Further characterization of the replicative complex of vesicular stomatitis virus.

Replicating vesicular stomatitis virus ribonucleoprotein (RNP) complexes were isolated in nonequilibrium Renografin density gradients. These nascent RNPs had the same buoyant density as virion nucleocapsids in both isopycnic Renografin and CsCl gradients. Both transcribing and replicating RNP complexes were shown to be stable in sucrose gradients, whereas only replicating RNP complexes were stable in Renografin gradients. Size analysis of the 5-min-pulse-labeled RNA species from the replicating RNPs using methylmercury gels revealed that the nascent strands were primarily less than full-length molecules. Longer times of radiolabeling demonstrated that the nascent RNA accumulated as 42S RNA, which was primarily of the same sense as the virion strand when it was radiolabeled at 5 h postinfection. The percentage of this radiolabeled RNA which was plus stranded was higher at 2.5 h postinfection, reflective of the shift in plus- to minus-stranded full-length 42S RNA synthesis which occurs in the cell. Addition of cycloheximide to the infected cells before the addition of the radiolabel prevented the formation of these RNP complexes. Both the change in the percentage of minus strands found in the RNP complexes at the different times postinfection and the sensitivity to cycloheximide indicate that the RNP complex which was isolated was indeed the replicative complex.     

Density Gradient Centrifugation of Rubella Virus

Rubella virus was centrifuged in sucrose density gradients. One of two densities could be ascribed to the virus, depending upon the suspending medium used. The virus was found at a density of 1.16 g/cm³ after centrifugation for 18 hr in sucrose gradients prepared in distilled water. By contrast, when the sucrose gradients were prepared in tris(hydroxymethyl)aminomethane (Tris)buffer containing ethylenediaminetetraacetic acid (EDTA), the virus was found at a density of 1.18 g/cm³ after 18 hr of centrifugation. The virus banded at this higher density after only 2 hr of centrifugation when pretreated by overnight incubation in the Tris-EDTA buffer. A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1.16 g/cm³ after 2 hr of centrifugation to the higher 1.18 g/cm³ density after 18 hr. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.16 g/cm³ density. The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. However, it is possible that the two densities observed were a reflection of the existence of rubella virus in different hydration states in the presence and absence of Tris buffer containing EDTA.     

Structure of the Ribonucleoprotein of Influenza Virus

The ribonucleoprotein (RNP) internal components of influenza virus were separated into distinct size classes by sedimentation in glycerol gradients and examined by electron microscopy by using positive staining with uranyl acetate. The large RNP have a peak in length distribution at 90 to 110 nm, the medium, at 60 to 90 nm, and the small, at 30 to 50 nm. These lengths can be correlated with the estimated molecular weights of the ribonucleic acids contained in the various RNP size classes. The RNP structure appears to consist of a strand which is folded back on itself and coiled in a regular double-helical arrangement.     

On the synthesis of viral ribonucleic acids and ribonucleoproteins in the submitochondrial system completely free of interfering cytoplasmic contaminations

Summary The synthesis of virus-specific macromolecules was studied in the reconstituted system containing inner membrane-matrix fraction from rat liver mitochondria and infectious RNA of Venezuelian equine encephalomyelitis (VEE) virus. In a series of preliminary experiments it was shown that isolated submitochondrial fraction was completely free of interfering cytoplasmic contaminations and particularly, of cytoplasmic 80S ribosomes. VEE RNA when added to submitochondrial system caused significant stimulation of RNA and protein synthesis. These processes were resistant to actinomycin D which inhibited profoundly the synthesis of proper mitochondrial macromolecules. The stimulating effect of VEE RNA in experiments with submitochondrial system was about three times higher than that with intact mitochondria. The stimulation of¹⁴C-amino acid incorporation increased as a function of incubation time; a certain lag-period being observed. The newly formed virus-specific RNA's and ribonucleoproteins were identified with the aid of sedimentation analysis. In particular, radioactive RNA's with sedimentation coefficients 40S and 26-18S were isolated from the incubated system. These RNA's are similar respectively to VEE genome RNA and doublestranded VEE replicative RNA. In double labelling experiments with³H-uridine and¹⁴Camino acids it was shown that VEE RNA induced synthesis of ribonucleoproteins containing newly formed RNA and protein. These RNP possessed sedimentation coefficients 60-80S, 140S and 300S in sucrose gradient and buoyant densities 1.32 and 1.50 g/cm³ in cesium chloride gradients. These properties of ribonucleoproteins synthesized de novo in submitochondrial system are close to those of RNP intermediates of VEE virus reproduction in the infected cells. We concluded that viral RNA could program virus-specific synthesis in the submitochondrial system under conditions that eliminated the contribution of cytoplasmic ribosomes.     

