources directed the synthesis of multiple polypeptides with similar distributions of electrophoretic mobilities in sodium dodecyl sulfate-containing polyacrylamide gels, including one conponent of the same size as the major A-particle structural protein (73,000 daltons). Analysis of the RNAs by electrophoresis in methyl mercury-containing agarose gels revealed a 35S component common to A-particles from both cell types. This was a major component of the N4 preparations, whereas a 28S species predominated in the case of MOPC-104E. These two RNAs (35S from N4 cells and 28S from MOPC-104E), when isolated on isokinetic sucrose gradients, each directed the synthesis of a 73,000-molecular-weight polypeptide that comigrated on gels with authentic A-particle structural protein. Idnetity of the cell-free product was confirmed by two-dimensional analysis of the [35S]methionine-labeled tryptic peptides. The N4 RNA preparations also contained a major32S component which did not code effectively for the A-particle structural protein.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.     

Packaging of hepatitis delta virus RNA via the RNA-binding domain of hepatitis delta antigens: different roles for the small and large delta antigens.

Hepatitis delta virus (HDV) is composed of four specific components. The first component is envelope protein which contains hepatitis B surface antigens. The second and third components are nucleocapsid proteins, referred to as small and large hepatitis delta antigens (HDAgs). The final component is a single-stranded circular RNA molecule known as the viral genome. In order to study the mechanism of HDV RNA packaging, a four-plasmid cotransfection system in which each viral component was provided by a separate plasmid was employed. Virus-like particles released from Huh-7 cells receiving such a cotransfection were found to contain HDV RNA along with three proteins. Therefore, the four-plasmid cotransfection system could lead to successful HDV RNA packaging in vitro. The system was then used to show that the large HDAg alone was able to achieve a low level of HDV RNA packaging. Analysis of a variety of large HDAg mutants revealed that the RNA-binding domain was essential for viral RNA packaging. By increasing the incorporation of small HDAg into virus-like particles, we found a three- to fourfold enhancement of HDV RNA packaging. This effect was probably through a direct binding of HDV RNA, independent from that of large HDAg, with the small HDAg. The subsequent RNA-protein complex was packaged into particles. The results provided insight into the roles and functional domains of small and large HDAgs in HDV RNA packaging.     

Occurrence and properties of low molecular weight RNA components from cells at different taxonomic levels.

1. The occurrence and gel electrophoretic properties of low molecular weight RNA components (LMW RNA) have been studied in species at different taxonomic levels. The LMW RNA components apart from tRNA, 5S RNA and 5.5S RNA are called LMW*RNA. 2. The major components of LMW*RNA in mammalian cells are L, A, C and D, accounting for 0.1-0.7% of cellular RNA. The gel electrophoretic migration of components L, C, and D is similar in different mammals but the migration of component A shows differences. 3. Amphibia, reptiles and birds contain L, A, C and D in about the same amounts as mammals but slight differences in migration are seen for L, C and D. Component A is absent from the nucleated red blood cells of the chicken and the frog. 4. Sea urchins contain three LMW*RNA components with migrations different from L, A, C and D. These components account for about 0.1% of the cellular RNA. 5. Insects contain only one LMW*RNA component, migrating as component L. 6. Tetrahymena, Physarum and Mycoplasmas have one component which may be a counterpart to component L in higher cells. Yeast shows no LMW*RNA components. 7. In the multicellular species the occurrence and gel electrophoretic migration of LMW*RNA components are not related to tumorigenicity, developmental stage or origin of tissue.     

