Alice S. Huang

Ribonucleic acid synthesis of vesicular stomatitis virus: III. Multiple complementary messenger RNA molecules☆

Abstract Infection of Chinese hamster ovary cells by vesicular stomatitis virus (VSV), in the absence of any detectable interference by defective particles, leads to a replacement of host polyribosomes by a distinct class of smaller polyribosomes. These small polyribosomes synthesize only virus-specific polypeptides and are as active in protein synthesis as polyribosomes from uninfected cells. The 28 S and 13 S single-stranded RNA species made during viral replication are associated with the viral polyribosomes after separation of the polyribosomes by rate zonal and isopycnic centrifugations. Release of the 28 S and 13 S RNA species from polyribosomes by EDTA and hybridization of these RNA species to virion RNA shows that they have nucleotide sequences which are complementary to those of the virion RNA. The finding of multiple pieces of messenger RNA, all smaller than 40 S virion RNA, suggests that VSV polypeptides are synthesized from individual molecules of RNA, rather than from one large messenger RNA. These studies also demonstrate the presence of newly synthesized nucleocapsid-like structures which contain heterogeneous 28–40 S RNA.

Interaction of HeLa cell proteins with RNA

Abstract When any RNA is added to a cytoplasmic extract from HeLa cells there is a large increase in the rate of sedimentation of the RNA (Girard & Baltimore, 1966). Cytoplasmic extracts also cause RNA to be retained by a membrane filter. The same material appears to be responsible for these two effects; it has been called “binding factor” A number of lines of evidence indicate that binding factor is a heterogeneous collection of soluble proteins which are bound to RNA by ionic forces and are probably not very basic: (1) centrifugation for 60 minutes at 105,000 g does not sediment the binding factor; (2) the active material can be bound to, and eluted from, DEAE-cellulose but fractions eluted at different salt concentrations have approximately the same specific activity; (3) it is sensitive to pronase and trypsin; (4) it is not active in 0.5 m -NaCl; (5) it causes RNA to band at a buoyant density of 1.40 in CsCl; (6) the same material will bind to different RNAs and (7) basic proteins will cause RNA to be retained by a filter but they precipitate the RNA rather than form a soluble complex like that formed by binding factor and RNA.

Isopycnic Separation of Subcellular Components from Poliovirus-Infected and Normal HeLa Cells

A rapid method for determining the density of ribonucleoprotein particles and complexes has been developed. The method involves glutaraldehyde fixation of sucrose gradient fractions and immediate centrifugation for 5 hours through preformed cesium chloride gradients. There is little or no aggregation of particles, so that components which co-sediment in sucrose gradients are resolved by the cesium chloride gradient. By this method the densities of HeLa cell ribosomes, polyribosomes, and subribosomal particles have been determined. Furthermore, the poliovirus replication complex has been separated from polyribosomes and its density has been found to be unaffected by treatment with ethylene-diaminetetraacetic acid.

Initiation of polyribosome formation in poliovirus-infected HeLa cells

Abstract Poliovirus RNA is synthesized in the replication complex and is released free of ribosomes. Much of the RNA then goes on to become part of polyribosomes, a process requiring at least ten minutes. As isolated in low ionic strength buffer, the viral RNA recovered from polyribosomes is coated with protein and has a buoyant density in CsCl of 1.40. Dissociation of polyribosomes with puromycin leads to two predominant complexes banding at buoyant densities of 1.44 and 1.47. Similar complexes are formed by viral RNA synthesized in the presence of cycloheximide and can be shown to be precursors of polyribosomes.

Complementarity between messenger RNA and nuclear RNA from HeLa cells

Abstract Hybridization of labeled HeLa cell messenger RNA to HeLa cell nuclear RNA occurs as determined by ribonuclease resistance of annealed mixtures of the two RNAs. Hybrids were characterized by melting temperature and by centrifugation to equilibrium in Cs 2 SO 4 gradients. No annealing to nucleolar RNA, f2 phage RNA or homopolymers, was detected. The apparent complementarity of nuclear RNA and messenger RNA probably reflects an aspect of messenger RNA synthesis.