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.     

Subnuclear particles containing a small nuclear RNA and heterogeneous nuclear RNA

Five of the stable low molecular weight RNA species in the HeLa cell nucleus have been localized in RNP complexes in the cell nucleus. The two abundant species C and D and the three minor species F, G′ and H are found in RNP particles following two different methods of preparation. Sonication of nuclei releases the five small RNAs and also the hnRNA in RNPs that sediment in a range from 10 to 150 S. Alternatively, incubation of intact nuclei at elevated temperature and pH releases four of the small RNAs and degraded hnRNA in more slowly sedimenting structures.When nuclear RNPs obtained by sonication are digested with RNAase in the presence of EDTA, the hnRNA is degraded and the hnRNPs sediment at 30 S. The structures containing the small RNA species D are similarly shifted to 30 S particles by RNAase and EDTA but not by either agent alone. In contrast, the sedimentation of complexes containing species G′ and H are not altered by exposure to RNAase/EDTA and small RNA species C and F are unstable under these conditions.In isopycnic metrizamide/²H₂O gradients species D and hnRNA accumulate at a density characteristic of RNP particles. They have a similar but not identical distribution.Species D is released from large RNPs by salt concentrations of 0.1 m-NaCl or greater, while the hnRNA remains in large RNP particles. In contrast, the structures containing species G′ and H are stable in 0.3 m-NaCl. All five of the small nuclear RNA species and the hnRNAs are released from rapidly sedimenting complexes by the ionic detergent sodium deoxycholate.It is suggested that the low molecular weight RNA species play a structural role in RNP particles in the cell nucleus and that a subpopulation of species D may be associated with the particles that package the hnRNA.     

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.     

The elementary RNP fiber — not the higher order structure — determines the all-or-none disintegration behaviour of balbiani ring pre-messenger RNP particles upon RNase A treatment

Summary— Balbiani ring premessenger ribonucleoprotein (RNP) particles are built from a 7-nm RNP fiber which is tightly folded into a ring-shaped RNP ribbon. Isolated particles are known to disintegrate in an all-or-none fashion upon RNase A treatment. In the present study we investigated whether this mode of disintegration is dependent on an intact particle structure or is inherent in the 7-nm fiber. When treated at low ionic strength, the Balbiani ring (BR) particles lost their higher order structure and the 7-nm fiber was unpacked, as evidenced by sucrose gradient sedimentation and electron microscopy. When treated with RNase A, unfolded as well as intact particles disintegrated in the all-or-none fashion, with similar kinetics and without apparent intermediates. Proteinase K treatment, however, obliterated this pattern: the protein-free particle RNA degraded progressively. As the typical disintegration pattern of the particles was not altered by unfolding, but was lost by deproteinization, the all-or-none mode of disintegration is likely to be a property of the 7-nm RNP fiber.     

Identification of the protein cross-linked to 3′-terminus of 5 S RNA in rat liver ribosomal 60 S subunits

To cross-link the 3′-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with ¹²⁵I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the α-chymotrypsin digest of this ¹²⁵I-labeled protein-oligonucleotide complex was similar to that of the digest of ¹²⁵I-labeled L5 protein. The results indicate that L5 protein binds to the 3′-terminal region of 5 S RNA in rat liver 60 S subunits.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Mass isolation of pole cells from Drosophila melanogaster.

Crude cell suspensions were obtained from 3 × 10⁶ pregastrula staged embryos. These cells fractionated into three bands on Renografin density gradients. Using ultrastructural characteristics for identification, pole cells were localized in the two denser bands. EM analyses also revealed a striking difference in the frequency of lipid vacuoles found in the pole-cell cytoplasm versus the cytoplasm of other embryonic cells. This difference has enabled us to identify pole cells by light microscopy using a neutral lipid stain. Through detailed analyses of the Renografin fractions with this stain, we have shown that pole cells resolve into two to three density classes. Evidence is presented which suggests that these density classes reflect different developmental age classes. The size differences between the pole cells and other embryonic cells contained in the enriched pole-cell fractions from the density gradients has enabled us to use sedimentation velocity centrifugation for additional enrichment. A distinct lower band of cells was obtained which consisted of 80% pole cells. Using these procedures, 1–2 × 10⁷ pole cells can be obtained daily.     