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

Thirty temperature-sensitive mutants of encephalomyocarditis virus have been isolated and partially characterized. Fifteen of these mutants are phenotypically RNA+ thirteen are RNA-, and two are RNA +/-. Six RNA + mutants, one RNA- mutants, and one RNA +/- mutant have virions which are more thermosensitive at 56 degree C than the wild-type virions. Hela cells infected at the nonpermissive temperature with any of the RNA+ mutants produced neither infective nor noninfective viral particles. The cleavage of the precursor polypeptides in cells infected with 11 of the RNA+ mutants was defective at the nonpermissive temperature. This defect in cleavage occurred only in those precursor polypeptides leading to capsid proteins.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

Latent infection of a new alphanodavirus in an insect cell line

Insect BTI-TN-5B1-4 (Tn5) cells have been used extensively with recombinant baculoviruses to express foreign genes. When a recombinant baculovirus containing the hepatitis E virus capsid protein gene was used to infect Tn5 cells, unknown virus particles in addition to the anticipated hepatitis E virus-like particles were produced in the infected cells. The unknown virus particles were 35 nm in diameter and contained RNA that was highly homologous to full-length RNA1 (3,107 bp) and RNA2 (1,383 bp) genomic RNAs of flock house virus. Surprisingly, both RNAs seen in these induced nodavirus particles could be amplified from commercially available Tn5 cells without infection with or induction by a baculovirus. The nucleotide sequences from the purified nodavirus particles and the normal Tn5 cells were identical, demonstrating that the Tn5 cells themselves were latently infected with a nodavirus. However, the generation of nodavirus particles was significantly stimulated by infection with recombinant baculoviruses. Phylogenetic analysis suggested that this new nodavirus belongs to the genus Alphanodavirus in the family Nodaviridae.     

Defective interfering particles of poliovirus. 3. Interference and enrichment

Interference with standard poliovirus growth resulting from co-infection of cells with standard virus and defective interfering particles has been investigated. At all time following infection, co-infected cells produced less standard progeny than cells infected only by standard virus. The total yield of physical particles and the percentage of standard virus among these particles was a linear function of the percentage of standard virus in the inoculum. The actual yield of standard virus thus varied as the square of the percentage of standard virus in the inoculum. The extent of interference could also be controlled by varying the time interval between initial infection of cells by one type of particle and superinfection by the other.Identical amounts of viral RNA and virus-specific polyribosomes are formed in co-infected or singly infected cells. Interference apparently results from the partitioning of these limited synthetic capacities between standard and defective interfering-specific RNA and protein synthesis. Standard and DI RNA appear to serve equally well as messenger RNAs because standard and DI-specific viral proteins are synthesized in ratios proportional to the ratio of standard to DI particles in the inoculum. Only standard RNA can direct the formation of capsid protein, so co-infected cells contain reduced amounts of the virion protein precursor, the procapsid. Standard and DI RNA are encapsidated with approximately equal efficiency. Thus interference results from equal participation in the intracellular events of the infection cycle by both types of particles.The progeny yield from co-infected cells was always enriched about 5 to 8% in DI particles. Progeny were produced in the enriched ratio throughout the infection cycle.     

Isolation of intracisternal type A-particles and associated high-molecular-weight RNA after cell disruption by nitrogen cavitation.

The liberation of intracisternal A-type particles from the endoplasmic reticulum of mouse plasmacytoma cells (line MPC-11) without the use of detergents, has been achieved by the technique of nitrogen cavitation. By this method cell rupture is due to a sudden decompression of a cell suspension after being equilibrated with nitrogen at high pressure, and this results in a relatively efficient release of A-type particles from the cisternae as judged by electron microscopy and the amount of A-particle-associated RNA that may be isolated. Comparative studies showed that at least 50% of the particles are released from the endoplasmic reticulum during nitrogen cavitation. Since the release is achieved purely by mechanical means, the A-particle preparation obtained may be useful for assaying these particles for biological activity. A further release of particles from the endoplasmic reticulum may be achieved by a combination of detergent treatment and mechanical shearing. About 30% of the particle-associated RNA was polyadenylated. As determined by velocity centrifugation, the polyadenylated RNA in the preparation of A-particles released during nitrogen cavitation and that of the detergent-released A-particles were similar with respect to the high-molecular-weight RNA species present. A dominant 35 S RNA species with a 30 S shoulder was observed in addition to a 24 S component in both preparations, but no significant amount of 60–70 S RNA.     

Pancreatic microsomes; an integrated morphological and biochemical study

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, ( approximately 15 mmicro), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mmicro), limited by a thin membrane (7 mmicro) which bears small ( approximately 15 mmicro) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules ( approximately 150 mmicro) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small ( approximately 15 mmicro), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.     