Nucleostemin delays cellular senescence and negatively regulates TRF1 protein stability

Nucleostemin (NS) encodes a nucleolar GTP-binding protein highly enriched in the stem cells and cancer cells. To determine its biological activity in vivo, we generated NS loss- and gain-of-function mouse models. The embryogenesis of homozygous NS-null (NS^−/−) mice was aborted before the blastula stage. Although the growth and fertility of heterozygous NS-null (NS^+/−) mice appeared normal, NS^+/− mouse embryonic fibroblasts (MEFs) had fewer NS proteins, a lower population growth rate, and higher percentages of senescent cells from passage 5 (P5) to P7 than their wild-type littermates. Conversely, transgenic overexpression of NS could rescue the NS^−/− embryo in a dose-dependent manner, increase the population growth rate, and reduce the senescent percentage of MEFs. Cell cycle analyses revealed increased pre-G₁ percentages in the late-passage NS^+/− MEF cultures compared to the wild-type cultures. We demonstrated that NS could interact with telomeric repeat-binding factor 1 (TRF1) and enhance the degradation but not the ubiquitination of the TRF1 protein, which negatively regulates telomere length and is essential for early embryogenesis. This work demonstrates the roles of NS in establishing early embryogenesis and delaying cellular senescence of MEFs and reveals a mechanism of a NS-regulated degradation of TRF1.     

Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP

The influenza virus genome replicates in the host cell nucleus, and the progeny viral ribonucleoproteins (vRNPs) are exported to the cytoplasm prior to maturation. The influenza virus NS2 protein has a nuclear export signal (NES) and binds to M1. It is therefore postulated that vRNP is exported from the nucleus by binding to NS2 through M1. However, the significance of the association between NS2 and M1 for the nuclear export of vRNP is still poorly understood. We herein demonstrate that the C-terminal domain of NS2 (residues 81–100) is essential for M1 binding and the nuclear export of progeny vRNPs.

Structured summary

MINT-8057301, MINT-8057317: NS2 (uniprotkb:P03508) binds (MI:0407) to M1 (uniprotkb:P03485) by pull down (MI:0096)     

Polysome disassembly in cell extracts of cricket male accessory gland during sucrose gradient centrifugation

The stability of polysomes in cell extracts of cricket (Acheta domesticus) male accessory gland has been examined by sedimentation through a variety of step and linear sucrose gradients. After prolonged centrifugation there is a considerable decline in polysome content with a concurrent increase in monosomes. The extent of the reduction is more severe in step gradients, although the polysomes that remain show a typical profile on linear gradients.Evidence is presented which indicates that the reduction in polysome content is not due to nuclease action. The presence of detergents can affect the extent of disassembly but is not the principal cause. Comparison of [³H]leucine pulse-labelled gluteraldehyde-fixed and unfixed polysomes subjected to extended centrifugation reveals a release of nascent label near the top of the gradients in unfixed preparations. At least part of this displaced material is present as peptidyl-tRNA, suggesting that forced dissociation of polysomes rather than premature termination of nascent chains occurs as a consequence of sedimentation pressures. Comparison of the distribution of polyadenylic acid (poly(A)) sequences in sucrose gradients following short- and long-term centrifugation shows a shift of poly(A) containing RNA out of the polysome and into the pre-monomer region. It is concluded that sucrose gradient sedimentation results in the disassembly of a portion of the polysome population in the tissue examined. The implications with regard to the study of nonpolysomal messenger ribonucleoprotein and monomeric ribosomes are discussed.     

Repair of DNA strand breaks after gamma-irradiation of protoplasts isolated from cultured wild carrot cells

We have isolated protoplasts of cultured wild carrot (Daucus carota L.) cells, lysed them directly on top of alkaline sucrose gradients, and measured single-stranded DNA of molecular weight 1·10⁸ by velocity sedimentation. DNA sedimentation studies on γ-irradiated protoplasts indicate that the energy absorbed in DNA per strand break is 85 eV in air and 260 eV in nitrogen. Isolated wild carrot protoplasts can repair 50% of the DNA strand breaks within 5 min after a dose of 20 krad, and by 1 h none can be detected.     

Characterization of mycoplasmatales virus DNA

The DNA of the group L1 $\text{Mycoplasmatales}$ virus, MVL51, was analyzed using alkaline sucrose velocity sedimentation, neutral and alkaline CsCl isopycnic sedimentation, and treatment of the DNA with nucleases. These treatments show that the viral chromosome is a covalently linked single-stranded DNA circle of molecular weight 2×10⁶ daltons.