Biochemical evidence associating membrane-encapsidated particles with reverse transcription in human gastric cancer.

We provide biochemical evidence demonstrating that membrane encapsidated structures present in terminal gastric patients share similar features with retrovirus-like particles. Purified particles reveal few polypeptides, one of them glycosylated. A ribonucleoprotein core-like component is recovered which retains the DNA-polymerizing activity and contains at least one RNA component which can be efficiently translated in vitro.     

Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids.

We have shown previously that COS-1 cells infected with a vaccinia virus recombinant (vTF7-3) which expresses the T7 RNA polymerase gene and then transfected with four pGEM-derived plasmids encoding the influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA polypeptides) can express a synthetic influenza virus-like chloramphenicol [correction of chloramphenical] acetyltransferase (CAT) RNA (I. Mena, S. de la Luna, C. Albo, J. Martín, A. Nieto, J. Ortín, and A. Portela, J. Gen. Virol. 75:2109-2114, 1994). Here we demonstrate that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids. The released particles can transfer the enclosed CAT RNA to MDCK cultures and resemble true influenza virions in that they require trypsin treatment to deliver the RNA to fresh cells and are neutralized by a monoclonal antibody specific for the influenza A virus hemagglutinin. Moreover, analysis by electron microscopy showed that the culture medium harvested from the transfected cells contained vesicles that could be labeled with an anti-HA monoclonal antibody and that were similar in size and morphology to authentic influenza virus particles. It is also shown that detection of recombinant particles capable of transmitting the CAT RNA does not require expression of the influenza virus nonstructural protein NS1. All of these data indicate that influenza virus-like particles enclosing a synthetic virus-like RNA can be assembled in cells expressing all viral structural components from recombinant plasmids.     

7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles

The virion incorporation of 7SL, the RNA component of the host signal recognition particle (SRP), has been shown for several simple retroviruses. Data here demonstrate that 7SL is also packaged by HIV-1, in sevenfold molar excess of genomic RNA. Viral determinants of HIV-1 genome and primer tRNA packaging were not required for 7SL incorporation, as virus-like particles with only minimal assembly components efficiently packaged 7SL. The majority of 7SL within cells resides in ribonucleoprotein complexes bound by SRP proteins, and most SRP protein exists in signal recognition particles. However, Western blot comparison of virion and cell samples revealed that there is at least 25-fold less SRP p54 protein per 7SL RNA in HIV-1 particles than in cells. Comparing 7SL:actin mRNA ratios in virions and cells revealed that 7SL RNA appears selectively enriched in virions.     

Comparison of In Vitro and Cell-Mediated Alteration of a Human Rhinovirus and Its Inhibition by Sodium Dodecyl Sulfate

After human rhinovirus type 2 (HRV-2) attaches to HeLa cells, two types of subviral particles are formed which closely resemble particles produced in vitro by acid or heat. One type of particle contains RNA whereas the second sediments as an empty capsid and is RNA-deficient. Sodium dodecyl sulfate (SDS) at 10(-4) M inhibits the cell-mediated formation of these particles from HRV-2 virions and the ability of HRV-2 to form plaques, but it does not inhibit the formation of plaques by human rhinovirus 14 (HRV-14). SDS also stabilizes HRV-2 against inactivation by acid or heat to a much greater extent than it does HRV-14. In a similar manner, SDS protects against the acid inactivation of the subpopulation of HRV-2 natural top component particles which attach to virus-specific cellular receptors. This suggests that the loss of native properties of natural top component particles and of virion are related processes. The basis for this alteration and also its role in infection are discussed.     

Identification of a 60-kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes

Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at greater than 1500S; (b) in ribonucleoprotein particles that sediment at 20-120S and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120-360S. We have compared the RNA and protein components of the first two of these RNP fractions. The polyadenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril-associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibril-associated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre-mRNP). RNA-binding experiments demonstrate that the polypeptide most tightly bound in pre-mRNP is a 54-kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60-kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre-mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre-mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA-associated protein in blocking translation are discussed.